By Julia Karow
Sequencing genes implicated in genome-wide association studies of common diseases can uncover additional rare variants that contribute to the risk of the disease independently and can help explain both disease biology and heritability, a team led by the Broad Institute and Massachusetts General Hospital has found.
The results indicate that common SNP associations do not simply point to underlying rare variants with strong effects, but that both common and rare variants in the same genetic loci contribute to common diseases independently. "If these observations become commonplace through available technology, they may help make the debates about common versus rare variation biologically irrelevant," the researchers wrote.
In a pilot project, published online in Nature Genetics this week, the scientists sequenced a subset of genes associated through GWAS with Crohn's disease in several hundred cases and controls and validated rare variants they discovered by genotyping them in a larger cohort. They discovered several new rare risk and protective variants, one of them with potential therapeutic value, that were independent of the GWAS signal. Based on the results, they have now expanded their study to larger numbers of genes and patients and to other types of common diseases.
The targeted approach of sequencing GWAS-implicated genes "will give us returns from sequencing in complex diseases in a way that has proven challenging for truly unbiased exome searches to find new genes because we already know that the genes we are sequencing are involved in these processes," said Mark Daly, chief of the analytic and translational genetics unit at MGH and the senior author of the study.
GWAS have so far identified more than 70 genetic susceptibility loci for Crohn's disease, but the common SNPs identified in those studies usually only have a modest effect and explain only about a quarter of the inherited Crohn's disease risk. Many of them also do not have any known function and implicate regions with several genes.
For their study, the researchers sequenced 56 genes in genomic regions that have been associated with Crohn's disease in 350 disease cases and 350 controls. They PCR-amplified the exons — a total target size of 108 kilobases — and sequenced DNA pools of 50 samples at a time using the Illumina Genome Analyzer. The data was analyzed using a new SNP-calling software for pooled sequencing projects called Suzygy.
The researchers identified 115 non-synonymous, nonsense, or splice-site variants that occurred in at least two copies up to a frequency of 5 percent. Excluding those variants that were already known, they genotyped 70 variants in nine independent case-control cohorts, totaling about 16,000 Crohn's disease cases, 12,100 ulcerative colitis cases, and 18,000 healthy controls, using either Sequenom iPLEX or Illumina GoldenGate technology.
The analysis showed that variants in four genes — CARD9, NOD2, CUL2, and IL18RAP — are highly significant and contribute to risk independently of the previously identified GWAS variants. They also found protective variants in the IL32R gene and "nominally significant" variants in three other genes.
Interestingly, the researchers found no evidence for the hypothesis that the common SNP associations merely point to underlying rare variants with a strong effect. "Basically, the signals had nothing to do with each other," Daly explained. While the common variants are often located in regulatory regions upstream of a gene, the rare variants they found are located in coding regions of the same genes.
"Whether you study common variants with genome-wide association, or whether you are seeing rare variants with direct sequencing of coding regions, each of those approaches will discover some of the genetic variation involved in the disease," he said. "Happily, those studies point at the same genes and pathways because they tell us truly what the biology of the disease is."
One variant in particular — in the CARD9 gene — may have therapeutic implications. The researchers identified a splice-site variant in CARD9 that appears to have a strong protective effect against the disease.
"This gives us a potential model to mimic therapeutically, because if a naturally arising splice variant knocks out a copy of CARD9, and this is strongly protective against the development of disease, this tells us that perhaps knocking down chemically the function of CARD9 may be effective against disease, but has also already been validated as being a relatively safe thing to do," Daly said. "There are no guarantees in drug development, obviously, but it gets us over a few potential pitfalls that we might otherwise have to encounter later on in the development process."
The results increase the fraction of Crohn's disease heritability that can be explained, though only by a small percentage. However, that fraction might grow further as more GWAS loci implicated in Crohn's are sequenced. "When we complete these types of studies in much larger samples, we will be able to, presumably, explain a similar amount [of risk] to what's been explained by the common SNPs in the GWAS," Daly said.
He and his colleagues have already "greatly expanded" their Crohn's study and are now sequencing many more associated genes in "thousands" of patients.
They are also now using the same approach to study other common diseases or phenotypes where GWAS have been successful, including serum lipids, type 2 diabetes, and type 1 diabetes.
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