Author Summary Isolated populations have contributed to the discovery of loci with simple Mendelian segregation and large effects on disease risk or trait variation. We hypothesized that the use of isolated populations might also facilitate the discovery of common alleles contributing to complex traits with relatively larger effects. However, the use of association analyses to map common loci influencing trait variation in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the as- sumptions of independence upon which traditional associ- ation test statistics are based. We developed an analytic strategy to perform genome-wide association studies in an inbred family containing over 2,800 individuals from the island of Kosrae, Federated States of Micronesia. No alleles with large effect were observed with strong statistical support in any of the 15 traits examined, suggesting that the contribution of individual common variants to complex trait variation in Kosraens is typically not much greater than that observed in other populations. We show that the effects of many loci previously identified in Caucasian populations are indistinguishable in Caucasians and Kos- raens, despite very different population genetics and environmental influences. We have been studying the native population of Kosrae, Federated States of Micronesia, under the hypothesis that power to detect mutant alleles might be enhanced by reduced allelic heterogeneity, and that different genes (and thus biological insights) might be obtained. Our initial analyses of genotyping data from 30 Kosraen trios and ,110,000 genome-wide SNPs showed that Kosraens exhibit strikingly reduced haplotype diversity and extended LD, likely resulting from a strong founder effect and repeated population bottlenecks [4–6]. These features were much more dramatic than in commonly cited ‘‘founder’’ populations such as Finland and Iceland. Our prior analyses, including resequencing on Kosrae, suggested that fixed marker sets such as the Affymetrix SNP genotyping products would provide better coverage for common variants in Kosraens than in any HapMap population [4]. We also previously observed that native Kosraens exhibit elevated rates of obesity and diabetes, as seen in other indigenous populations [7–10]. It is likely that many common mechanisms underlie the rising prevalence of obesity and metabolic disease in both Caucasian and native populations. However, given the reduced genetic diversity of isolated populations, the high prevalence of metabolic disease raises the possibilities that population-specific disease loci and fewer disease loci of relatively larger effect segregate in Kosraens. The genetic architecture of isolated populations introduces analytic challenges which confound traditional association tests [11]. Inbreeding and the historical lack of random mating in a small population violate assumptions such as Hardy-Weinberg equilibrium which underlie many association test statistics. Members of isolated populations descend from a small number of founders, thus are related, typically in large families. In addition to ‘‘known’’ relationships, cryptic relatedness further confounds the test statistic, as more distant relationships may be unreported or incorrectly specified during patient interview. We ascertained over 3,100 Kosraen adults in three screens spanning a decade and performed genome-wide association studies for 15 quantitative traits in this cohort. To do so, we developed analytic strategies to address the complexities of GWAS on the Island of Kosrae studying a population in which the majority of subjects are related. Our work includes: extensive validation of the extended Kosrae pedigree; identifying an analytic approach to maximize power; calibrating the association score to correct for relatedness in the cohort; and application of this method to the analysis of 15 quantitative traits. Results from the genome-wide association analyses validate our approach by detecting previously known loci for LDL-C, HDL-C, triglycerides and C-reactive protein. Additionally, our data suggest novel loci contributing to phenotypic variation in thyroid stimulating hormone (TSH) and fasting plasma glucose (FPG). While empirical power calculations suggest our study is well-powered to detect common variants of relatively large effect ($5% variance explained) with genome-wide significance, no such effects were observed in our data with convincing statistical support. Results Sample Ascertainment We performed a population-based screen of native Kosraens over three separate visits to the island (Table 1). The 1994 cohort was described previously [7,12]. Self-reported family relationships were recorded for use in constructing pedigrees and blood was collected for DNA extraction and genotyping. A rich phenotypic dataset was collected for a majority of the adult population of the island, including measurements of height, weight, body mass index (BMI), waist circumference, plasma leptin, percent body fat, fasting plasma glucose, blood pressure, plasma lipids (ApoA1, HDL-C, ApoB, LDL-C, total cholesterol, triglycerides), thyroid stimulating hormone (TSH), and plasma C-reactive protein (CRP). Phenotypic data were carefully reviewed for errors in data entry, unit conversion and spurious measurements, and to verify that measurements of related traits are correlated (e.g., r2.0.7 between BMI and waist circumference). Any values that could not be reconciled were excluded from the analysis. Heritability estimates for each trait are typically within published ranges; mean values, distribution, number of phenotyped individuals, and heritability estimates for each trait can be found in Dataset S1. Genotyping A total of 2,906 individuals were successfully genotyped using the Affymetrix 500 k mapping assay (minimum per-chip call rate 95%) (Table S1). SNPs were excluded from the analysis for the following reasons: mapping to multiple genomic locations (n = 3,462); missing .5% data (n = 43,849); or more than 10 Mendelian errors observed (n = 5,887) (Figure S1). Hardy-Wein- berg equilibrium was not used as a quality filter, as it is difficult to assess in our highly related cohort using standard formulae. For the purposes of SNP quality control, allele frequencies were estimated assuming all 2,906 genotyped individuals are unrelated. After excluding monomorphic SNPs (n = 30,581), 408,775 SNPs passed technical quality filters, including 78,862 SNPs of very low frequency in Kosraens (0,MAF,0.01). We next used data from 2,906 individuals genotyped for 400,301 polymorphic, autosomal SNPs to validate the Kosrae pedigree. Refining the Kosrae Pedigree with Genome-Wide Genetic Data Genetic accuracy of the Kosrae pedigree was assessed using pairwise identity-by-descent (IBD) estimates generated in PLINK [13]. For three types of known relationships (parent-child, full sibling, and half-sibling), pairs of genotyped individuals were evaluated to determine whether estimates of the proportion IBD PLoS Genetics | www.plosgenetics.org 2 February 2009 | Volume 5 | Issue 2 | e1000365