screening for organic acid disorders among egyptian...

Research Journal of Medicine and Medical Sciences, 4(2): 369-385, 2009

© 2009, INSInet Publication

Corresponding Author: Marian Yousry Girgis MD., Neurology Department, Cairo University.

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Screening for Organic Acid Disorders among Egyptian Children with ClinicallySuspected Neurometabolic Disorders

Laila Abdel Moteleb Selim MD, Sawsan Abdel Hadi Hassan MD, Fadia Ali salem MD, Fayza1 2 2 5

Abdel Hamid Hassan MD, Fatma El Mogy MD, Sahar Abdel Atty MD, Iman Mandour MD, Marian5 5 5 5

Fathy MD, Azza El Orabi amin MD, Iman gamal el din MD, Manal sadek El defrawy MD, Ali el1 3 4 3

ayat MD, Amira El Badawy Msc, Marian yousry MD, Mohammed Abdel Monem MSc, Dina3 1 5 5

Mehaney MSc

Neurology Department, Cairo University Children Hospital, Genetic Department, Cairo University1 2

Children Hospital, Metabolic Unit, Cairo University Children Hospital, Pediatric Hepatology and3 4

Metabolic Unit, Benha Faculty of Medicine and Clinical and Chemical Pathology Department, Faculty5

of medicine, Cairo University

Abs tr ac t: Ba c kground: Organic acid disorders are a group of disorders characterized by the excretion ofnon-amino organic acids in urine. Expanded newborn s c re ening methods by tandem mass spectrometry

(MS/MS) can detect organic acide mias early in infancy with a better outcome when diagnosed in the firs t

ten days of life. Aim: This s tudy aims at re v ie wing the clinical, biochemical and neuroradiological dataof patients diagnosed as organic acidemias and to highlight the importance of including organic acidemias

in Newborn screening programs us ing MS/MS. Methods : Eight hundred p a t ients attending the

neurometabolic clinic at Cairo Un iv e rs ity Children Hospital (CUCH) screened for inborn errors ofmetabolism (IEM) by MS/MS. Organic acid pro file in urine, by gas chromatography mass spectrometry

(GC-MS/MS), was performed for selected cases . Results : Nineteen patie n t s out the 800 cases werediagnosed as organic acidemias (1/42).Three cas e s (15.8%) were diagnosed as methyl malonic acidemias

(MMA), 3 (15.8%) as â-ketothiolase deficiency (BKT), 2 (10.5%) as 3-me t h y lcrotonylglycinuria (MCG),

3 (15.8%%)as biotinidase deficiency , 2 (10.5%) as Canavan disease, one patient (5.3% )for each of thefollowing: glutaric aciduria type I, D-2 hydroxyglutaric a c id u ria ,is o v a le ric a c id u ria (IVA),

asargininosuccinic aciduria (ASA),3-methyl glutaconic aciduria and propionic ac id e mia (PA ).

Developmental delay was a dominant symptom being present in 16/19 (84.2%) followed by metabolicacidos is in 15/19(78.9% ), vomit in g in 12(63.2%), encephalopathy with dis turbed conscious level in 11

(57.8%), seizures in 8 (42%), overwhelming illness [encephalopathy, re s p ira t ory dis tress , seizures ,

septicemia, pers is tent vomitings] in 8 (42%) namely in MMA, PA and BKT and diarrhea in 6(31% )cases .Central nervous sys tem abnormalit ie s in c luding cranial nerve affection in 8(42%) extrapyramidal

ma n ifes tations namely dys tonia in 4 (21%), ataxia in 2(10.5%), long tract s igns in 7(36.8%), h y p o t o n ia

in 11 (57.8%) patients and dysmorphism in 11 cases ( 57.8%). Conclus ion: Screening for organicacidopathies by MS/MS us ing dried blood spot technique is a rapid and efficient method in detecting cases

with suspected organic acidemias , requiring confirmatory tes ts by GC-MS. Including organic acidopathiesin newborn sc re e n in g wo u ld help in rapid and properly timed therapeutic intervention to prevent

devas tating neurological outcomes . However, confirmatory tes ts by GC-MS are mandatory particularly for

clinically suspected cases with normal acyl carnitine profile

Key words: Organic acidemia, methylmalonic acidemia, tandem mass spectrometry.

INTRODUCTION

Inborn errors of metab o lism (IEMs) are commonthroughout the Middle Eas t, presumably because of the

relatively high rates of consanguinity (38.5%) . Many[1 ,2]

of the IEM carry serious clinical consequences to theaffected neonates , including mild to severe mental

retardation, ph y s ical handicap and even fatality.

Organic acidemias (OA ) caused by mitochondrialenzyme defects in the cata b o lism of branched chain

amino acids and fatty acids oxidation defects cons titutea grou p o f more than 20 disorders . These disorders

include propionic acidemia (PA), me t h y lma lo n ic

acidemia (MMA), isovaleric acidemia (IVA ), biotin-un re s p o n s iv e 3-me t h y lcrotonyl-CoA carboxylase

deficiency, 3-hydroxy-3-me t h y lglutaryl-CoA (HMG-

Res. J. Medicine & Med. Sci., 4(2): 369-385, 2009

370

CoA) lyase deficiency, â-ke t o t hiolase deficiency andglutaric acidemia type I (GA t ype I), . In these etc...

in th ese disorders acyl-coA esters accumulate in the

mitochondria. Carnitin e plays a key role in removingth e potentially toxic Acyl-coA es ters through the

formation of acylcarnitine es ters an d t h ereby releas ingcoenzyme A and res toring mitochondrial homeos tas is [3 ,4]

This results in increased concentrations of circulating

acylcarnitine, increas ing e xcretion of acylcarnitine inurine and secondary carnitine deficiency [5]

The aim of this descriptive s tudy is to find out the

prevalence of OA disorders detectab le by MS/MSamong all patients attending the Neurome t a bolic Clinic

at Cairo Univers ity Children Hospital (CUCH), located

at the Centre of Social an d Preventive Medicine(CSPM) and to re view the clinical, biochemical and

neuroradio lo g ical data of the Egyptian patientsdiagnosed as organic acidemias and t o highlight the

importance of including organic acidemias in newborn

screening programs with MS/MS.

MATERIALS AND METHODS

Eight hundred pa t ie n t s re fe rre d to the

neurometabolic clin ic , during the period from January

2008 to September 2008, suspected of having inbornerrors of metabolism were subjected to the following:

Clinical evaluation including his tory, general a nd

neu ro lo g ic a l e xa min a t io n a n d family pedigreecons truction

Basic metabolic investigations including completeblood picture (CBC), blood glucose and serum

electrolytes , blood gases , anion gap, liver function tes ts ,

kidney function tes ts , urine analys is , urine ketones andreducing subs tances , plasma ammonia and lactate.

Special Metabolic Investigations Including: Metabolicscreening for amino acids and acylcarnitine

profiles by MS/MS.

Organic acid profile in urine us ing GC-MS wasperformed for patients with abnormal acylcarnit ine

profiles us ing M S/MS, or those with unexplainedmental retardation or compensated metabolic acidos is

with normal MS/MS profile.

Molecular diagnos is was done to one patie nt and hisfamily.

MRI brain was done to mos t of patients .

Neu ro physiological testing including EMG/NCV, ERG,VEP, ABR were performed according to the diagnos tic

needs .

Patients were managed symptomatically anda p p ro p riate antiepileptic drugs

were ins tituted. W hen inves tigations poin t e d towards

specific disorders , treatment was ins tituted accordingly.All patients were regularly followed up.

MS/MS Laboratory Methods: A mas s spectrometer isa device that ionizes molecules and s e parates the

charge d products , or ions according to their mass-to-

charge ratios (m/z). A n acylcarnitine is a fatty acyles ter of L-carnitine . T h e family of acylcarnitines

analyzed by M S/MS includes fatty acyl groups forsaturated and u n s a t urated species ranging from the 2-

carbon acetylcarnitine (C2) through the 20-carbon

aliphatic groups . Blood s a mples were spotted onW hatman filter paper cards (Schleicher and Sch u e ll

903; Dassel, Germany) and sent to t h e s c reening lab,

Centre o f Social and Preventive Medicine (CSPM),Cairo Un iv e rs ity Children Hospital (CUCH), for

screening by MS/MS. The blood spots were punched

us ing automatic puncher (BSD 400 automated puncher(Perkin Elmer)), extracted with 200 ìl methanol (Merck

(Da rms t a d t , Ge rma n y )), c o n t a i n i n g k n o wnconcentrations of a mixture of isotopically labeled

internal s tandards of diagnos tic acylcarnitines and

amino acids (Cambridge Isotope Laboratories , USA).Samples were then evaporated to dryness under a

s tream of nitrog e n (TurboVap 96, Caliper lifesciences ,

MA, USA) and the res idue was derivatized with 80 ìlof butanolic HCl (Regis Technologies , USA) and

heated at 65 ° C for 15 min. The derivatize d e xtract

was evaporated again and the re s idue was finallyre c o n s t ituted in 80 ìl of the mo b ile p h a s e

(acetonitrile/water, 80/ 20 v/v). A 20ìl sample was

directly injected into the mobile phase flowing to ESI-LC-MS/MS (Quattro LC; Micromass , Manches te r, UK)

at a flow rate of 0.2 ml/min. A parent scan o f m/z 85for ac y lcarnitine butyles ters profile was examined for

the detection of OA disorders .

GC-MS/MS: Urinary acylcarnitine profiles by GC-

M S/MS was done for selected cases . Samples

belonging t o cases clinically sugges tive of havingorg a n ic acidemias , but showing normal MS/MS profile

as well as those wit h pos itive ESI-LC-MS/MS results

were diagnosed and confirmed by gas chromatographymass spectrometry us ing the GCQ, Finnigan Mat, USA

RESULTS AND DISCUSSIONS

Among 800 pat ients attending the clinic ofinherited metabolic dis o rd ers at CUCH, 19 (2,37%)

patients were d iagnosed as organic acidurias after

confirmation of their initial results obtained by MS/MSby performing urine organic acid profile us ing gas

chromatography mass spectrometry (GC-MS)

Onset was in the firs t mo n t h of life in 1/19 patient(5.2%), within firs t year in 12/19(63.1%) and second

yea r in 4/19 (21%). Clinical presentation was acute in

10/19 (52.6), usually trig g e red by infection, chronic in9/19 (47.3%) namely in patients with cerebral organic


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acidurias . Mean age at presentation was 7.7months and21 months at dia g n o s is (the mean was calculated

excluding case 15 and 16 due to their unusual late

presentation and late diagnos is ) i.e. mean time elaps ingbetween presentation and diagnos is was 13.3 months ,

wh ic h is attributed to lack of access ible laboratoryfacilities for diagnos is , as MS/MS was available in o u r

lab only in January 2008.

Developmental delay was a dominant symptombeing pre s e n t in 16/19 (84.2%) followed by metabolic

acidos is in 15/19(78.9%), vomiting in 12(63.2%),

encephalopathy with dis turbed conscious level in 11(57.8%), seizures in 8 (42%), overwhelming illness

[e n c e p h a lo p a t h y , re s p iratory dis tress , seizures ,

septicemia, pers is tent vomitings] in 8 (42%) namely inMMA, PA and BKT and diarrhea in 6(31%)cases .

Central nervous sys tem abnormalities including c ra nialn e r v e a f f e c t io n in 8(42% ), e xt ra p y ra mid a l

manifes tations namely dystonia in 4 (21%), a t a xia in

2(10.5%), long tract s igns in 7(36.8%), hypotonia in 11(57.8%) and dysmorphism in 11 cases ( 57.8%).

Hy p e ramonemia was observed in 7/19 (36.8% ).

(table1,2,his togram)

Laboratory Results of the OA Cases: Thre e o u t of 19

cases (15.8%) were diagnosed as beta ketothiolase(BKT), 3 (15.8%) as methylmalonic acidemia (M MA),

3 (15.8%) a s biotinidase deficiency, 2 (10.5%) as

methyl crotonyl glycinuria (MCG), 2 (10.5%) patientsas Canavan disease, one patien t (5.3%) for each of the

following: glutaric aciduria t ype I (GA-1), D-2 OH-glutaric aciduria, IVA, propionic ac a demia (PA),

arginosuccinic aciduria(ASA) and 3 methyl glutac o nic

acidurias .Diagno s t ic markers demons trated by LC- MS/MS

and GC-MS are shown in table 3.

Neuroradi ol og i c al Findings: Brain MRI/CT was

performed in 15/19 patients with OA. It revealed brain

atrophic change s in 6 patients (40%) more prominentin patient with GA type I (case 13,fig 3c), extens iv e

wh ite matter demyelination in 2 patients (cases 12 a n d14 with Canavan disease; fig 5b), patchy demyelination

of white ma t t er (case 17 with 3-OH-GA), high s ignal

intens ity le s ions in the basal ganglia (case I with BKT,normal findings in 2 cases (cases 3,9), co rpus callosum

agenes is (case 18 with ASA), incomplete myelination

in 1 case(case 17),while generalized demyelination insuperficial and deep white matter in c a s e 11 with total

biotinidase deficiency (table4).

Di scuss ion: The introduction of ESI-MS/MS as a

diagnos tic tool in the metabolic lab has h a d a great

impact on the number of cases diagnosed as org a n icacidemias or aminoacidopathies from eith e r referred

s ick children or screened newbo rn s . In pediatricp ractice, early diagnos is of IEM in newborns a n d

young infants is s ignificantly important to avoid serious

con s e q u e n c e s s uch as mental retardation anddevelopmental delay.

T h e term "organic acidemia" or "organic aciduria "(OA) applies to a diverse group of disorders

cha racterized by the excretion of non-amino organic

acids in u rin e. The organic acidemias share manyclinical s imilarities .

Metabolic acidos is was a salient clin ical feature in

15 patients with orga n ic acidemias , triggeringmetabolic cris is and necess itating hospital admiss ion in

PICU.

A- Class ical Organic Acidemias:

1- Methyl Malonic and P r opionic Acidemias: Theshort-ch a in acylcarnitine C3 is an extremely important

diagnostic marker fo r certain organic acidemias . An

increase in C3 concentration may indica t e the presence

o f e it her a propionic acidemia (PA), where the primary

enzyme deficient is the porpionyl-CoA carboxylase, or

a methylmalonic acidemia (MMA) or c o b olamindefect[6]

In this s tudy, the acyl carnitine profile o f 4 / 800

cases (0.5%) attending the outpatient clinic showedhigh C3, detected at m/z 274 by use of a Pre 85 scan

function. Three cases were confirmed as methylmalonic

and one case as propionic acidemia by urinary organicacid profile us ing GC-MS.

Although the prev alence of methylmalonicacademia (MMA) is difficult to define precis e ly, one

survey in California sugges ted an incidence of 1:32,000

infants with organic acidemias A much greater[7]

prevalence of between 1:1,000 and 1:2,000 has been

reported in Middle Eas tern popula t io ns . However in[8]

the present work 3 cases wit h M M A were detectedamong 800 high risk group, patie nts attending the OP

clinic, which is attributed to the widespread tradition of

consanguineous marriages . A ll 3 patients with MMA had episodes of

metabolic acidos is associated with dis turbed consciouslevel triggering hospital admiss io n and was the event

leading to diagnos is . One out of three had fair

complexion and 1/3 had mild ren al impairment (case5). One had a s ibling who d ied undiagnosed at the age

of 15 days highlighting the importance o f early

detection of organic acidemia by newborn screeningwith MS/MS. None had ab n o rma l movements and all

presented with generalized hypotonia and hyporeflexia.

One out of 800 cases (0.125%) was d iagnosed aspropionic acid emia. This case presented at the age of

24 months and diagnos is was set at the age of

36mont h s . He presented with fever, vomiting anddiarrhea followed by metab o lic decompensation


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Table 1: Frequency of clinical signs and symptoms among 19 patients with organic acidurias

Clinical symptoms/signs OA patients (n=19) Frequency %

Male preponderance 11 57.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Positive consanguinity 17 89.4

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Affection of other siblings 10 52.6

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Repeated abortions/ unexplained deaths 8 42.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Global developmental delay 16 84.2

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Metabolic acidosis 15 78.9

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Vomiting 12 63.2

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Disturbed conscious level 11 57.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Dysmorphic features 11 57.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Skin or hair changes 11 57.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Febrile episodes 10 52.6

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Seizures 8 42.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Overwhelming illness 8 42.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Diarrhea 6 31.5

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Organomegaly 4 21.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Renal impairment 2 10.5

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Hepatic impairment 2 10.5

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Cranial nerve affection 8 42.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Microcephaly 5 26.3

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Macrocephaly 3 15.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Hypotonia 11 57.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Pyramidal tract affection 7 36.8

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Extrapyramidal manifestation 4 21.1

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Ataxia 2 10.5

Histogram showing frequency of signs & symptoms among 19 patients with organic acidemia


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Table 2: Summary of Clinical Findings in 19 cases with Organic AcidemiasSerial Diagnosis GDD seizures Micro macroceph Dysmorphism Hair & Metabol DCL pyramidal Extra Hypotonia Ataxia number cephaly skin acidosis pyramidal

changesCase1 BKT + – – – + + +++ + – – ++ –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 2 BKT – – – – + + +++ +++ – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------##Case 3 BKT – – – – + + +++ ++ – – – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 4 MMA – – – – – – +++ + – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 5 MMA ++ – – – + – +++ +++ – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 6 MMA + + – – – + +++ ++ – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 7 3 MCG ++ – + – ++ + – – ++ ++ – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 8 3 MCG ++ – + – ++ + – – + ++ – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 9 IVA ++ – + – + – +++ + – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 10 PA ++ ++ – – – ++ +++ ++ – – – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case11 biotinidase + +++ – – – +++ +++ + – – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 12 canavan ++ ++ – ++ – – + – + – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 13 Glut 1 ++ ++ – ++ + + + ++ + ++ – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 14 canavan +++ – – ++ – – + – + – + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 15 Partial biotinidase ++ – + – ++ ++ – – ++ – – ++--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 16 Partial biotinidase ++ – + – ++ ++ – – ++ – – ++--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case17 D2glutaric aciduria + +++ – – – – + – – – – –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 18 Arginosuccinic aciduria ++ + – – + – + – – + + –--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Case 19 3-MGA ++ + – – – – + ++ – – ++ –

Table 3: Summary of biochemical data of the 19 cases with organic acidemias

Organic Mean age Mean age MS/MS Urine organic acid profile by GC-MS Lactic Hypera-

acid disorder at presentation at diagnosis Marker acidosis mmonemia

(months) (months) component

BKT Cases 1,2&3 13.3 15 Increased 82-methyl-3-hydroxybutyrylcarnitine - +

C5-OH & tiglyglicine

C5:1 883 hydroxy butyric acid &

acetoacetate. â- ketothiolase,

3 hydroxy isovaleric triglycine

MMA Cases 4,5&6 11.2 19 Increased C3 8methyl malonic & methyl citrate, + +

fig 1 C3:C2 trace of 3hydroxy propionic acid

3 MCG Cases 7&8 12 90 increased 83 hydroxy isovaleric & highly - -

fig 4 C5-OH elevated 3 methyl crotonyl

glycinuria (3 methyl co A

carboxylase deficiency)(MCCD)

IVA Case 9 10 days 24 increasedC5 88isovalerylglycine (mono and - +

fig 2 di derivative)

PA case 10 24 36 Increased C3 8uinary glycine, 3-hydroxypropionic - -

acid, methylcitrate, tiglic acid,

tiglyglycine butanone & propionyl

glycine

Biotinidase 4 6 Normal Not Remarkable - -

deficiency Case 11

Partial biotinidase 11 years 13years Normal Not remarkable - -

deficiency Cases

15&16

Canavan 4 8.5 Normal 8 8 N_acetyl aspartic acid - -

cases12&14

GA I \ Case 13 6 7 increased 88glutaric acid elevated - -

C5-DC 3- hydroxyglutaric acid (diagnostic

marker for GA type 1)

2 OH-GA Case 17 3 5 -mild increase ##882-hydroxyglutaric acid, - -

fig 6 in c3 succinic acid & lactic acid with

low 2- oxoglutaric acid. Data

consistent with D 2-hydroxyglutaric

aciduria


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Table 3: Continue

ASA Case 18 1 day 6.5 High citrulline - -

3MGA Case19 12 90 increased Increased execretion of - -

C5-OH 3-methyl glutaconic and

3-methylglutaric acids

Abbreviations: C5OH: 3 Hydroxy Is o va l er yl Or 3 Hydroxy 2 Methylbuteryl Carnitine; C3: Propionyl Carnitine; C5:1: Tiglyglycine or 3

Methylcrotonyl Carnitine; C5 –DC: Glutaryl C a r n i t i ne; C5: Isovaleryl or 2-Methylbuteryl Carnitine; MCCD: Methyl CoA Carboxylase

Deficiency BKT:Betaketothiolase; MMA: Methylmalonic A ci d em i a ;3MCG: 3-Methylcrotonylglycine; IVA: isovaleric acidemia; PA : Propionic

Acidemia;GA1: Glutaric Aciduria type I; 3-OH-GA: 3hydroxy Gl u t a r i c Aciduria; ASA: Argininosuccinic Aciduria; 3MGA: 3-Methylglutaconic

Aciduria

Table 4: MRI/CT findings in organic aciduria cases

Diagnosis MRI/CT findings

BKT Case 1 bilateral and symmetrical lesions in caudate and p u t am en , appearing as faint high signal intensity in T W 2 and FLAIR

and low signal intensity in T W 1(case1)Case 3 Normal findings in case 3

MMA Case 6 Brain atrophy evidenced by prominent cerebral ventricles,cortical sulci,anterior interhemispheric and both sylvian

fissures.wide extra-axialCSF spaces at the frontotemporoparietal region fig 1.

3 MCG Cases 7 &8 C ereb ral atrophic changes appearing as prominent cortical sulci with associated prominent both sylvian fi s s u res an d

ventricular systems,abnormal gyral pattern fig 4c.

IVA Case 9 MRI shows no focal cerebral lesion, (normal)

Biotinidase MRI shows generalized demyelination involving both superficial and deep white matter

deficiency Cases

11, 15 &16

#CT brain shows cortical brain atrophy at the frontotemporal regions(case 15&16,partial biotinidase deficiency)

Canavan disease Extensive demyelinating brain disease involving both the deep peri v en t r i cu l ar w h ite matter as well as the subcorticalCases (12 & 14) white matter and involving U fibers,the corpus callosum as well as the internal capsule fig 5.

GA 1 Case 13 prominent cortical brain sulci,basal cisterns and both sylvian fissures with widening of the subdural space at both

frontotemporoparieto-occipitalregions,denoting

severe brain atrophy(fig 3)

D-2 glutaric aciduria M R I s h o w s periventricular ill defined patchy area of abnormal signals, likely related to incomplete myelination process .

Case 17 fig 6.

ASA Case 18 Delayed myelination of dentate nuclei and p o s t er i o r l imbs of internal capsules, colpocephaly,dilatation of the right

lateral ventricle and partial agenesis of the corpus callosum

3 MGA Case 19 Frontotemporal brain atrophy, more on the right side and mild bilateral frontotemporal subdural effusion and right

frontal heamatoma fig 7.

characterized b y in t ra c t a b le metabolic acidos is ,

associated with encephalopathy and dis turbed consciousle v e l. A b d o min a l e xa min a t io n re v e a le d mild

hepatosplenomegaly and there was extens ive exfoliativedermatitis .

Analys is by LC/MS/MS showed an increased C3and ketotic h yperglycinemia and diagnos is was

confirmed by urine organic a c id profile us ing GC-MSfig 1.

Although Chace and co workers s tated thatextremely high conce n t rations of C3 (>10 µmol/L)

gen e rally indicate acute PA and Moderate increases inC3 (>6–10 µmol/L) u sually indicate a MMA, this was

not the case in our s tudy as two c a s e s with very highC3 (59.6 and 22.4) were confirmed to be MMA by

GC-MS. This may prove that th is rule is anapproximation, and organic acid analys is and other

confirmatory tes ts are required to accurately diagnoseany of these disorders .[9]

2- Beta Ketothiolase: Three case s o u t of 800 (0.375%)

attending the outpatient clinic showed elevated wholeblood C5OH were detected b y M S/MS and further

confirmed as BKT by GC- MS. All 3 cases suffered from severe metabolic acidos is

triggered by an a t t a ck of gas troenteritis and fever and

c u lminating into dis turbed conscious level a n d

admiss ion in ICU. All patients exhibited minordysmorphic facial features in the fo rm of synophyris ,

and high arched palate,2/3 developed hypotonia,1/3developped shock and required artificial ventilation

(case no. 2),and case 3 re q uired peritoneal dialys is forcorrection of intractable metabolic acidos is .

3- Is ovaleric Acidemia: W e have recorded only one

patient out of 800 patients (0.125%) seen a t theneurometabolic clinic who was diagnosed as isov a leric

acidemia, showing an elevated C5= 6.7umol/l (fig 2).C5 is the primary marker fo r IVA and the flag was set

at 0.7umol/L. Because C5 acylcarn it ine represents amixt u re o f is o me r s ( is o v a le ry lc a rn it in e , 2-

methylbutyrylcarnitine, and pivaloylcarnitine) a nd theclinical picture overlaps other o rganic acidemias and

urea cycle defects further diagnos tic evaluation byperforming urine organic acid p rofile by GC-MS was

performed to confirm diagnos is .

The patient presented with several ep is odes ofmetabolic acidos is and encephalopathy with dis turbed

consc io u s level with repeated attacks of vomiting,dehydration and refusal o f feeding necess itatingfreque n t hospital admiss ions . Examination revealed


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Fig. 1: 1a showing the photography of case no6 wit h me thyl malonic academia(MMA);1b, a serial of TW I&TW 2

brain MRI showing moderate atrophic brain changes mos tly apparent in fronto t e mp o ral regions ;1c,theLC-MS/MS curve of the same case showing increas e d C3(the marker component of MMA/PA), and 1d,

urinary acylcarnitine profile by lC-MS/MS showing high methylmalonyl carnit in e m/ z 374.5 (case No 6).


376

Fig. 2a: photography of case9 with Isovaleric academia(IVA)

Fig. 2b: Lc-MS/MS curve of case 9 showing high C5 (Isovaleryl Carnitine)

Fig. 3: 3a,photography of case 13 with glutaric aciduria type 1showing severe dys tonic pos turing


377

Fig. 3b: Lc-MS/Ms & GC-MS results of dried blood s p o t a n d urine organic acid profile of Case No13 with

Glutaric Aciduria Type I.

Fig. 3c: TW I Brain MRI showing prominent cortical brain sulci, basal cis terns and both s y lv ian fis sures with

widening of the subdural space at both frontotemporoparieto-occip it a lre gions denoting severe brainatrophy(case 13, Glutaric Aciduria type1).

mic ro c e p h a ly with generalized hypot o n ia a n d

hyporeflexia, dysmorphism in the form of everted earlobules and synophyris , fig 2 mild normocytic anemia

and thrombocytopenia pancytopenia, as well as isolatedneutropenia and th ro mbocytopenia may occur in IVA

and is a ttributed to bone marrow suppress ion [10]

Hyperammonemia was also observed in the patient and

is presume d to be due to inhibition of N_acetylglutama t e synthetase by isovaleryl CO-A(leading to

reduced N ac e t y l glutamate and impairement of urea

cycle . Similar clinical presentations were previous ly[11]

reported by others [12 ,13 ,14 ,15 ,16 ,17]

4- 3 Methyl Glutaconic Aciduria: 3-Methylglutac o n ic

aciduria is a rare hereditary metabolic d isorderch a ra cterized by increased urinary excretion of 3-

methylglutaconic and 3-methylglutaric acids . Fourclinical forms are recognized .[18]


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Fig. 4: 4a, case no7 with methylcrotonyl glycinuria(MCG)

Fig. 4b: Lc-MS/MS curve demons trating high C5OH in case 7(MCG)

Fig. 4c: TW I brain MRI from case 7 showing both cortical & central atrophic brain changes


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Fig. 5: fig 5a , urine GCMS profile from case 12 with canavan disease showing increased N-acetyl aspartate

Fig. 5b: T2W brain MRI from case 12 with canavan disease showing extens ive demyelination in v o lv in g both

the deep periventricular & the subcortical white matter, U fibers , and corpus callosum .

One female patient (0.125%) was diagnosed as 3methylglutaconic aciduria. The patient p resented

initia lly with delayed mental and motor developmentalmiles tones and recurrent ches t infections . Examination

rev e a led obes ity, generalized hypotonia and moderatehepatos p lenomegaly, mild cardiomegaly with left to

right shunt demons trable on echocardiography. Patientexhibited one attack of metabolic decompensation wit h

metabolic acidos is and dis turbed conscious level. Theclinical presentations of this c a s e are cons is tent with

those of 3 methylglutaconic aciduria type II (BarthSynd ro me ). MS/MS revealed an elevated C5OH[19 ,20]


380

Fig. 6: Urine organic acid profile from case no 17 by GC-MS showing marked increased of d 2 is o me r o fglutaric aciduria .

Fig. 7: TW 1 brain MRI from Case 19 showin g ma rked frontotemporoparietal atrophic brain changes , bilateralfrontoparietal subdural effus ion and right frontal hematoma.


381

and GC-MS analy s is showed increased 3-methylglutaconic acid and 3 methyl glutaric acid.

B- Cerebral Organic Acidurias: Cerebral organic

acidurias are a group of disorders in which neurologicalmanifes tations can be the leading presenting feature,sometimes in the absence o f metabolic derangement.

Characteris tic clinical findings are ataxia, myoclonus ,e xt ra p y ramidal symptoms, metabolic s tro ke a n d

megalencephaly. As a group these "cerebral" organic[21]

acid disorders are often undiagnosed and their true

incidence is much less well-known than that of the"class ical" organic acid disorders .

1- Methyl Cr otonyl Glycinuria: Two male s iblings

(descendant of consanguineous parents) were diagnosedas methyl crotonyl glycin uria. Extremely high C5OH

on MS/MS analys is (60 umol/l, Cut off value = 1.0umol/L) (fig 4b). Highly ele v ated 3 hydroxyisovaleric

and 3 methyl crotonyl glycinuria were the biochemicalmarkers found on urine o rg a n ic acid analys is by GC-

MS result in g from deficiency of 3 methyl Co-Ac a rb o xy la se deficiency (MCCD). Both p a t ie n t s

presented with glo bal developmental delay, profoundmental retardation, growth retard a t ion, involuntary

movements , squint, microcephaly, hypertonia a n dspas tic gait. Cerebral palsy-like symptoms accompanied

by extrapyramidal s igns called for extens ive me tabolicevaluation including the determination of urinary 3-

methyl crotonyl glycin u ria fig 4. There was no his toryof metabolic decompensation or encephalo pathy.Controvers ial clinical presentations were reported in the

literature. The main clinical features of p a tients[22 ,23 ,24]

with MCCD reported in the literature included

muscular hypotonia and atrophy , failure to thrive,[25]

seizures and pro fo und irrevers ible metabolic acidos is

, i n v o l u n t a ry mo v e me n t s , me t a b o l i c[ 2 6 , 2 7 ]

decompensation , quadriplegia, hemipares is and focal[28]

seizures Brain MRI results in the 2 p a t ients with MCG

showed both cortical and central a trophic brainchanges . Marked atrophic brain c h a nges and multiple

loci of leukodys trophy on brain MRI were also foundby Murayama et al., 1997 and De Kremer et al.,[29 ,30]

2 - D2 Hy dr o x y G l u t a r i c Ac i dur i a: D2-

Hydroxyglutaric aciduria is a neurometabolic diseasecharacterized by the accumulation of abnormal amounts

of D-2-hydroxyglutarate in cerebrospinal fluid, bloodand urine firs t described by Chalmers et al.,[31]

A 5 months old female patient presented withintractable seizures as th e leading symptom.. She also

had - mild ps y c h omotor retardation and compensatedmetabolic acidos is , with no dysmorphic fa cial features ,

a picture s imilar t o t h a t reported by Van der Knaap eta l . , The brain MRI of our patient showed[3 2 ]

periventricular ill defined patchy area of abnormals ignals , likely related to incomple t e myelination

process . Comparab le findings were reported by W ajneret al[33]

MS/ M S screening showed an elevated C3, whichwa s not remarkable. Urine organic acid profile sho we dhighly elevated D 2-hydroxyglutaric acid, s u c cinic acid

and lactic acid with low 2- o xo g lutaric acid. Furtherc o nfirmation and id e n t ific a t io n o f t h e e xa c t

configuration of 2-hydroxyglutaric acid was performedand data were cons is te n t with D 2-hydroxyglutaric

aciduria (fig 6).

3- Glutaric Aciduria: One p a tient was diagnosed withglutaric aciduria by a typical increase in the C5DC,

which is a dic a rb oxylic acylcarnitine that was detectedat m/z 388 by a Pre 85 scan. Increased C5DC is

indicative of GA-I. Although relatively rare in t h egeneral popu la t io n , it o ccurs more frequently in

populations that are more commonly consanguineous .[34]

The reported p atient presented at the age of 6

months with tonic clonic se izu res , opis thotonicdys tonia. neck retroflexion, progress ive macrocephaly,

and a compensated metabolic acidos is , with sparse hair,ves icular eruption on the back, dysmo rphic facial

features in the form of upturned n o s e , and frontalb o ss ing(fig 3a). Subtle early findings , such as tru n c a l

hypotonia and relative macrocephaly as seen in thispatien t , are often present but eas ily overlooked. Many

are misdiagnosed as cerebral pals y or infectiousencephalopathy. Comparable findings are also[3 5 , 36 ,37]

described by M orton et al., . Brain MRI shows[38]

prominent cortical brain sulci, basal cis terns a nd bothsylvian fis sures with widening of the subdural space at

both frontotemporoparieto-occipital regions , a picture ofsevere cortical brain atrophy(fig 3c). These findings are

s imilar to reported literature. [39]

4 - Canavan Disease: Canavan disease is one of themost common cereb ral degenerative diseases of

infancy . It is one of a group of genetic disordersknown as leukodys trophies . In this disorder the white[40]

matter of the brain degenerates into spongy tissue. It iscaused by a mutation in the g e n e fo r aspartoacylase

enzyme (ASPA).[41]

Two patients (0.250%) were diagnosed as canavandisease (4 & 12months respectively). The salient

clinical features were atonia of neck muscles ,truncalhypotonia, upper a nd lower limb hypotonia in the

young infant while the older presented with hypertonia,o c u la r ma n ifes tations in the form o f v is u a l

inattentiveness and oculoclonia, severe mental defectand megalancephaly. The ne u rologic findings are due

to demyelination a nd leukodys trophy as reported byMatalon et al., and Matalon et al.,[42 ,43]


382

Both patients had a normal metabolic screeningu s in g M S/ M S, h o we v er, organic acid profile

demons trated marked increase in N acetyl aspartic acid(NAA) in urine(fig5a). This is in a c c o rd ance with

Matalon et al., who d emons trated excess ive urinary[44]

NAA e xc retion almos t 200 times the amounts found innormal age-matched individuals . Neither enzymatic

assay nor brain biopsy was performed in our cases . Exte n s ive demyelination involving both superficial

and deep white matter as well as u fibres and internalcapsule were shown on brain MRI in both cases (fig5b).

Molecular diagnos is was carried out on one patient bygene sequencin g o f t he 6 exons and of intron –exon

junction of t h e A SPA for the proband case as well ashis s is ter, and both parents . Results proved that the

p a t ient is homozygous for the protein sequenc evariant of ASPA gene located on chromosome 17:

p.Arg233fs / p.Arg233fs while the s is ter as well a s bothparents was heterozygous for the protein sequ e n c ing

variants of the ASPA gene.

5 - Biotinidase Deficiency: Three patients in this s tud ywere diagnosed with biotinidase deficiency. Their

MS/ M S scan was normal. MS/MS can be normal in upto 20% of patients with biotinidase deficien c y a n d

therefo re t h e b e s t method of screening is asemiquantitative co lo rimetric assessment of biotinidase

act iv ity that can be performed on whole blood spotted

on filter paper.[45]

Marked deficiency in the activity of biotinid aseenzyme (<10%) was re p o rt ed in one patient (case 11)and 2 cases (s iblings) with partial biot in id a s e

deficiency (10- 30%).The case with marked enzyme deficiency presented

with lack of head support and in t ractable seizures .Patient had extens ive seborrheic dermatitis , with total

a lo p e c ia , c o n ju n c t iv it is with periorb it a l s kininflammation and very sparse eye brows , hypotonia and

deve lo p me n t a l delay. Similar presentations wereprevious ly reported by others . Other features cited[46 ,47]

in the literature by W o lf e t al., and W olf et al., ,[48 ,49]

included ataxia , s ensory neural hearing loss were

encountered in the two s iblings with partial biotinidased e ficiency and in addition they had bilateral pyramid a l

tract affe c tion with very rough and sparse hair. Theformer case showed extens ive demyelination including

subco rtical and periventricular white matter while thela t t e r two cases showed cortical atrophic changes on

MRI.Although many previous sc reening programs

emphasized only o n t h e value of amino acidabnormalities , this s tudy h ig hlights the importance of

screening high risk group s fo r organic acidopathies ,which are evidently highly prevalent fro m this s tudy

among our cons a n guineous community. It also drawsattention to the importance of implementing expanded

metabolic screening to avert serious co n sequences ofeither mental retardation or even death. The availability

of second further c o n firmatory tes ts provided by theGC-MS is of utmo s t importance for differentiating

subgroups of abnormal acylcarnitne profiles detected inNBS by MS/MS.

Conclus ion: Organic acidemias are not rare aspre v ious ly thought especially in countries with high

rates of consanguin e o u s ma rriages like Egypt.Expanded screening fo r organic acidopathies by

MS/MS us ing dried blood spot technique is a rapid andeffic ie nt method in detecting cases with poss ible

organic acid emias requiring confirmatory tes ts by GC-MS. Including organic acidopathies in newborn

s c reening programs would be of tremendous help inearly detection of pos itive cases , t hus a rapid and

properly timed therapeutic inte rv e n t io n can beundertaken to prevent devas tating neurological outcome.

Advances in n ewborn screening technology, coupledwith recent advances in the diagnos is and treatment of

rare but serious congen it a l conditions that affectnewb o rn infants , provide increased opportunities for

pos itively affectin g t he lives of children and theirfamilies . These ad v a n tages , however, also pose new

challenges in response to the management of affectedinfants

ACKNOWLEDGMENT

W e express our de epes t gratitude to Prof. Dr.Mohammed Abdel Hamid, Faculty of Pharmacy Kuwait

Univers it y ; Prof. Dr. Mohamed Rashed, King FaisalHospital, Riyadh; for their continuous support and help

for urinary GC/MS analys is , throughout our work byoffering their experie n ces and collaboration. W e also

would like to thank Catherine Sabane,hospital neckerDe s e nfants malades …. for s incere help with genetic

s tudies performed.

Footnotes: Nonstandard abbreviations : MS/MS, tandemmass spectrometry; C3 , p ro p io n y lc a rnitine; C5,

isovalerylcarnitine ; C5OH, 3-hydroxydecanoylcarnitine;PA, propionic acidemia; MM A , me t h y lma lo n ic

a c id e mia ; GC-MS, g a s c h ro ma t o g ra p h y / ma s ss p e c t r o me t r y ; ES I / M S / M S , e l e c t r o s p r a y

io nization/tandem mass spectrometry; PICU, pediatricintens ive care unit; IEM, inborn errors of metabolism;

CUCH, Cairo Univers ity Children Hospital.

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