next-generation metabolic screening: the application of ... → 3-hydroxyisovalerylcarnitine mcad...

Next-generation metabolic screening: the

application of untargeted mass spectrometry-

based metabolomics in the diagnostics of Inborn

Errors of Metabolism (IEM) in the individual

patient

Dr. Leo AJ Kluijtmans, Laboratory specialist Clinical Genetics

Dept of Laboratory Medicine, Laboratory Genetic, Endocrine and Metabolic

Diseases, (LGEM) Radboudumc Nijmegen

Radboudumc • One of eight UMCs in the Netherlands • Core activities:

• Patient care • Research • Education

• 11.000 colleagues • 50 departments • 3.000 students • 1.000 beds (ambition to close 500 by improving

healthcare)

• Mission: “To have a significant impact on healthcare” • Strategic focus on Personalized Healthcare

Personalized Healthcare @ Radboudumc

People are different Stratification by multilevel diagnosis

+ Patient’s preference of treatment

Exchange experiences in care communities

Select personalized therapy

Expansion of our IEM knowledge >1000 IEMs

Scriver: The Metabolic & Molecular Bases of

Inherited Disease

Where’s Wally?

Current targeted analysis in diagnostics of IEM

Selected tests

Amino acids

Amino acid analyser

Carnitine-ester profile

LC-MS/MS

Purines & pyrimidines

- HPLC & LC-MS/MS

Monosaccharides

GC-MS

2012

Patient Targeted

Metabolic

screen

Targeted

gene

analysis

Diagnosis

+ follow-up

2013 / 2014

Patient

Whole

exome

sequencing Targeted

confirmatory

metabolite +

enzyme

testing

Diagnosis

+ follow-up

Targeted assays vs holistic approach

Next

generation

metabolic

screening

Times are changing… whole exome analysis

Aims Metabolomics

• Development of validated LC-MS based metabolic profiling assay in body

fluids

• Identify normal profiles and normal “concentration” ranges of metabolites

• Application of LC-MS based metabolomics in the diagnostics of IEM in an

individual patient

A plasma profile by UPLC-QTOF

A typical plasma sample

shows ~10.000 “features”

Maybe

Human plasma

Control vs. IEM

Agilent QTOF MS-data

- Reverse phase liquid chromatography - Positive and negative mode - Features

•Accurate mass (165.07898) • Retention time •Intensity

(New) biomarkers for diseases

XCMS

Alignment

Peak comparison

> 10000 Features

Metabolomics – analysis of “all” metabolites

Proof of Principle: PKU

Proof of principle: UHPLC-TOF analysis of plasma

Which one is the biomarker?

10480

10480 Features Mass, Retention time, Intensity

Experiment

Alignment

Peak Comparison

Raw data

t-test with B.C.

Intensity Ranking

Da

ta a

na

lys

es

Identification T20

Verification

Da

ta P

rep

roc

es

sin

g

& P

retr

ea

tme

nt

Da

ta

inte

rpre

tati

on

sG sT Feature

Patiënt 7 Feature

Intensity P-value Mass Ret 1 74 1909 24490807 2E-17 166.08634 3.67 2 51 1695 8386343 8E-21 120.08065 3.67 3 47 1683 2915430 6E-21 167.08956 3.67 4 62 1780 751272 2E-19 121.08385 3.67 5 56 1765 556095 3E-20 103.05433 3.67 6 38 1611 480141 1E-22 120.12527 3.67 7 57 1956 460491 3E-20 166.19191 3.67 8 55 1761 403223 3E-20 131.04910 3.67 9 35 1559 355404 2E-23 188.06796 3.67

10 3 1137 303853 8E-40 295.12893 5.88 11 65 4994 291156 5E-19 188.12476 3.66 12 49 1762 269007 7E-21 107.04902 3.67 13 43 1659 252382 6E-22 168.09171 3.67 14 52 1727 250238 8E-21 149.05937 3.67 15 32 1492 140562 6E-25 120.15785 3.67 16 76 2854 135373 2E-17 189.07304 1.04 17 16 1018 126136 7E-29 328.13918 4.01 18 23 1516 96662 1E-26 120.17050 3.67 19 135 265 93406 1E-10 104.10735 12.85 20 96 670 77111 4E-14 397.23525 14.31 21 128 294 76090 3E-11 104.10733 12.67 22 120 79 64437 3E-12 467.25918 7.45 23 66 1719 61501 6E-19 204.04192 3.67 24 125 89 60054 2E-11 467.00845 7.45 25 121 91 57801 3E-12 622.67550 7.45 26 108 87 57092 9E-13 622.34113 7.45 27 4 1139 51484 2E-37 296.13220 5.88 28 97 65 51462 5E-14 405.22508 12.55

▼

Feature 1909

Control

P7 P7

PKU

Feature 1137

Control

P7 P7

PKU

Patiënt 7

sG sT Feature

Patiënt 7 Feature



▼

Patiënt 7

Feature 2854

Control

P7 P7

PKU

Feature 670

Control

P7 P7

PKU

rI rP Feature

Patiënt 7 Feature



▼

Compound Adduct In-source L-Phenylalanine M+H

L-Phenylalanine + L-Phenylalanine 13C

L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + L-Phenylalanine M+Na

Glutamylphenylalanine M+Na

L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + L-Phenylalanine + Not in all PKU

? L-Phenylalanine +

? ? ? ?

L-Phenylalanine M+K

? ? ?

Glutamylphenylalanine 13C

?

Patiënt 7: Phe=359 (ref 17-140)

Use of My Coumpound ID to annotate

Proof of Principle

• All diagnostic metabolites were identified for the 3 diseases:

PKU → phenylalanine

3MCC → 3-methylcrotonylglycine / 3-hydroxyisovaleric acid

MCAD → octanoylcarnitine, 9-decenoylcarnitine and hexanoylcarnitine

• Additional biomarkers:

PKU → glutamylphenylalanine

3MCC → 3-hydroxyisovalerylcarnitine

MCAD → capryloylglycine, nonanoylcarnitine, suberylglycine, hexanoylglycine

and heptanoylcarnitine

• Unidentified metabolites:

PKU → F1018 (m328.13918; r4.01), possibly addition of hexose to phenylalanine

3MCC → F2197 (m262.164945; rt3.62)

MCAD → Several

A blind study

Plasma sample choice : Dr. C.D.G Huigen

Analytical chemistry : Ing. E. van der Heeft

Chemometrics : Dr. U.F.H. Engelke

Diagnosis : Prof. dr. R.A. Wevers;

Dr. L.A.J. Kluijtmans

10 samples from 10 patients

with 5 different IEM’s

21 controls

The blind study

MSUD (2) → leucine, isoleucine, valine, 3-methyl-2-oxovaleric acid

Aminoacylase I deficiency (2) → N-acetylglutamine, N-acetylglutamic acid,

N-acetylalanine, N-acetylserine, N-acetylasparagine, N-acetylglycine

Prolinemia type II (2) → proline, 1-pyrroline-5-carboxylic acid

Hyperlysinemia (2) → pipecolic acid, lysine, homoarginine, homocitrulline

3-Hydroxy-3-methylglutaryl-CoA lyase deficiency (2) → 3-methylglutaryl-

carnitine, 3-methylglutaconic acid, 3-hydroxy-2-methylbutanoic acid, 3-hydroxy-3-

methylglutaric acid

Diagnostic metabolites found in blood plasma

CONCLUSION:

Five different IEM’s

Correct diagnosis in all 10 patients

Validated method SOP

Targeted metabolomics approach

• Young child, date of birth: 30-06-2013

• Presenting clinical feature @ 08-09-2013: kidney stones

• Usual tests in metabolic screening: organic acids, purines/pyrimidines and

amino acids.

• Xanthine: 413 µmol/mmol (N<50)

• Uric acid: 15 µmol/mmol (N: 200-3000)

• Highly suspected of xanthinuria

• Type I: isolated xanthine oxidase deficiency • Type II: combined XO and aldehyde oxidase deficiency (discrimination by allopurinol loading test; only patients with type I

can metabolize allopurinol via aldehyde oxidase)

• Analysis in plasma sample without allopurinol loading

PRPP

2 Glutamine

Glycine

Aspartate

N5,N10-methenyl-THF

N10-formyl-THF

4 ATP

HCO3-

IMP

Ino

Hypoxanthine

Succinyl-AMP

AMP

Ado

ADP

ATP

dADP

dATP

dAMP

dAdo

RR

XMP

GMP

Guo

Guanine

Xanthosine

Xanthine

Uric acid

GDP

GTP dGTP

dGDP

dGMP

dGuo

RR

Adenine

IMPDH

DNADNA RNARNA

1 2 2

3333

4

5677

89 9 9 9 9 9 10

18

10

15

18

1111

11

11

12

13

14

14

15

16

17

Figure 3: Purine metabolism

Enzymes

IMPDH: IMP dehydrogenase

RR: Ribonucleotide reductase

1: GMP synthetase

2: (d)GMP kinase

3: Nucleoside diphosphate kinase

4: GMP reductase

5: Adenylosuccinate synthetase

6: AMP lyase

7: AMP kinase (myokinase)

8: AMP deaminase

9: 5’-Nucleotidase

10: dGuo kinase

11: Purine nucleoside phosphorylase

12: Hypoxanthine-guanine phosphorybosyl transferase (HGPRT)

13: Guanine deaminase

14: Xanthine oxidase

15: Ado kinase

16: Ado deaminase

17: Adenine phosphoribosyl transferase (APRT)

18: dCyd kinase

12

Purine metabolisme

Targeted: Marker compounds common to type I and type II xanthinuria

Hypoxanthine

Xanthine Uric acid

Xanthosine

Targeted: Marker compounds specific for type II xanthinuria

Hydantoin 5-propionate

N-(3-acetamidopropyl) pyrrolidin-2-one



pyridoxal

Conclusions • We have developed a reversed-phase LC-QTOF assay that is applicable in a

diagnostic setting, both in an untargeted (several IEMs) and in a targeted approach

(xanthinuria type II). C

• CV in RTs: <0,5%; CV in signal intensity: <15%.

• Assay applicable to plasma, urine (neg mode: 1 mM kreat; pos mode 5 mM kreat)

and CSF samples.

• Diagnosis has been established in patients with the following diseases: PKU, MCAD,

VLCAD, MCC, amino acylase I, MSUD, MAT I/III, HMG-CoA lyase, Prolinemia type II,

Xanthinuria type II and Hyperlysinemia

• Challenges:

• extension of assay to more polar compounds (HILIC) and apolar compounds

(lipidomics)

• Application of QTOF assay in 50 WES patients with ‘metabolic filter (543 genes)

and comparison metabolomics and exome data: functional exome analysis!

Where’s Wally?

Acknowledgments

• Laboratory Genetic Endocrine and Metabolic Diseases, Dept of Laboratory Medicine, Radboudumc

Udo Engelke

Ed van der Heeft

Siebolt de Boer

Marleen Huigen

Ron Wevers

• Dept. of Analytical Chemistry/Chemometrics; Institute of Molecules and Materials, Radboud University

Jasper Engel

Interaction of genome and environment shape the metabolome


N-Methylnicotinamide Pyridone carboxamide

Mass 153.0407

QTOF plasma metabolomics ESI+

5 x10

0

0.5

1

1.5

2

2.5

3

3.5

5 x10

0

0.5

1

1.5

2

2.5

3

3.5

5 x10

0

0.5

1

1.5

2

2.5

3

3.5

Counts vs. Acquisition Time (min)

1.7 1.72 1.74 1.76 1.78 1.8 1.82 1.84 1.86 1.88 1.9 1.92 1.94 1.96 1.98 2 2.02 2.04 2.06 2.08 2.1 2.12 2.14 2.16 2.18 2.2 2.22 2.24 2.26 2.28 2.3 2.32 2.34 2.36 2.38 2.4 2.42 2.44 2.46 2.48 2.5

Control

Patient with xanthineria, type II

“Control” using allopurinol

Xanthine

Xanthine (11 µmol/l)

Xanthine (10 µmol/l)

Oxypurinol (47 µmol/l)

Oxypurinol

M=152.03343

Xanthine

M=152.03343

Ion-exchange chromatography

concentration (µM)

UH

PL

C-Q

TO

F-M

S

XC

MS

in

ten

sitie

s (

arb

itra

ry s

ca

le)

Phenylalanine (n=10) Octanoylcarnitine (n=10)

LC-MS/MS

concentration (µM)

Correlation of phenylalanine and octanoylcarnitine determination in plasma between

UHPLC-QTOF-MS and “dedicated” methods

UH

PL

C-Q

TO

F-M

S

XC

MS

in

ten

sitie

s (

arb

itra

ry s

ca

le)

Exact mass: 165.0789786 Exact mass: 287,2096584

PKU1 66

PKU5 197

PKU6 64

PKU3 73

PKU4 90

PKU7 80

PKU2 195

7 metabolites - Phenylalanine - Glutamylphenylalanine - Phenylalanine + C6H10O5

- Mass 278.17518 Unknown

- Prolyl-L-phenylalanine + H2

- N-Acetyl-L-phenylalanine - Mass 424.17163 Unknown

Plasma biomarkers for PKU patients

Accuracy of Q-tof mass analysis

Deviation from actual mass for 19 metabolites

(Mass range 90.0552 – 428.3737 Dalton)

mass number of

metabolites

0.0000 2

0.0001 10

0.0002 4

0.0003 2

0.0004 1

19

Data Interpretation

From mass 176.1030 to metabolite identification

HMBD database

176.1 ± 0.1 9 metabolite options

176.1030 ± 0.0005 C6H13N3O3: 2 metabolites

Citrulline

176.1030

Argininic acid

176.1030

Citrulline and argininic acid have

different retention times on the

column and different mass spectra

Q-TOF sensitivity

Pipecolic acid spiked in urine (diluted 50x)

Sensitivity

Q-TOF

low nanomolar range

NMR

low micromolar range

Factor 1000!

Pipecolic acid

S/N=20

500 nmol/L

50 nmol/L

5 nmol/L

no addition

next-generation metabolic screening: the application of ... → 3-hydroxyisovalerylcarnitine mcad...

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