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. 2009 Aug 18;106(33):13886-91.
doi: 10.1073/pnas.0907336106. Epub 2009 Aug 10.

Systematic Haplotype Analysis Resolves a Complex Plasma Plant Sterol Locus on the Micronesian Island of Kosrae

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Free PMC article

Systematic Haplotype Analysis Resolves a Complex Plasma Plant Sterol Locus on the Micronesian Island of Kosrae

Eimear E Kenny et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Pinpointing culprit causal variants along signal peaks of genome-wide association studies (GWAS) is challenging. To overcome confounding effects of multiple independent variants at such a locus and narrow the interval for causal allele capture, we developed an approach that maps local shared haplotypes harboring a putative causal variant. We demonstrate our method in an extreme isolate founder population, the pacific Island of Kosrae. We analyzed plasma plant sterol (PPS) levels, a surrogate measure of cholesterol absorption from the intestine, where previous studies have implicated 2p21 mutations in the ATP binding cassette subfamily G members 5 or 8 (ABCG5 or ABCG8) genes. We have previously reported that 11.1% of the islanders are carriers of a frameshift ABCG8 mutation increasing PPS levels in carriers by 50%. GWAS adjusted for this mutation revealed genomewide significant signals along 11 Mb around it. To fine-map this signal, we detected pairwise identity-by-descent haplotypes using our tool GERMLINE and implemented a clustering algorithm to identify haplotypes shared across multiple samples with their unique shared boundaries. A single 526-kb haplotype mapped strongly to PPS levels, dramatically refining the mapped interval. This haplotype spans the ABCG5/ABCG8 genes, is carried by 1.8% of the islanders, and results in a striking 100% increase of PPS in carriers. Resequencing of ABCG5 in these carriers found a D450H missense mutation along the associated haplotype. These findings exemplify the power of haplotype analysis for mapping mutations in isolated populations and specifically for dissecting effects of multiple variants of the same locus.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Stepwise conditional analysis of PPS levels on the Island of Kosrae. (A) Unconditioned genome-wide association of PPS levels. Association analysis was performed as described in the methods section. The red line marks the threshold of empirical genome-wide significance. (B) Unconditioned association of PPS in an 11-Mb interval on chromosome 2 surrounding the genome-wide peak signal and the ABCG5/ABCG8 genes. Maroon square indicates the ABCG8 nonsense mutation with the best signal. Red shaded area marks the ABCG8 locus. (C) Association of PPS levels in the same chromosome 2 interval after conditioning for the ABCG8 nonsense mutation. Blue square indicates the SNP (rs12185607) with the best signal. (D) Association of PPS levels in the same chromosome 2 interval after conditioning for both the ABCG8 nonsense mutation and rs12185607 genotypes.
Fig. 2.
Fig. 2.
Schema of the analysis pipeline for PPS haplotype discovery, refinement, and validation. BEAGLE phased haplotypes for the chromosome 2 region (36–47 Mb) were excised and analyzed with GERMLINE to detect matching pairwise IBD segments. Overlapping segments that matched across the population were clustered, and then the mean phenotype of cluster members was assessed. One cluster containing a 1.3 Mb haplotype strongly associated to high PPS levels. Careful comparison of haplotypes in that cluster revealed a unique ≈526-kb shared segment carried by 52 individuals. The CRANEFOOT software was used to visualize the ≈526-kb haplotype segregating in three closely related kindreds.
Fig. 3.
Fig. 3.
Location of PPS haplotype and effect on PPS levels. (A) Annotated genes on the background of the 526-kb haplotype. The PPS haplotype (indicated as a gray bar) extends over a 526-kb region on chromosome 2p21 that includes the ABCG8 and ABCG5 candidate genes (indicated by a black dashed square) and also fully or partially five other annotated loci (Ensembl Homo sapiens version 54.36p [NCBI 36] Chromosome 2: 43,000,000; 45,000,000 [54]).(B) Effect of the ABCG8 nonsense mutation and 526-kb haplotype carrier state on PPS, fasting plasma levels of campesterol and sitosterol were determined as described in the Methods section. Values shown are mean ± SEM.

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