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METABOLOMICS & Biomarker discovery

METABOLOMICS & Biomarker discovery. Anika Vaarhorst (a.a.m.vaarhorst@lumc.nl) Section of Molecular Epidemiology Leiden University Medical Centre Leiden, The Netherlands. What is Metabolomics. The nonbiased identification and quantification of all metabolites in a biological system

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METABOLOMICS & Biomarker discovery

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  1. METABOLOMICS&Biomarker discovery Anika Vaarhorst (a.a.m.vaarhorst@lumc.nl) Section of Molecular Epidemiology Leiden University Medical Centre Leiden, The Netherlands

  2. What is Metabolomics • The nonbiased identification and quantification of all metabolites in a biological system • Metabolites are the biochemicals including lipids, sugars, nucleotides, amino acids and related amines of < 2000 Dalton to be found in biological fluids • All metabolites combined make the human metabolome

  3. Why metabolomics • More than 4000 metabolitescanbemeasuredby different platforms in blood. Notall at high throughputyet. • Blood is the highway fordegraded, secreted, discardedandsynthesizedmolecules. • Indicates tissues lesions, organdysfunctionandpathological state • As -omicstechnology is close tobiomedicalphenotypes.

  4. Epigenome

  5. Pathman.smpdb.ca

  6. Metabolites marking diabetes in patients Suhre et al. PLoS ONE | November 2010 | Volume 5 | Issue 11

  7. environment Suhre et al., Nat 2011 Genetically Determined Metabotypes 37 genetic loci accounting for 10-60 variance in level Wang et al., Nat Med 2011: markers of 4 x increased T2D risk branched chain amino acids, tyrosine and phenylalanine Metabolome Genotype Phenotype Administration of branched amino acids increased insulin resistance

  8. Psychogios et al. 2011 PloS One

  9. A step to step approach Biological experiment Sample extraction NMR analysis Raw data Data preprocessing Clean data Data pretreatment Data fit for analysis Data analysis Rank the important metabolites Van den Berg et al. 2006 BMC Genomics

  10. Sample analysis 1H-NMR spectroscopy The sample is in the tube, which is in the probe, whichis in the core of the magnetic field. vacuum Liquid nitrogen Liquid helium coil core

  11. Metabolomics, NMR 1, imidazole; 2, urea; 3,D-glucose; 4, L-lactic acid; 5, glycerol; 6, L-glutamine; 7, L-alanine; 8, DSS; 9, glycine; 10, L-glutamic acid; 11, L-valine; 12, L-proline; 13, L-lysine; 14, Lhistidine;15, L-threonine; 16, propylene glycol; 17, L-leucine; 18, L-tyrosine; 19, L-phenylalanine; 20, methanol; 21,creatinine; 22, 3-hydroxybutyric acid; 23, ornithine; 24, L-isoleucine; 25, citric acid; 26, acetic acid; 27, carnitine; 28, 2-hydroxybutyric acid; 29, creatine; 30, betaine; 31, formic acid; 32,isopropyl alcohol; 33, pyruvic acid; 34, choline; 35, acetone; 36, glycerol. Analyse known variables 50

  12. Data pretreatment • Check for outliers • Check for distribution • Centering • Scaling • Transformations

  13. Data analysis • Univariate analysis • Univariate analysis combined with step wise regression • multicollinearity • LASSO regression, elastic net, ridge regression, PLS-DA

  14. Multiple testing • Bonferoni correction • 100 tests, test with a significance level of 0.05 • P after Bonferoni correction: 0.05/100 = 0.0005 • For metabolomics to conservative • Replicate your findings in independent studies • Cross-validation Storey and Tibshirani 2003, PNAS

  15. Confounding • Confounder variable: a variable other than the predictor variables that potentially affects the outcome variable • Prevent confounding: • Matching • Stratification • Controlling for confounding • Include the known confounders as covariates in your model Metabolite Outcome variable Confounder

  16. Problems: Confounding • Brindle JT et al., 2002. Nat Med. 8(12), 1439-45. → NMR spectroscopy is diagnostic for the occurrence and severity of CAD • But accordingto: Kirschenlohr et al. 2006. Nat Med. 12(6), 705-10. • Gender & statin treatment affect the ‘biomarkers’ of disease → groups must bestratified • NMR analysis of plasma is a weak predictor for CAD

  17. BBMRI Rainbow RP4 Metabolomics Applying Metabolomics in Dutch cohorts

  18. Reference populations • Leiden Longevity Study (LLS) • Netherlands Twin Register (NTR) • Erasmus Rucphen Family study (ERF) • Selection based on existing metabolomics data • Extensive phenotypic data High throughput / high resolution NMR LUMC Deelder et al. Mass spectrometry: Nederlands Metabolomics Centre, lipid platform Hankemeier et al. Mass spectrometry: Biocrates platform Gieger et al.

  19. 327 metabolites measured Biocrates N=163 146 1H-NMR N=52 Lipidomics N=129 12 5 40 124

  20. The practical Long-lived siblings Spouses as controls Offspring of long-lived siblings Which metabolites differ between controls and offspring of long-lived siblings

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