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Identifying multiple causative genes at a single GWAS locus.

ABSTRACT: Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the Agtrap-Plod1 locus (Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1) for blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (five out of six) can impact hypertension by modifying BP and renal phenotypes. Mutations of Nppa, Plod1, and Mthfr increased disease susceptibility, whereas Agtrap and Clcn6 mutations decreased hypertension risk. Reanalysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.

SUBMITTER: Flister MJ

PROVIDER: S-EPMC3847770 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Identifying multiple causative genes at a single GWAS locus.

Flister Michael J MJ Tsaih Shirng-Wern SW O'Meara Caitlin C CC Endres Bradley B Hoffman Matthew J MJ Geurts Aron M AM Dwinell Melinda R MR Lazar Jozef J Jacob Howard J HJ Moreno Carol C

Genome research 20130904 12

Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locu ...[more]

PMID: 24006081

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Project description:Background: DNA in the nucleus of a living cell carries out its functions in the context of a complex, three-dimensional chromatin architecture. Several recently developed methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of loci in yeast, fruit fly, human and mouse. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication. Here we describe a novel assay to map genome-wide chromatin contacts, tethered multiple 3C (TM3C), that involves a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by deep DNA paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. Results: We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, as well as a low noise-to-signal ratio between inter- and intrachromosomal contacts. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Ph+ formation in KBM7. Furthermore, we develop a two-phase mapping strategy that separately maps chimeric subsequences within a single read, allowing us to identify contacts involving three or four loci simultaneously, potentially corresponding to combinatorial regulation events. This mapping strategy also greatly increases the number of distinct binary contacts identified and, therefore, the coverage obtained for a fixed number of mapped reads. We confirm a subset of the triplet contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Assaying the genome architecture of a near-haploid cell line allows us to create 3D models of a human cell line without averaging signal from two homologous copies of a chromosome. Our 3D models of KBM7 show clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines. Conclusion: TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and complex chromatin loops that may play a role in regulating reciprocal expression of the IGF2 and H19 genes. Analysis of the spatial organization of two human cell lines (KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line) using tethered multiple 3C (TM3C), a novel and simple protocol for ascertaining genome architecture which can be used to identify simultaneous contacts among three or four loci in addition to binary contacts that can be identified using traditional chromosome conformation capture coupled with next generation sequencing (Hi-C).

Project description:BackgroundSeveral recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.ResultsWe describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.ConclusionTM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

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Identifying multiple causative genes at a single GWAS locus.

Publications

Identifying multiple causative genes at a single GWAS locus.

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