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Next Generation Metabolic Screening
11th Middle East Metabolic
Group meeting - MEMG22-25 October 2014, Marrakech, Morocco
Prof. dr. Ron Wevers, Department of Laboratory Medicine,
Radboudumc, Nijmegen, The Netherlands
1997
There is no single analytical platform that canmeasure all metabolites
The metabolome
NMR based metabolomics
BODY FLUID NMR SPECTROSCOPY IN INBORN ERRORS OF METABOLISM
Body fluid NMR• Diagnosis of 106 IEM (1D
and 2D COSY)• 1H resonances from 158
metabolites involved IEM• Seven novel inborn errors
could be defined
LimitationSensitivity: low micromolarrange
Molecular complexity in Life Science
Number of Dynamic molecular entities range__
Genomics 104 103
Proteomics 106 1010+
Metabolomics 104 106+
Typical eukaryotic organisms contain between 4000 and 50000 metabolites (Kegg: 16896; HMDB 2012: 7900; HMDB 3.6: 41808)
METABOLIC SCREENING
IN THE INDIVIDUAL PATIENT
Metabolomics – analysis of “all” metabolites
Human plasma, CSF(urine)
Controls vs. patient
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
AlignmentPeak comparison> 10,000 Features
Q-tof mass spectrometry
Molecular mass: 118.0266
C4H6O4
Methylmalonic acid
Accuracy of Q-tof analysis
Deviation from actual mass for 19 metabolites(Mass range 90.0552 – 428.3737 Dalton)
mass number of metabolites
0.0000 20.0001 100.0002 40.0003 20.0004 1
19
Q-tof sensitivity
Sensitivity
Q-TOF low nanomolar range
NMR low micromolar range
Factor 1000 more sensitive thanNMR!
Pipecolic acid
S/N=20
500 nmol/L
50 nmol/L
5 nmol/L
no addition
Pipecolic acid added to urine (diluted 50x)
Validation Next Generation Metabolic Screening (NGMS)
• Comparison signal intensity withconcentratios classical assays
• Mass accuracy on QTOF:95%: ΔM < 0,0003 Da(range: 90-425 Da; n=19)
• Sensitivity of UHPLC-QTOF MS assay in low nM range!(~1000 fold more sensitive than NMR)
• Intra: CV in RT: <0.5%; CV in signal intensity: <15%Inter: CV in RT: <1%; CV in signal intensity: <25-30%
• Clinical validation: diagnosis established in16 individual patients with different IEMs
Standard operation procedure for plasma and CSF samples introduced in patient care in 2014
Where is/are the biomarker(s)?
10,480 features
10,480 Features (incl. adducts etc)Mass, Retention time, Intensity
INPUT
Endogenous metabolites
Diet derived
Intestinal flora input
Medication
ANALYTICAL
Original small molecules
Adduct information (Na+, K+, NH4+)
In source fragments
13C variants
How to explain 10.000 features in plasma?
Which features are the IEM biomarkers
10,480 features
Experiment
Alignment
Peak Comparison
Raw data
Corrected t-test
Intensity/P Ranking
Dat
a an
alys
es
Identification T20
Verification
Dat
a P
rep
roce
ssin
g&
Pre
tre
atm
ent
Dat
ain
terp
reta
tio
n
sG sT Feature
Patient 7 Feature
Intensity P-value Mass Ret1 74 1909 24490807 2E-17 166.08634 3.672 51 1695 8386343 8E-21 120.08065 3.673 47 1683 2915430 6E-21 167.08956 3.674 62 1780 751272 2E-19 121.08385 3.675 56 1765 556095 3E-20 103.05433 3.676 38 1611 480141 1E-22 120.12527 3.677 57 1956 460491 3E-20 166.19191 3.678 55 1761 403223 3E-20 131.04910 3.679 35 1559 355404 2E-23 188.06796 3.67
10 3 1137 303853 8E-40 295.12893 5.8811 65 4994 291156 5E-19 188.12476 3.6612 49 1762 269007 7E-21 107.04902 3.6713 43 1659 252382 6E-22 168.09171 3.6714 52 1727 250238 8E-21 149.05937 3.6715 32 1492 140562 6E-25 120.15785 3.6716 76 2854 135373 2E-17 189.07304 1.0417 16 1018 126136 7E-29 328.13918 4.0118 23 1516 96662 1E-26 120.17050 3.6719 135 265 93406 1E-10 104.10735 12.8520 96 670 77111 4E-14 397.23525 14.3121 128 294 76090 3E-11 104.10733 12.6722 120 79 64437 3E-12 467.25918 7.4523 66 1719 61501 6E-19 204.04192 3.6724 125 89 60054 2E-11 467.00845 7.4525 121 91 57801 3E-12 622.67550 7.4526 108 87 57092 9E-13 622.34113 7.4527 4 1139 51484 2E-37 296.13220 5.8828 97 65 51462 5E-14 405.22508 12.55
▼
Patiënt 7
Feature 1909
Controls
P7P7
PKU
HMDB: phenylalanine
Feature 1137
Controls
P7P7
PKU
HMDB: glutamylphenylalanine
PKU: two biomarkers
Phenylketonuria
Patiënt Metaboliet rI rP
PKU-1 (BB07/01056)
Phe=204 (ref 17-140)
L-Phenylalanine
F8091 (m152.94715; 18.34)
F1018 (m328.13918; r4.01)
Glutamylphenylalanine
N-Acetyl-L-phenylalanine
1
5
15
16
134
285
141
28
51
162
PKU-2 (BB09/01551)
Phe=202 (ref 17-140)
L-Phenylalanine
Glutamylphenylalanine
F1018 (m328.13918; r4.01)
N-Acetyl-L-phenylalanine
1
16
20
126
447
42
28
86
PKU-3 (BB09/01183)
Phe=355 (ref 17-140)
L-Phenylalanine
Glutamylphenylalanine
F1018 (m328.13918; r4.01)
N-Acetyl-L-phenylalanine
1
14
15
128
100
9
8
116
PKU-4 (BB09/00590)
Phe=528 (ref 17-140)
L-Phenylalanine
Glutamylphenylalanine
F1018 (m328.13918; r4.01)
N-Acetyl-L-phenylalanine
1
9
12
42
1
10
8
12
PKU-5 (BB09/01951)
Phe=384 (ref 17-140)
L-Phenylalanine
Glutamylphenylalanine
F1018 (m328.13918; r4.01)
N-Acetyl-L-phenylalanine
1
58
59
142
204
93
66
67
PKU-6 (BB09/02152)
Phe=241 (ref 17-140)
L-Phenylalanine
F1018 (m328.13918; r4.01)
Glutamylphenylalanine
1
13
14
101
19
32
PKU-7 (BB10/00030)
Phe=359 (ref 17-140)
L-Phenylalanine
Glutamylphenylalanine
F1018 (m328.13918; r4.01)
N-Acetyl-L-phenylalanine
1
10
17
160
74
3
16
117
Control
PKU
P6P6
N-Acetyl-L-phenylalanine[F1031; m208.0968, r7.65]
I
F1018 is an as yet unidentified biomarker for PKU
II
PKU: but also two as yet unknown biomarkers
rI rP Feature
Patient 7 Feature
Intensity P-value Mass Ret1 74 1909 24490807 2E-17 166.08634 3.672 51 1695 8386343 8E-21 120.08065 3.673 47 1683 2915430 6E-21 167.08956 3.674 62 1780 751272 2E-19 121.08385 3.675 56 1765 556095 3E-20 103.05433 3.676 38 1611 480141 1E-22 120.12527 3.677 57 1956 460491 3E-20 166.19191 3.678 55 1761 403223 3E-20 131.04910 3.679 35 1559 355404 2E-23 188.06796 3.67
10 3 1137 303853 8E-40 295.12893 5.8811 65 4994 291156 5E-19 188.12476 3.6612 49 1762 269007 7E-21 107.04902 3.6713 43 1659 252382 6E-22 168.09171 3.6714 52 1727 250238 8E-21 149.05937 3.6715 32 1492 140562 6E-25 120.15785 3.6716 76 2854 135373 2E-17 189.07304 1.0417 16 1018 126136 7E-29 328.13918 4.0118 23 1516 96662 1E-26 120.17050 3.6719 135 265 93406 1E-10 104.10735 12.8520 96 670 77111 4E-14 397.23525 14.3121 128 294 76090 3E-11 104.10733 12.6722 120 79 64437 3E-12 467.25918 7.4523 66 1719 61501 6E-19 204.04192 3.6724 125 89 60054 2E-11 467.00845 7.4525 121 91 57801 3E-12 622.67550 7.4526 108 87 57092 9E-13 622.34113 7.4527 4 1139 51484 2E-37 296.13220 5.8828 97 65 51462 5E-14 405.22508 12.55
▼
222
Compound Adduct In-sourceL-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 +
?? N-(1-Deoxy-1-fructosyl)phenylalanine
L-Phenylalanine +????
L-Phenylalanine M+K
???
Glutamylphenylalanine 13C
?
Patient 7: Phe=359 µmol/L (ref 17-140)
PKU166
PKU5197
PKU664
PKU373
PKU490
PKU780
PKU2195
45 features(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
Untargeted metabolomics: Xanthinuria
Xanthine Uric acid
Child, 2.5 months with kidney stones; urine metabolome analysis
Xanthine
Xanthine oxidase
Uric acid
CONCLUSIONS:• Xanthinuria I (isolated xanthine oxidase)• Xanthinuria II (combined xanthine oxidase
+ aldehyde oxidase defect)
Targeted analysis of markers specific for xanthinuria type II
CONCLUSION:
Xanthinuria type II (diagnosed in urine sample without allopurinol loading)
Hydantoin 5-propionate Pyridoxal
The clinical validation
Amino acid disorders
Fatty acid oxidation
Organic acidurias Miscellaneous
PKU MCAD MSUD Xanthinuria II
Hyperprolinemia II VLCAD HMG-CoA lyase Amino acylase I
Hyperlysinemia MCC Antiquitin (ATQ) def.
MAT I/III IVA Beta-ketothiolase
Alcaptonuria Dimethylglycinuria
Ureidopropionase
Diagnosis on plasma samples
Current status: 23 inborn errors
Where is Wally?
2012
PatientTargeted
Metabolic screen
Targeted gene
analysis
Diagnosis+ follow-up
2013 / 2014
Patient
Whole exome
sequencingTargeted
confirmatory metabolite +
enzyme testing
Diagnosis+ follow-up
Targeted assays vs holistic approach
Next generation metabolic screening
Times are changing… whole exome analysis
Nijmegen four day march
The bridge between the exome and the metabolome
The Nijmegen approach
Conclusions
• Next Generation Metabolic Screening (NGMS) introduced
in diagnostics of the individual patient
• NGMS will change the metabolic laboratory
• NGMS bridges whole exome sequencing and metabolic
diagnostics: integrative biology in a functional genomics
setting
• The technique we have developed is also of interest outside
the field of inborn errors
METABOLIC SCREENING
The individual patient suspected for an IEM WES and NMGS in parallel together with dept. Genetics Radboudumc
C.D.G Huigen
E. van der Heeft
U.F.H. Engelke
R.A. Wevers
L.A.J. Kluijtmans
Nijmegen metabolomics
C. van Karnebeek,
Vancouver
J. Engel
S. Wortmann-Hagemann
The human metabolome HMDB
Food constituents FooDB
Medication Drugbank
Genetic disease OMIM
Pathway analysis Pathman-SMP
(Kegg)
Challenge - 1
Integrate databases
Challenge - 2
The issue
ApolarLC-Qtof This lecture (reversed phase)* column options Zhang T; Anal Chem 2012
PolarGC-(tof)MS Fiehn O; Trends in Anal Chem 2008 Capillary Electrophoresis-(tof)MS Ramataur R; Mol Biosyst 2011HILIC Nijmegen, 2014
Challenge - 3
The big data
• Pathway analysis• How to integrate Whole Exome Sequencing data with the
metabolomics data?
METABOLIC SCREENING
IN THE INDIVIDUAL PATIENT
DNA SEQUENCING
IN THE INDIVIDUAL PATIENT
Integratingsoftware
The next step
The coding data of the humangenes
10
,48
0
Challenge - 4
The intracellular metabolome
HILIC-ToF-MS analysis urine samples (XBridge amide)
Reproducibility retention times
Standard solution polar compounds (n = 4)
Verbinding Retentietijd
gemiddelde %CV
Arginine 10.07 0.08
Asparagine 8.59 0.10
Aspartic acid 9.47 0.41
Glutamic acid 8.69 0.33
Glutamine 8.37 0.18
Histidine 9.84 0.46
Isoleucine 5.77 0.48
Leucine 5.50 0.57
Lysine 10.33 0.14
Methionine 6.07 0.37
Phenylalanine 5.35 0.34
Proline 6.52 0.49
Serine 8.46 0.20
Threonine 7.81 0.17
Tryptophan 5.31 0.11
Tyrosine 6.44 0.00
Valine 6.39 0.08
Quality requirement RT: CV ≤ 0.5%
HILIC-ToF-MS analyse van urinemonsters. Kolom: XBridge amide
Herhaalbaarheid retentietijden
Analyse standaardoplossing polaire metabolieten (n = 2)
Eis: retentietijd %CV ≤ 0.5%Verbinding Retentietijd
gemiddelde ∆rt
Cytosine 4.33 0.04
Kreatinine 3.61 0.05
Betaine 5.92 0.09
Nicotinic acid 3.94 0.17
Kreatine 7.47 0.06
Adenine 3.28 0.01
Hypoxanthine 3.60 0.06
Xanthine 3.97 0.04
Carnitine 6.75 0.09
2,8-Dihydroxyadenine 7.36 0.03
Cafeïne 1.42 -0.01
Cytidine 5.41 0.08
2'-deoxyadenosine 3.08 0.01
2'-deoxyinosine 3.96 0.06
2'-deoxyguanosine 4.93 0.04
Adenosine 3.68 0.02
Inosine 4.78 0.08
Guanosine 5.76 0.07
Xanthosine 5.52 0.12