Genome wide association studies of complex traits in outbred and isolated populations CM van Duijn Genetic Epidemiology Unit Erasmus MC, Rotterdam, The Netherlands

Topics To what extend do isolates differ from outbred populations ? What is the role of isolates in gene wide association studies ?

Population dynamics in isolates Founder effect Genetic drift

Genetic Research in Isolated Populations (GRIP) program  Recent genetically isolated population in the South-West of the Netherlands  At least 150 founders in 1750  Substantial population growth, with minimal new immigration until last decades  Genealogical data Rotterdam GRIP

Comparison of genetic variability GRIP, Iceland, CEPH Mean number of alleles CEPH > Iceland > GRIP > > (p<0.01) Mean heterozygosity CEPH > Iceland > GRIP > > 0.757(p<0.05) Inbreeding Pardo et al, Ann Hum Genet 2005

Pedigree of the GRIP population What is the effect of isolation on genome?

Expected fluctuations alleles proportions by allele frequency P 0 * Probability loss of allele Probability allele proportion is > 5P 0 * P 0 * : Allele proportion original population Probability allele proportion is > 10P 0 * Pardo et al, Ann Hum Genet 2005

ProteinGeneComplex disease Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Rare mutations in an outbred population

ProteinGeneComplex disease Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Rare mutations in an isolated population

ProteinGeneComplex disease Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Protein Gene Common mutations in an isolated population

Comparison of linkage disequilibrium of recently isolated populations Antioquia, Colombia ((ANT) Ashkenazi (ASH) Azores (AZO) Central Valley of Costa Rica (CR) GRIP population Netherlands (NL) Early settlement Finland (FIC) Finnish subisolate of Kuusamo (FIK) Finland nationwide (FIP) Newfoundland (NFL) Afrikaner (SAF) Province of Nuoro in Sardiana (SAR) Caucasian general population (CAU) Service et al, Nature Genet 2006

Linkage disequilibrium units (LDU) for chromosome 22 Service et al, Nature Genet 2006

To what extend do isolates differ from outbred populations? Have a reduced variability for rare genetic variants May show extended linkage disequilibrium Are often characterised by consanguinity

Topics To what extend do isolates differ from outbred populations ? What is the role of isolates in gene wide association studies ?

Finding genes in isolates Rare variants Multiple common variants Became very prevalent or disappeared Will be found also in isolated populations Linkage is most powerful approach Association is most powerful approach

Finding genes in isolates Rare variants with major effects Multiple common variants Linkage: example Alzheimer disease Association: eye colour

 111 probable late onset (75 ± 5.3 years) Alzheimer patients  103 patients were connected based on genealogy  Genome wide screen with 420 micro-satelilite markers (Perkin Elmer) Pedigree Alzheimer’s disease (AD)

Results genome wide screen ChromosomeMarkercMLODHLODAlpha 1D1S D3S D3S D3S D6S D10S D10S APOE* Liu et al, AJHG in press

Haplotype analysis chromosome 3

Finding genes in isolates From linkage to association? From association to linkage?

Genome wide association analysis Population 200 distantly related individuals Phenotype Cognitive function Genotyping Affymetrix 250K SNP array Data Analysis Single SNP association: Type 1 error (p): nominal and permutation tests

Genome wide analysis Stroop test in 200 individuals (ERF)

rs rs rs Learning Recall Recognition Stroop TMT-B Semantic Fluency Phonological Fluency Block Test Total evidence 7* * * Adjusted p-value CYP2C19 - CYP2C9 Chromosome 10 region Liu et al, AJHG in press

Finding genes in isolates Rare variants with major effects Multiple common variants Linkage: example Alzheimer disease Association: eye colour

 Co-dominant trait  Brown dominates over green, green over blue  Strong association for blue/nonblue eye color was found with three OCA2 SNPs (Duffy et al. 2007) Genetics of eye color

3-SNP haplotype was observed with a frequency of in those with blue eyes and in those with brown eyes OCA2 and eye colour Duffy et al, AJHG 2007, 80:

GWA in 192 participants of GRIP (Affymetrix 250k)  Significant association (p=1.2×10 -9 ) to the OCA2 region at chromosome 15q13.1  Adjusted for multiple testing by 100 genome-wide permutations

Rotterdam Study Population-based study: outbred Caucasian population 12,000 subjects aged 55 + years who have been followed for 15 years Sampling DNA, RNA, serum/plasma Screening for major diseases and risk factors ever 5 years Genome wide association: Affymetrix 500K

GWA analysis in 481 persons of the Rotterdam study

Linkage of OCA2 to eye color in 4 families GRIP Family 14 LOD=1.6 Family 12 LOD=1.1 Family 5 LOD=1.1 Family 7 LOD=1.4 Overall LOD 3000 relatives = 22.0

Finding genes in isolates From linkage to association From association to linkage Powerful replication

Diseases studied in GRIP NeuropsychiatricCardiovascular Parkinson disease Hemochromatosis Alzheimer's disease Diabetes type 2 Stroke/ White Matter Disease Hypertension Multiple Sclerosis Pre-eclampsia ADHD Fertility: PCOS DepressionAtherosclerosis AnxietyHyperlipidemia

OphthalmologicMacular degeneration Glaucoma Auto-immuneType 1 diabetes BoneOsteoporosis Osteoarthritis Rheumatoid arthritis LungCOPD PharmacologyResponse antihypertensives Diseases studied in GRIP

Cardiovascular assessments Plaques, intima media thickness, pulsewave velocity

Ophthalmologic studies Age related macular degeneration Glaucoma

Neuropsychiatric testing

Thanks !