1. MECHE-NANS Progress UPDATE
03/11/2011

2. ANALYSIS OF PLASMA AND URINE
Daytona analysis basic biochemistry
(Cholesterol, HDL, TGs, Glucose, NEFAs, APOs)
NANS: Completed
MECHE: Underway (~50%) Completion: End November
Delays to MECHE work
Staffing issue mid-June to September, now rectified
NEFA and APO sample instability, now resolved
Material delays (APO CII, CIII kits) from Randox

3. MECHE: 64 REPEAT OLTTS – 65 REPEAT OGTTS -- 71 OLTT–OGTT SETS

4. Protein profiling & Proteomics
MECHE
Evidence investigator platform
Metabolic array almost complete (85%)
Insulin-Leptin-Resistin-PAI1-Cpeptide
Cytokine array work complete Il’s-VEGF-TNFa-Ifg-MCP1
Adhesion molecules array underway ICAM-VCAM-E/P/L-selectin
Expected completion date for all: End November

5. Protein profiling & Proteomics NANS
Evidence investigator platform
Metabolic array almost complete
Insulin-Leptin-Resistin-PAI1-Cpeptide
Cytokine array work Completed Il’s-VEGF-TNFa-Ifg-MCP1
Adhesion molecules array to begin ICAM-VCAM-E/P/L-selectin
Expected completion date for all: End November

6. ELISA Analysis MECHE & NANS MECHE only NANS only Adiponectin  FABP
RBP
Visfatin
Glucagon
Ghrelin
LSR
Il-RA
SAA
Amylin
Complement 3
NPY
Galinin
Peptide YY
Osteopontin
Hepcidin

7. ELISA Analysis: Issues
Suppliers to confirm: Analysis on individual kit/bulk analysis basis? Cost vs. Quality issue
Sample volume and type required: Amount and type (EDTA/serum) has implications for biobanking supply for future
Staffing to complete work: 1 person- 5 ½ months (MECHE) and 9 months (NANS)
Order of priority of work to establish

8. Metabolomics analysis
NMR 235 fasting urine samples postprandial urine samples fasting plasma samples postprandial plasma samples
GCMS (targeted analysis)
91 fasting urine samples fasting plasma samples
Expected completion dates
MECHE: Mid November
NANS: initial 600: January 2012, Extra samples: April 2012

9. Metabolomics analysis Collaborative work
Imperial college London
Bile acids: under progress
Untargeted lipidomics: under progress
Biocrates
Targeted lipidomics: samples dispatched

10. Genotyping (Item 5) Completed by Kbiosciences
COHORT
No.
SNPs
No. Samples
Quote on Cost calculator (€)
Actual Cost @ O% VAT (€)
MECHE
150
200
16,875.00
10,000.00
NANS
100
1200
22,875.00
15,000.00
TUDA 34
6000
23,587.50
20,000.00
63,337.50
45, Saving €18,337.5
MECHE: Currently DNA for extra 40 Snps Interest/need for further genotyping? Available budget?

11. Transcriptomic analysis : MECHE
4 cohorts established, 3 analysis pipelines Cohort 1 (n=9, lean, 18-30yr) Arrays completed, network analysis completed. Publication forthcoming. Cohorts 1-3 (n=8, 18-30/>30kg.m2, 30-60yr) Arrays completed. Single gene analysis completed. Novel network analysis underway. Cohort 4 (n=9, adipose tissue biopsy group) Arrays completed. Analysis pipeline under construction

12. Physical function and fitness data
Database completed: Bod Pod, DXA, VO2 max, RMR
Additional information: Body strength, segmental DXA analysis Completion date: End November

13. EPIGENETICS
100 DNA samples were sent to Sean Ennis group for epigenetic analysis
DNA samples were subjected to initial bisulphite treatment, of which approx. 70% survived and were applied to the Infinium HumanMethylation27 array, examining methylation status of 27,000 sites on 14,000 genes.
EILEEN will send on tomorrow

15. Proposed analysis
Identify CpG sites which show a significant difference in methylation values between low and high groups of the key metabolites, as described above
Name
Function
CpG (CpG island associated or not)
Distance to transcription start site

16. Proposed analysis
As per Fernandez et al and Uddin et al (2011) identify CpG sites which were
methylated (>80%) or unmethylated (<20%) in all the samples
Name
Function
CpG (CpG island associated or not)
Distance to transcription start site

17. Proposed analysis
Identify genes that were uniquely methylated or unmethylated among low v high group2.

18. Timeplan Initial meeting with C-Star (Tim Grant)
Will draw up strategy by end on Nov – second meeting
Start initial analysis Dec
Paper early 2012

19. FATTY ACID Analysis All samples have been sent to Jack Bloomfield
38 individual fatty acids
Analysis underway
Forecasted completion date : December
C6:0
C8:0
C10:0
C11:0
C12:0
C13:0
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1n9c
C18:2n6c
C18:3n6
C18:3n3
C18:4n3
C20:0
C20:1n9
C20:2
C20:3n6
C21:0
C20:4n6
C20:3n3
C20:4n3
C20:5n3
C22:0
C22:1n9
C22:2
C23:0
C22:4n6
C22:5n3
C24:0
C22:6n3
C24:1n9