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Ingenious strategy
Swedish & British subjects
8k
Cases – Hypertensive
Age <60 years
BP>160/100
NORDIL & ASCOT
Controls – Age >50 years
BP <120/80
Not on antiHTN
No prevalent CVD (MI,CVA)
No incident CVD on follow-up until
2001
8k
1M Illumina BeadChip=SNPs & CNVs
Hypercontrols
Increase odds ratio
Increase power
Better LD coverage using HapMap2
BRIGHT & InGenious
HyperCare
Investigators
Cardiovascular Gene Centric Association Study of
Metabolic Syndrome and Ambulatory Blood Pressure
in the PAMELA Study
Sandosh Padmanabhan, Cristina Menni, Wai K Lee, Stuart Laing, Paola
Brambilla, Roberto Sega, Roberto Perego, Giancarlo Cesana, Giuseppe Mancia,
Anna F Dominiczak
BHF Glasgow Cardiovascular Research Centre, University of Glasgow
University of Milano - Bicocca
PAMELA STUDY
M
O
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PAMELA STUDY
Pressioni Arteriose Monitorate E Loro Associazioni
• 2051 people 25-64 males and females
200 from each decade of life randomly
recruited from Monza
• extensively phenotyped for BP
• DNA available
•Metabolic syndrome in 16.2%
1991
10 yrs follow up
• 24H, CLINIC, HOME BP & HR
• Waist, Hips, Weight, Height
• Echo
• Blood analysis
• Drug treatment
2001
• 24H, CLINIC, HOME BP & HR
• Waist, Hips, Weight, Height
• Echo
• Blood analysis
• Drug treatment
Metabolic Syndrome: NCEP ATP III Criteria
Three or more of the following:
Abdominal obesity
waist: male >102 cm, female >88 cm
Triglycerides 150 mg/dL (1.7 mmol/L)
HDL cholesterol
Male <40 mg/dL (1 mmol/L), female <50 mg/dL
(1.3 mmol/L)
SBP 130 mm Hg or DBP  85 mm Hg
Fasting glucose 110 mg/dL (6.1 mmol/L)
NCEP=National Cholesterol Education Program.
ATP III=Third Report of NCEP Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults.
Metabolic Syndrome
Does Metabolic Syndrome exist or is it a sum of its risk components?
•Components of the system occur more often than
expected by chance
–Common underlying process
•Heritability of Metabolic Syndrome
–26% - 48%
•Linkage loci
–Chromosomes 1,2,5,6,17,18
•Candidate Genes
–LDLR, PPARG, APOA5, CYP3A4, C1QTNF5
Gene Selection
Biological plausibility
biochemical pathways that have been implicated in the
development and progression of cardiovascular disease
RAAS
SNS
OXIDATIVE
SODIUM
LIPID
STRESS
BALANCE
METABOLISM
OTHERS
Prior evidence of potential association
1536 SNPs across 98 genes selected
Illumina TagSNP selection and Genotyping
Determine chromosomal
location of target gene
(+ 10kb)
Raw Data
submitted to
BeadStudio
Software
Identify available SNP
resources (HapMap,
SeattleSNPs, PARC,)
Genotype calls for locus 4517
2.40
2.20
2.00
1.80
AA
1.60
AB
1.40
1.20
BB
1
Colon normal
0.80
0.60
Colon tumor
0.40
0.20
0
-0.20
Samples are genotyped
using GoldenGate AssayTm
and processed samples are
visualised using BeadArray
Scanner
Download SNP
Identifiers,&
submit to
Illumina for
scoring
9
0
0.20
23
0.40
0.60
0.80
71
Suitable Tagged SNPs
submitted to Illumina for
manufacture of Oligo Pool
used in GoldenGate AssayTm
Scoring by Illumina
essential as it will
determine as to
whether scored
SNP is suitable for
GoldenGateTm
Assay
Illumina scored
SNPs >0.6, MAF
> 5% submitted
to Tagger
Software
Statistical Analysis
Quality Control
Standard QC protocol involving manual review of all cluster
plots, genotype frequency, Hardy Weinberg Equilibrium, call
rates
Association Analysis
1df Trend test for dichotomous traits and Linear regression
analysis using an additive model for continuous traits
All association analysis performed using PLINK
BHF GLASGOW CARDIOVASCULAR RESEARCH CENTRE
Acknowledgements
All participants of the PAMELA
Study
Paolo Signorini, Rossana Cecere –
Desio Hospital, Milan