confirmacion de las alteraciones halladas en el cribado ... · • the laboratory must use qc and...
TRANSCRIPT
Confirmacion de las alteraciones halladas
en el cribado neonatal ampliado
Cristiano Rizzo Division of Metabolism and Research Unit of Biochemical Genetics
Bambino Gesugrave Childrenrsquos Hospital IRCCS - Rome - Italy
Newborn screening
2nd tier testing
Confirmatory testing
Test looking for the same analyte identified among NBS analytes with a high specificy
Test looking for additional diagnostic markers not detectable among NBS analytes
Metabolites hours
Enzymatic activity week
Genetic analysis weeks
second-tier tests test looking for the same analyte identified in NBS analyses
blood spot acylcarnitines
Second-tier tests test looking for additional diagnostic markers not detectable among NBS analytes
blood organic acids or aminoacids
XIC of -MRM (10 pairs) 11700073000 Da ID MMA-SUCCINIC from Sample 1 (SAMPLE-1)
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
117 147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA Ethylmalonic
2nd tier testing
The four most common analyses for confirmatory testing
AMINOACIDS Plasma
ORGANIC ACID Urine and plasma
ACYLGLICINES Urine
ACYLCARNITINES Plasma
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Newborn screening
2nd tier testing
Confirmatory testing
Test looking for the same analyte identified among NBS analytes with a high specificy
Test looking for additional diagnostic markers not detectable among NBS analytes
Metabolites hours
Enzymatic activity week
Genetic analysis weeks
second-tier tests test looking for the same analyte identified in NBS analyses
blood spot acylcarnitines
Second-tier tests test looking for additional diagnostic markers not detectable among NBS analytes
blood organic acids or aminoacids
XIC of -MRM (10 pairs) 11700073000 Da ID MMA-SUCCINIC from Sample 1 (SAMPLE-1)
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
117 147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA Ethylmalonic
2nd tier testing
The four most common analyses for confirmatory testing
AMINOACIDS Plasma
ORGANIC ACID Urine and plasma
ACYLGLICINES Urine
ACYLCARNITINES Plasma
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Metabolites hours
Enzymatic activity week
Genetic analysis weeks
second-tier tests test looking for the same analyte identified in NBS analyses
blood spot acylcarnitines
Second-tier tests test looking for additional diagnostic markers not detectable among NBS analytes
blood organic acids or aminoacids
XIC of -MRM (10 pairs) 11700073000 Da ID MMA-SUCCINIC from Sample 1 (SAMPLE-1)
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
117 147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA Ethylmalonic
2nd tier testing
The four most common analyses for confirmatory testing
AMINOACIDS Plasma
ORGANIC ACID Urine and plasma
ACYLGLICINES Urine
ACYLCARNITINES Plasma
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
second-tier tests test looking for the same analyte identified in NBS analyses
blood spot acylcarnitines
Second-tier tests test looking for additional diagnostic markers not detectable among NBS analytes
blood organic acids or aminoacids
XIC of -MRM (10 pairs) 11700073000 Da ID MMA-SUCCINIC from Sample 1 (SAMPLE-1)
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
117 147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA Ethylmalonic
2nd tier testing
The four most common analyses for confirmatory testing
AMINOACIDS Plasma
ORGANIC ACID Urine and plasma
ACYLGLICINES Urine
ACYLCARNITINES Plasma
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
The four most common analyses for confirmatory testing
AMINOACIDS Plasma
ORGANIC ACID Urine and plasma
ACYLGLICINES Urine
ACYLCARNITINES Plasma
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
bull The laboratory must be equipped with appropriate instrumentation and trained personnel bull The laboratory must also guarantee that the diagnostic confirmation tests are carried out in case of an emergency (24H) ensuring the connection with the Clinical Center and timely delivery of the final results bull Modalities and time of sample delivery must be well established bull The laboratory must use QC and should partecipate in external quality control programs (CDC ERNDIM proficiency testing programs)
Dietzen D et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
Diagnostic confirmation tests amp laboratory requirements
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Instrumentation
IECDAD HPLC- fluorometerDAD
HPLC- TMS GC-MS
Aminoacids Aminoacids Orotic acids Pterins
Aminoacids Orotic acids Acycarnitines Organic acids
Organic acids
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Sample Amount Transport Storage
Urine 4 ml 4Cdeg-or frozen -20Cdeg
Blood heparinedta
3 ml +4Cdeg Centrifuge remove plasmaserum
Plasmaserum 1 ml Frozen -20Cdeg
Proper collection of the biological sample is a crucial step to detect metabolic disorders
Collect samples possibly before any treatment
Specimen collection
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Clinical status
Treatments Nutritional
Sample collection preparation storage
Interferences
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
II tier test for Maple syrup urine disease
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples Alodaib A Carpenter K Wiley V Sim K Christodoulou J Wilcken B Ann Clin Biochem 2011 48(Pt 5)468-70 Direct analysis of dried blood spots by in-line desorption combined with high-resolution chromatography and mass spectrometry for quantification of maple syrup urine disease biomarkers leucine and isoleucine Miller JH 4th Poston PA Karnes HT Anal Bioanal Chem 2011 Apr400(1)237-44 Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)Oglesbee D et al Clin Chem 2008 54(3)542-9
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
The GC-MS needs a high level of chemical expertise and experience with instrumentation It is essential to have specific expertise in pattern recognition to provide a reliable interpretation of the organic acid profile
The excretion profile depends on the age of the patient his diet and clinical status
1000120014001600180020002200240026002800300032003400
2000000
4000000
6000000
8000000
1e+07
12e+07
14e+07
16e+07
T ime--gt
Abundanc e
T IC AO17867D datams
Organic acidurias include a large number of inherited disorders
Interpretation
Organic acids
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
TIC 065
05 10 15 20 25 30 35 40 45 Time min
Inte
nsity
cps
ndashMRM
890430 1030570 1150710 1170590 1310870 1470590 15701130 16701230 1770890 19701370 Q1Q3 masses amu
Inte
nsity
cps
ndashMRM
1130430 1290420 1350920 15101080 15701130 24301110 26601340 26701350 Q1Q3 masses amu
Inte
nsity
cps
ndashPrecursor (74)
100 120 140 160 180 200 220 240 260 280 mz amu
Inte
nsity
cps
Organic acid
Purine and Pyrimidine
Acylglycine
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
000
Inte
nsity
cps
117
147
118
152
105
3-OH-Prop
Succinic
Lactate Ethylmalonic-D3
MMA-D3
Glutaric MMA
Ethylmalonic
UHPLC-MSMS methods for specific organic acids
Organic acids by LC-MSMS
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Acylglycines are the hallmarks of some inborn errors of metabolism
Species Disorders
Propionylglycine PA MMA
Butyrylglycine SCAD GA II
Isobutyrylglycine Isobutyryl-CoA dehydrogenase GA II ETHE1
Tiglylglycine PAMMA -KT
3-methyl-crotonylglycine 3-MCC MCD
Isovalerylglycine IVA
2-methylbutyrylglycine 2-Methylbutyryl-CoA dehydrogenase GA II ETHE1
Hexanoylglycine MCAD GA II
Octanoylglycine MCAD
Suberylglycine MCAD GA II
Phenylproprionylglycine MCAD
Acylglycines
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Acylglycines are often analyzed in conjunction with organic acids however its analysis is more sensitive and specific to identify mildintermittent biochemical fenotipes or asymptomatic patients that may be missed by organic acid analysis alone
GC -MS and HPLC-MSMS techniques based on stable isotope dilution have been developed for the specific determination of acylglycineslt
bullHPLC-MSMS is more practical and easy to use than GCndashMS bullHPLC-MSMS methods are less expensive time-sparing and require simple sample preparation
Quantitative liquid chromatography coupled with tandem mass spectrometryanalysis of urinary acylglycines Application to the diagnosis of inborn errorsof metabolism Daniela Ombrone Francesco Salvatore Margherita Ruoppolo Analytical Biochemistry 417 (2011) 122ndash128
Acylglycines
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Gucciardi A et al A rapid UPLC-MSMS method for simultaneous separation of 48 acylcarnitines in dried blood spots and plasma useful as a second-tier test for expanded newborn screening Anal Bioanal Chem 2012 Aug404(3)741-51
bull Plasma is the preferred matrix for acylcarnitine confirmatory analysis bullButylation for derivatization enhances ionization and analytic specificity bullIn the presence of interfering substances chromatographic separation of acylcarnitines is needed to increase accuracy
Plasma acylcarnitines
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Ion exchange chromagraphy DAD detector
HPLC- fluorimeter DAD detector
45 AA Run time 3 h
25 AA Run time 40 min
Aminoacid analysis
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Commercial kit with derivatization Commercial kit without derivatization
Method without derivatization Giordano G et al Quantification of underivatised amino acids on dry blood spot plasma and urine by HPLC-ESI-MSMSMethods Mol Biol 2012828219-42
Warning It is necessary to verify the separation of alloisoleucine leucine isoleucine for the diagnosis of MSUD
Aminoacid analysis by LC-MSMS
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
+Prec (8510) Exp 1 0319 to 0744 min from Sample 7 (22614) of SP22610-SP22629wiff (Turbo Spray) Max 44e5 cps
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600mz Da
00
20e4
40e4
60e4
80e4
10e5
12e5
14e5
16e5
18e5
20e5
22e5
24e5
26e5
28e5
30e5
32e5
34e5
36e5
38e5
40e5
42e5
44e5
Intensity cps
2743
2603
4875
45952182
2633 48254565
2272 4315403437542773 31943113 3474 48052042 2212 3944
Propionyl-carnitine
Methylmalonic acidurias
Propionic aciduria Biotine recycling defects Non-genetic causes (dietary deficency B12 abnormal absorption) Artifacts
Fowler et al JIMD 2008
Elevated propionyl-carnitine (C3)
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
TCA cycle
fumarate malate
oxaloacetate
citrate
isocitrate
2-ketoglutarate
valine isoleucine methionine odd chain fatty acids cholesterol
succinyl-CoA
propionyl-CoA D-methylmalonyl-CoA L-methylmalonyl-CoA
mutase adocbl cobalamin
succinate SCS
methylcitrate
methionine homocysteine Mecbl
propionyl-carnitine
methylmalonic acid
x
3-OH-propionic acid
propionyl-glycine
x
Biotine cycle
x
x
Dietzen DJ et al National academy of clinical biochemistry laboratory medicine practice guidelines follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry executive summary Clin Chem 2009 Sep55(9)1615-26
The biomarkers of methylmalonic amp propionic aciduria
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Fibro 14C-propionate incorporation methionine serine MeCbl AdoCbl
synthesis
Genetic testing single gene analysis NGS
C3 amp C4DC carnitine (P) organic acids (U P)
3-OH-propionate uarr propionylglicine uarr tiglylglycine uarr methylcitrate uarr
2nd tier testing positive
MMAuarr 3-OH-propionate uarr methylcitrate uarr
tHcys (PU)
Met (p)
Vit B12 in serum
MMA uarr Methymalonylcarnitine N Succinilcarnitineuarr
Succinate-CoA ligase def SUCLA2 SUCLG1 Propionic acidemia MMAs
C3 elevated laboratory investigations
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min
119
1
1
8
152
1
0
5
3-OH-Prop
Succinic
Lactate
Ethylmalonic-D3
MMA-D3
Prop-Gly
147
02 04 06 08 10 12 14 16 18 20 22 24 26 28 30 32 34 Time min 00
147
MMA
MMA-D3
Propionic aciduria
Methylmalonic aciduria
Organic acids (plasmablood) by LCMSMS
1000 1500 2000 2500 30000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time--gt
Abundance
3OHPAPG
TG
MC
Organic acids (urine) by GCMS
MMA
MC 3OHPA
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
11e+07
12e+07
13e+07
Time -- gt
Abundance
Organic acid analysis methylmalonic amp propionic aciduria
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
MMA-mutase def MMA epimerasi def
cblC cblD cblF cBlJ cblX
CBS def
SUCLA2 SUCLG1
TC receptor def TC II def
cblA cblB
MMA
HCY MMA + HCY
cblG cblE
MMA+MA
ACSF3 In methylmalonic acidosis with homocystinuria MMACHC gene analysis is the first step analysis (quicker easier and cheaper than fibro studies)
Methylmalonic aciduria(s) amp homocistinuria(s)
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
bull 243100 = 504 of the total neonatal population bull 79 cases identified (AA+AO+FAO) bull Overall incidence 13077 bull 11 cases of maternal disease
NBS in Italy year 2015
bull 42 cases of maternal vit B12 deficency
httpwwwsismmeititdocumentsrt_screeningindexhtml
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
PA
GALT
PKU
Tir I Tir II
MSUD
HCY MTHFR
BIOPT biosyntesis
BIOPT regeneration
CUD CPT1 CPT2 CACT
VLCAD TFP MCAD LCHAD
MSCHAD GA2
10 -oxidation def
12 Organic acidemias 8 Aminoacidopathies
4 UCD
CIT II
ASA ARG
CIT I
BTD
GA I IVA BKT HMG
(MMA MUT CblA CblB CBlc CblD)
2-MBG MAL MCD
PA
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
2E-3-OH-propionate
1000 1400 1800 2200 2600 3000 3400
2-CH3-butyryglycine
1000 1500 2000 2500 3000
IVA-G
IVA-GLU
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
532 C3-gly-D3
C6-gly-D3
2-CH3-butiryl-glycine
05 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time min 00
542
C3-gly-D3 C6-gly-D3
Isovaleryl-glycine
uarr C5 carnitine
SBCAD def 2-ethyl-3-OH-propionate 2-CH3-butyrylglycine
Isovaleric aciduria 3-OH isovalerate isovaleryl-glutamate isovaleryl-glycine
Organic acids GCMS Acylglycines LCMS-MS
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
PC N IVAC uarr
2MBC N
IVAG uarr 2MBG N
PG N
Pivalic acid therapy
IVA SBCAD
932CgtT mutation
IVD Gene sequencing
Genetic testing confirms the suspected diagnosis
and helps in treatment decisions
PC N IVAC N
2MBC uarr
IVAG N 2MBG uarr
PG N
SBCAD Gene sequencing
ldquopivalic derivates are also used in the cosmetic industry as emollient under the termneopentanoateldquo Boemer F et al Surprising causes of C5-carnitine false positive results in newborn screening Mol Genet Metab 2014 Jan111(1)52-4
AO ACG AC
PC uarr IVAC N 2MBC N
IVAG N 2MBG N
PG uarr
C5 elevated laboratory investigations
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
ldquo individuals with IVD deficiency discovered by NBS and who have at least one copy of a 932CrT (A282V) mutant allele can exhibit a mild phenotype or be free of symptoms throughout childhoodrdquo ldquo there are obvious implications for the management and genetic counseling of individuals identified through NBS which include the need for genotyping of the IVD gene in the early stages of follow-uprdquo
Genetic testing for IVA
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Disorder Organic acids Acylcarnitines Acylglycines
CUD n alldarrP ( U) N
CPT1 n C0 N
CACT DCA C16C18 n
CPT2 n C16C18 n
VLCAD DCA C141C16C161 n
MCAD DCA C8 C81C101 HexG SuGPHePG
SCAD EMA C4 BuG
MTPLCHAD 3-OH-DCA C16OHC18OHC181OH
n
SCHAD 3-OH-GLU C4OH n
MADD DCAIVAEMAGLU
C4C8C10C12 HexG SuGPHePG
differential diagnosis RFVT2 (BVVLs) amp RFVT3 (FLs)
-oxidation defects differential diagnosis
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
molecular genetic testing of ACADVL gene
Additional diagnostic tests -VLCAD enzyme activity in fibroblasts or lymphocytes - LC- Fatty acid oxidation in fibroblasts
Acylcarnitine analysis by TMS in plasmadried blood spot C141C142C14C121
NBS may also detect heterozygotes
VLCAD deficiency diagnosis
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
C141 (DBS amp P) gt cut-off
VLCAD enzyme activity
VLCAD healty heterozygous
VLCAD gene sequencing
Results in 2-6 weeks
Results in max 8 working days
optional
VLCAD enzyme activity in leukocytes fibroblasts (liver heart skeletal muscle amniocytes) can be used to confirm the diagnosis of VLCAD deficiency
Genotype-Phenotype Correlations A strong genotype-phenotype correlation exists in VLCAD deficiency bull Severe disease is associated with no residual enzyme activity often resulting from null variants Approximately 81 of pathogenic truncating variants are associated with the severe early onset form [Andresen et al 1999] bull Milder childhood and adult forms are often associated with residual enzyme activity resulting from one or two pathogenic missense variants The common pVal283Ala variant has usually been associated with the mild phenotypes [Spiekerkoetter et al 2009]
VLCAD deficiency diagnosis
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Distribuzione dell`attivita residua VLCAD nei campioni analizzati
31 middot 1 September 2017
Sani
VLCAD
Eterozigoti nessuna rilevanza clinica
Attivita residua
Diagnosi accurata e precisa in tempi rapidi Efficace valutazione del rischio di sviluppare la patologia
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Urine
Orotic acid
LysArgOrn
HCitr
Gal
3-CH3-glutaconic
uarr
uarr
NAGS CPS OTC
uarr uarr uarr
darr darr darr
darr darr darr
nuarr
ASS ASL ARG HHH
uarr uarr uarr uarr
uarruarr uarr darr
darr uarruarr
uarr
nuarr
uarr uarr uarr uarr
Arg Orn
uarr
CITR2 LPI
uarr uarr
uarr uarr
darr darr
darr
darr
uarr
uarr
uarr
all uarr
uarr
Plasma
Gln
Cit
Arg
ASA
Orn
Lys
PheTyrMet
LDH
Urea cycle defects differential diagnosis
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
Recent studies have demonstrated that NGS is a useful tool for the molecular diagnosis of IMD Next-Generation Sequencing (NGS) technology can be used to perform supplemental newborn screening reflexconfirmatory testing and carrier screening The multigene panel for NBS should be designed with specific considerations of genetic epidemiologic characteristics of the target disease and population
Miseq NextSeq550
Bambino Gesugrave Childrenrsquos Hospital NGS panel 36 metabolic disorders 8 aminoacidopathies 12 organic acidurias 10 b-oxidation def 4 UCD GALT BTD
Next generation sequencing (NGS)
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
bull Genetic testing confirms the suspected diagnosis and may of help for treatment and management decisions
bull To improve the knowledge of the pathophysiology of the disease
bull Specific genotypes may be associated with the prognosis
bull Useful for a genetic counseling of family members and other relatives for risk assessment
Why it is important to find the disease-causing mutation(s)
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
According to international guidelines we strongly advocate starting pre-emptive treatment in the setting of a metabolic centre as soon as the suspicion of an organic aciduria arises from newborn screening and before the diagnosis is finally confirmed
Hoerster F et al Newborn Screening Programmes in Europe Arguments and Efforts Regarding Harmonisation Focus on Organic Acidurias JIMD Rep 201732105-115
Why it is important to find the disease-causing mutation(s)
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
The informations obtained with the analysis of urinary organic acids is complementary to those derived from urinary acylglicines plasma acylcarnitines and plasma amino acids profiles
Itrsquos fundamental to carry out enzimatic assay and genetic testing to obtain the correct disease classification and to drive the correct treatment
Conclusions
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank
The Lab team Dr Cristiano Rizzo Dr Bianca Maria Goffredo Dr Sara Boenzi Dr Michela Semeraro Tec Giulio Catesini Tec Alessia Vitale Tec Giacomo Antonetti Tec Elisa Sacchetti
Head of the Unit Dr Carlo Dionisi-Vici
I wish to thank