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

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

3. Population dynamics in isolates Founder effect Genetic drift

4. 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

5. Comparison of genetic variability GRIP, Iceland, CEPH Mean number of alleles CEPH > Iceland > GRIP 10.059 > 7.623 > 7.240 (p<0.01) Mean heterozygosity CEPH > Iceland > GRIP 0.768 > 0.758 > 0.757(p<0.05) Inbreeding Pardo et al, Ann Hum Genet 2005

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

7. Expected fluctuations alleles proportions by allele frequency P 0 * Probability loss of allele Probability allele proportion is > 5P 0 * 0.0001 0.954 0.026 0.001 0.615 0.02 0.01 0.0083 0 0.025 0 0 0.05 0 0 0.1 0 0 0.25 0 0 0.5 0 0.046 0.0483 0.0068 0 0 0 0 0 0 P 0 * : Allele proportion original population Probability allele proportion is > 10P 0 * Pardo et al, Ann Hum Genet 2005

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

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

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

11. 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

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

13. 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

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

15. 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

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

17.  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)

18. Results genome wide screen ChromosomeMarkercMLODHLODAlpha 1D1S4841704.2 0.81 3D3S15491512.83.60.81 D3S15691584.34.40.82 D3S36261641.92.60.64 6D6S1610542.8 1.00 10D10S16861052.6 1.00 D10S1851162.5 0.94 19APOE*691.41.50.80 Liu et al, AJHG in press

19. Haplotype analysis chromosome 3

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

21. 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

22. Genome wide analysis Stroop test in 200 individuals (ERF)

23. rs17129622rs11185978rs7071717 Learning 0.23 0.36 0.30 Recall 0.55 0.60 0.67 Recognition 0.48 0.60 0.71 Stroop 0.00003 0.000020.000005 TMT-B 0.00023 0.01 0.01 Semantic Fluency 0.10 0.05 0.04 Phonological Fluency 0.002 0.001 0.01 Block Test 0.80 0.61 0.90 Total evidence 7*10 -13 9*10 -13 3*10 -14 Adjusted p-value 0.02 0.010.002 CYP2C19 - CYP2C9 Chromosome 10 region Liu et al, AJHG in press

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

25.  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

26. 3-SNP haplotype was observed with a frequency of 0.905 in those with blue eyes and 0.095 in those with brown eyes OCA2 and eye colour Duffy et al, AJHG 2007, 80:241-252

27. 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

28. 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

29. GWA analysis in 481 persons of the Rotterdam study

31. 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

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

33. 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

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

37. Cardiovascular assessments Plaques, intima media thickness, pulsewave velocity

38. Ophthalmologic studies Age related macular degeneration Glaucoma

39. Neuropsychiatric testing

40. Thanks !