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INHERITANCE Project

INHERITANCE Project. P7: Jules Griffin jlg40@mole.bio.cam.ac.uk ; Department of Biochemistry, University of Cambridge. Pavia, January 2010. Objectives.

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INHERITANCE Project

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  1. INHERITANCE Project P7: Jules Griffin jlg40@mole.bio.cam.ac.uk; Department of Biochemistry, University of Cambridge Pavia, January 2010

  2. Objectives • To define the metabolome and proteome of cardiac tissue associated with LMNA gene defects (examined at the transcriptional level in WP 4) from available heart tissue samples. • To develop a multivariate systems biology model of the -omic data obtained on LMNA gene defects to define the pathophysiological changes induced in the heart. • To expand the metabolomic description to include other disease genes in genotyped heart tissue to interface with transcriptomic studies in WP4. • To define the metabolome of zebrafish model of Dilated Cardiomyopathy (DCM). • To conduct metabolomic screening of blood plasma from DCM patients to identify novel biofluid markers.

  3. Metabolomics Metabolomicsis the measurement of the metabolite complement of a cell, tissue or organism in order to monitor a perturbation such as a genetic modification or pathophysiological stimulus. The concept has been around for several decades, but the advance in technology has allowed metabolic profiling to be carried out on a large scale.

  4. The challenges of metabolomics • How many metabolites? • Just considering one class there are a huge number of permutations • 40 common fatty acids • 40 FA acyl CoA • 64000 TAGs • 120 1-, 2-, 3- MAG • 4800 1,2-, 1,3-, 2,3- DAGs • Total = 69000 Conc. Range 109 Global profiles NMR GC-MS LC-MS Custom assays Polarity Log -6 to 14 Mass < 1500 amu

  5. Open or Closed? • Open analysis • An analysis of the total detectable content of the sample (e.g. an NMR spectrum of urine) • Primarily used for the detection of novel entities • Closed analysis • An analysis focused onto a specific molecule or molecules (e.g. measurement of a specific m/z) • Used for the measurement of known variables for a model

  6. Experimental protocol. • Extraction: • Chloroform/methanol extraction procedure; • Profiling techniques: • 1H-Nuclear Magnetic Resonance (NMR) spectroscopy and Gas Chromarography- Mass Spectrometry (GC-MS) will be used to profile the metabolite complement; • Multivariate statistics: • To characterise profiles associated with individual disease.

  7. Sample collection protocol. • Heart tissue (>30 mg wet weight) should be flash frozen in liquid nitrogen or on dry ice prior to storage at -80 C. It is imperative that any delay in freezing tissue is recorded as this will affect metabolism. Post-mortem samples should at the very least have a time associated with delay between time of death and subsequent freezing to assess sample degradation. Tissue samples to be transported to the UCAM on dry ice. For each tissue sample taken a full sample description should be provided. No preservatives are necessary. • Blood plasma (>400 µl) will be collected in hepinized tubes by spinning whole blood in a centrifuge and discarding the cell fraction. Blood plasma is stored at -80 C prior to shipping to UCAM on dry ice. For each blood plasma sample taken a full sample description should be provided (see below). For best results blood withdrawn from the coronary sinus (see Lewis GD et al., Metabolite profiling of blood from individuals undergoing planned myocardial infarction reveals early markers of myocardial injury. J Clin Invest. 2008 Oct;118(10):3503-12.

  8. Experimental subject description. Ethical approval details Geographical location/hospital/ Ethnic background Medical History (disease or clinical symptoms; criteria for disease presence) Age range Weight range and Height and/or BMI Gender Study design type (e.g. randomised trial, Disease biomarker and description of relevant control groups for samples). Diet - (e.g. vegetarian, vegan etc.) Further descriptors (Smoking, blood pressure, anomalies in habitual diet (e.g. vegetarian, vegan etc.), sporting activity and frequency, habitual alcohol consumption)

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