GWAS on the Island of Kosrae Table 3. cont. Rank 9 10 11 12 Chr Position (Mb) SNP A1 A2 MAF P-value b Var expl Nearest Gene(s) 12 12 2 12 119.7 119.7 23.9 119.8 rs6489780 rs10849788 rs2081302 rs3809314 G A A A C G C G 0.35 0.36 0.34 0.36 8.4E-06 0.20 9.0E-06 0.20 9.8E-06 0.20 1.8% 1.8% 1.8% sppl3 sppl3 ensg00000174074, sppl3 9.2E-06 20.22 2.2% ensg00000119778, ensg00000119771 A1 is the associated (minor) allele. MAF, minor allele frequency. b, effect size expressed as the number of standard deviations change in phenotype for each copy of the associated allele. ‘‘Var expl,’’ population phenotypic variance explained. Genes within 30 kb of the SNP are shown where applicable. For leptin and fasting plasma glucose, there were no results with p#1025; instead, the single best result is given. doi:10.1371/journal.pgen.1000365.t003 (MIM 602617). Missense mutations in TTF-2 have been reported in conjunction with thyroid agenesis and congenital hypothyroid- ism in humans [29–31]. The two best-associated SNPs for TSH, rs755109 and rs10983893, are located ,19 kb upstream and ,77 kb downstream of the TTF-2 coding region, respectively. Analyses conditioning on the best-associated SNP, rs755109, suggest that a single association signal underlies association between SNPs in this region and plasma levels of TSH in Kosraens (data not shown). In another example of association observed near a strong biological candidate, rs10998046 on chromosome 10 near MAWBP (MIM 612189) is modestly associated with fasting plasma glucose in Kosraens (p = 1.1261024). Upregulated expression of this gene has been reported in rat models of insulin resistance [32]. Meta-analysis of these data with publicly available results from the Diabetes Genetics Initiative [33] produces a combined p-value of 2.1061026, with 7 other neighboring SNPs producing combined p-values of 2.5361026 to 5.7361026. While extended linkage disequilibrium limits our ability to identify the causal variant in Kosraens or to exclude association to other genes in the associated region, these loci represent promising results for follow-up in other cohorts. Evidence for Many Variants of Small Effect Segregating on Kosrae Our study was motivated by the hypothesis that the reduction in allelic heterogeneity resulting from the founder effect on Kosrae, combined with drift and natural selection, might produce common variants with relatively large effects segregating through the population. Empirical power calculations demonstrated that effects $5% should be readily detected (95% power) at p#1026. Given these power calculations, and the evidence for a series of known associated loci as described above, the consistent lack of strong association across the majority of traits argues strongly that common variants of large effect are unusual on Kosrae (Table 3), as they are in the larger populations studied to date in GWAS. The best observed p-value for each trait at a novel locus ranged from 1.961025 for fasting plasma glucose (rs10745259) to 8.461027 for waist circumference (rs2222328). Only two of fifteen traits, TSH and waist circumference, have strong, novel associations with p#1026. No novel associations were detected in any trait with p,861027. Interestingly, these data indicate that on Kosrae, common variants in LD with SNPs on the genotyping array are of small effect in this founder population, similar to the genetic architecture observed in Caucasian populations. If the effect sizes for common variants are similar in Kosraens and Europeans, then the lack of strong associations is unsurprising given the modest size of our cohort. We observe modest evidence for SNPs near multiple other loci which have been convincingly replicated in other cohorts [21,22,27,33,34], including HDL-C and LPL (p = 0.025, rs17411024) or LIPC (p = 4.561023, rs11071386); triglycerides and GCKR (p = 0.015, rs780094), and LDL-C and APOB (1.661023, rs7575840). These and other common variants of small effect identified in Caucasian popula- tions may also influence trait variation on Kosrae, despite lack of genome-wide statistical significance for association. Comparison of Effects and Allele Frequencies in Caucasians and Kosraens for Known Associated Loci We examined data for previously associated SNPs not under the hypothesis of no effect on trait value, but rather under the alternative hypothesis that the effect seen in Europeans was also observed on Kosrae. We compared the effect sizes (b-coefficients) and allele frequencies for known loci in Caucasians to those observed in our study. Specifically, we identified 45 established associations for BMI, height, lipids, fasting plasma glucose, TSH and CRP (Table 4) where the best-associated SNP in the literature was directly genotyped in Kosraens or had a strong proxy (r2$0.95) in HapMap Caucasians and Asians [18–22,34–36]. A test of heterogeneity for the magnitude and direction of b- coefficients was performed for 39 SNPs with MAF$0.05. Six SNPs were omitted from the comparison of effect sizes, as there is little power to estimate the individual effects for SNPs observed at very low frequencies. Of the 39 SNPs examined for effect sizes on Kosrae, only 6 loci had significantly different (p#0.01) effects in Caucasians and Kosraens (p = 5.5610242 to 6.861024), of which 4 SNPs were associated with height in Caucasians. Over 70% of the loci evaluated (n = 28) had effects which were of indistinguishable magnitude and direction (p$0.1) in the two populations. We next considered whether differences in allele frequency could underlie the lack of association in Kosraens to loci with strong support in European studies. Of 45 established associations (Table 4), allele frequencies were compared for 36 SNPs directly typed on the Affymetrix array. For each risk allele, we identified SNPs on the Affymetrix array with frequencies in HapMap CEU within 2% of the risk allele frequency in HapMap CEU. For that set of SNPs, we generated a distribution of allele frequency differences between HapMap CEU and Kosrae. To determine whether a risk allele has an unusual difference in frequency between Kosraen and HapMap CEU, we examined the difference in frequency for the risk allele in the context of the complete distribution of allele frequencies. Over 85% of the SNPs evaluated (n = 32) have statistically indistinguishable frequencies in Kosraens and Europeans (empirical p$0.1) while none of the loci examined had significantly different (empirical p#0.01) frequencies in the two populations. PLoS Genetics | www.plosgenetics.org 10 February 2009 | Volume 5 | Issue 2 | e1000365