Ingenious strategy

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Ingenious strategy
Swedish British subjects
Controls Age gt50 years BP lt120/80 Not on
antiHTN No prevalent CVD (MI,CVA) No incident CVD
on follow-up until 2001
Cases Hypertensive Age lt60 years BPgt160/100 NORD
IL ASCOT
8k
8k
1M Illumina BeadChipSNPs CNVs
Hypercontrols Increase odds ratio Increase
power Better LD coverage using HapMap2
BRIGHT InGenious HyperCare Investigators
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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

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PAMELA STUDY
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

10 yrs follow up
1991
2001

• 24H, CLINIC, HOME BP HR
• Waist, Hips, Weight, Height
• Echo
• Blood analysis
• Drug treatment

• 24H, CLINIC, HOME BP HR
• Waist, Hips, Weight, Height
• Echo
• Blood analysis
• Drug treatment

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Metabolic Syndrome NCEP ATP III Criteria

• Three or more of the following
• Abdominal obesitywaist male gt102 cm, female gt88
  cm
• Triglycerides ?150 mg/dL (1.7 mmol/L)
• HDL cholesterolMale lt40 mg/dL (1 mmol/L), female
  lt50 mg/dL (1.3 mmol/L)
• SBP ?130 mm Hg or DBP ? 85 mm Hg
• Fasting glucose ?110 mg/dL (6.1 mmol/L)

NCEPNational Cholesterol Education Program.ATP
IIIThird Report of NCEP Expert Panel on
Detection, Evaluation, and Treatment of High
Blood Cholesterol in Adults.
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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

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Gene Selection

• Biological plausibility
• biochemical pathways that have been implicated in
  the development and progression of cardiovascular
  disease
• RAAS
• SNS
• OXIDATIVE STRESS
• SODIUM BALANCE
• LIPID METABOLISM
• OTHERS
• Prior evidence of potential association
• 1536 SNPs across 98 genes selected

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Illumina TagSNP selection and Genotyping
Download SNP Identifiers, submit to Illumina for
scoring
Determine chromosomal location of target gene (
10kb)
Identify available SNP resources (HapMap,
SeattleSNPs, PARC,)
Scoring by Illumina essential as it will
determine as to whether scored SNP is suitable
for GoldenGateTm Assay
Illumina scored SNPs gt0.6, MAF gt 5 submitted to
Tagger Software
Suitable Tagged SNPs submitted to Illumina for
manufacture of Oligo Pool used in GoldenGate
AssayTm
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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

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BHF GLASGOW CARDIOVASCULAR RESEARCH CENTRE
Acknowledgements All participants of the PAMELA
Study Paolo Signorini, Rossana Cecere Desio
Hospital, Milan
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