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Sequencing Genes Implicated in GWAS Uncovers Rare Variants in Disease Cohort

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By Monica Heger

Demonstrating the power of targeted resequencing to follow up on the results of a genome-wide association study, researchers at the University of Western Ontario identified 154 rare variants in four genes in 438 individuals with hypertriglyceridemia — a condition characterized by high blood levels of fatty acid — compared to only 53 such variants in 327 controls.

The researchers, who used Sanger sequencing, now plan to sequence more candidate genes that were indicated by the GWAS and eventually plan to move into next-generation sequencing, although they have not yet chosen a sequencing platform.

According to the authors, combining sequencing with GWAS could be a useful way to identify rare variants associated with disease.

"Sequencing is a logical next step to a GWAS study," Christopher O'Donnell, a co-author of the study and the associate director of the National Institutes of Health's Framingham heart study, told In Sequence. "GWAS is useful for localizing signals to a small region of the genome, but not in identifying the causal variations. Sequencing is needed to find and define the specific variations that may be underlying the causes that led to the signal."

In the study, published this week in Nature Genetics, the researchers first conducted a GWAS on 463 individuals with extreme phenotypes of hypertriglyceridemia and 1,197 controls. They then sequenced the coding regions of the top four genes identified by the GWAS in 438 of the affected individuals and 327 controls.

Despite selecting individuals with extreme cases of HTG, the researchers found that the genes and SNPs identified by the GWAS were the same ones identified in studies of previous population-based GWAS. But when they resequenced four of the genes, they found that the HTG individuals had a higher concentration of rare variants.

"This is one of the first reports to define a spectrum of both low-frequency and common variants that underlie a disease-related trait," said O'Donnell.

How exactly the rare and common variants affect the disease is still unclear, but the results show that the combination of GWAS and sequencing could be a good technique for studying disease. "If we hadn't done the sequencing, we wouldn't have found the rare variants," Robert Hegele, senior author of the paper and director of the Blackburn Cardiovascular Genetics Lab at the Robarts Research Institute at the University of Western Ontario, told In Sequence.

Also, in the individuals with HTG, "there was a heavier burden of rare coding sequence variants, including frameshift and nonsense mutations — things that looked functional — compared to the controls," he added.

The researchers found that the individuals affected by HTG had a rare variant carrier frequency of 28.1 percent, compared to 15.3 percent in controls. When they limited the variants to only those that had known functional consequences, affected individuals had a carrier frequency of 10.3 percent, while controls had a carrier frequency of 2.8 percent.

"The burden of rare variants found in individuals with HTG is highly suggestive of phenotype causation," the authors wrote. "Any given rare variant in affected individuals is not necessarily sufficient to cause HTG, but rare variants probably contribute to the biochemical heterogeneity observed among affected people," they noted.

The authors performed a statistical analysis to determine that the combination of clinical variables with both common and rare genetic variants explained nearly 42 percent of total variation in HTG diagnosis: clinical variables explained around 20 percent; common variants in seven HTG-associated loci explained around 21 percent, and rare genetic variants in four HTG-associated loci explained just over 1 percent.

These findings suggest that the rare variants found in the four genes implicated by the GWAS "incrementally contribute to the unexplained genetic variation contributing to HTG pathophysiology," they wrote. Hegele added that sequencing more of the genes indicated by the GWAS could further increase the contribution of rare variants to the unexplained genetic variation. He said that the team's next step is to sequence additional genes using Sanger sequencing, and to move into next-generation sequencing for exome and whole-genome sequencing within the next year.

Hegele said they are considering both outsourcing the sequencing to Complete Genomics and also purchasing their own machine, likely from Illumina. He said they are leaning towards acquiring their own sequencer because their genomics facility serves other scientists in the region, but it will depend on funding. In the meantime, he said they have already sent some patient samples to the Broad Institute for sequencing, but will perform the subsequent analyses in house.

Meanwhile, other groups are also combining GWAS with targeted sequencing to study disease. O'Donnell, a cofounder of the Cohorts for Heart and Aging Research in Genome Epidemiology, or CHARGE, consortium, said that group is using targeted sequencing to follow up on several dozen GWAS signals for a range of cardiovascular and blood traits. The sequencing is being led by Eric Boerwinkle at the University of Texas, who received a $12.3 million grant from the National Heart, Lung, and Blood Institute for the project last year as part of the federal stimulus package (IS 10/20/2009).

"We're taking this kind of concept and extending it to not just triglycerides but a whole range of quantitative traits that are implicated in cardiovascular, blood, and lung disease," said O'Donnell. "This paper provides reasonable evidence that the approach may be useful in uncovering a range of potentially damaging variants," he added.

However, O'Donnell said that one concern is whether the approach can be generalized to study such a wide range of traits. In the Nature Genetics paper, the authors studied a very specific and extreme phenotype, he said. But, it remains to be seen whether studying several different traits will yield such strong signals.

David Craig at the Translational Genomics Research Institute said his group has begun doing targeted sequencing studies based on the results of GWAS on diseases such as autism and Alzheimer's, but the projects are in their very early stages.

"Given the proliferation of next-gen sequencing and that there are now some decent targeted approaches for amplifying regions, it should make [targeted resequencing] the obvious next step of GWAS," Craig said.

The Wellcome Trust Case Control Consortium is also following a GWAS with targeted resequencing (IS 7/8/2008). Inês Barroso, the acting head of human genetics at the Sanger Institute, said "it's a nice balance of looking at common variants and following up on those loci with sequencing."

She agreed with Craig that the method would likely become more common. The study is "a nice prelude to what we might be seeing in the next few years with next-gen studies."

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