GWAS on the Island of Kosrae Table 1. Study participants successfully genotyped for the Affymetrix 500 k assay. 1994 2001 2003 Total Multiple exams N total 1,935 1,968 84 - 3,987 N unique % Male/Female Mean age Median age Age Range 903 889 33 1,081 2,906 49.6%/50.4% 40.8%/59.2% 51.5%/48.5% 35.3%/64.7% 41.6%/58.4% 43 28 24 47 40 39 24 22 46 38 20–86 16–80 16–53 17–89 16–89 Screenings took place in 1994, 2001, and 2003. For each screen, the total and unique number of individuals examined is shown, as some participants were examined in multiple screens. For subjects examined more than once, age (years) is reported from the most recent exam. doi:10.1371/journal.pgen.1000365.t001 zero, one or two copies were consistent with the relationship reported by the patients and their families. The pedigree was corrected to reflect the true genetic relationship between pairs of individuals whose IBD estimates were inconsistent with self- reported relationships (Table 2). For example, 2,553 parent-child pairs reported by study participants were validated by genetic data, while 141 parent-child pairs were identified using IBD estimates where the relationship was previously unknown, was misreported, or not reported by study participants. In some cases, individuals were added to the pedigree as ‘‘placeholders.’’ For example, if genetic data indicated that one individual of a reported sibship was actually a maternal half-sibling, an ungenotyped ‘‘placeholder’’ was added to the pedigree as the father of the newly-discovered half-sib. Discrepancies between the genealogical and genetic pedigrees on Kosrae are not unexpected given the inherent inaccuracies of self-reported relationships, and are also consistent with known adoption practices on the island. Changes to the pedigree were made based on data from a related pair in which both individuals were genotyped. However, successive iterations of pedigree validation and correction for fully genotyped, first-degree relatives produced a ‘‘ripple effect,’’ also improving the accuracy of relationships involving individuals not genotyped with the 500 k assay (Table 2), and second-degree and other higher-order relationships across the extended pedigree. After extensive comparisons with genetic data, the extended Kosrae pedigree spans eight generations and includes over 4,300 individuals (living or deceased), averaging four individuals per sibship (range 1–12). Nearly all (n = 2,900) of the subjects successfully genotyped with the Affymetrix 500 k assay can be joined in a single extended pedigree, with an additional six individuals forming three independent nuclear families. We count 58 consanguineous offspring as well as numerous marriage loops. Nearly 30% of all genotyped individuals have two genotyped parents. Fifty-six individuals appear distantly related or unrelated to any other study participants. Development of a Strategy for Association Analyses Our goal was to develop an analytic framework that accommodates the complex familial relationships in the Kosraen cohort while maximizing power to detect association. We were unable to identify or develop software capable of simultaneously computing over a complex pedigree of 2,900 individuals and .330,000 SNPs. Thus, our strategy became to break the pedigree into smaller units; a similar approach was recently taken by Przeworski and colleagues in their study of recombination in the Hutterites [14]. Below we also describe the data simulation framework used to perform controlled comparisons between analytic approaches, leading to the selection of an association test. We use empirical power calculations to determine an effective sample size for our highly related cohort and estimate the power of our study across a range of effect sizes. We applied our method for association analyses in a related cohort to the study of 15 quantitative traits in native Kosraens. Table 2. Evaluation and refinement of the extended Kosrae pedigree using identity-by-descent estimates. Relationship type Confirmed Conflicting Newly discovered Two individuals genotyped One individual genotyped No individuals genotyped Parent-Child Full sibling Half sibling Parent-Child Full sibling Half sibling Parent-Child Full sibling Half sibling 2,553 4,147 351 3,266 2,384 162 1,415 941 51 2 72 72 229 126 28 43 34 0 141 110 271 109 33 104 28 21 18 For each type of relationship, the number of related pairs is shown where the reported relationship and identity-by-descent estimates from genetic data were in agreement (‘‘Confirmed’’), conflicting, or added based on genetic data (‘‘Newly discovered’’). Estimates for genome-wide IBD sharing and sharing 0, 1, or 2 copies IBD were used to distinguish between the three relationship types. Individuals were added to the pedigree as necessary to represent genetic relationships, such as the addition of a ‘‘placeholder’’ father to reflect a newly-discovered maternal half-sib relationship. Corrections to the pedigree were made based on data from related pairs with two genotyped individuals, but impacted relationships throughout the extended pedigree. doi:10.1371/journal.pgen.1000365.t002 PLoS Genetics | www.plosgenetics.org 3 February 2009 | Volume 5 | Issue 2 | e1000365