By Monica Heger
Whole-genome sequencing in combination with genome-wide association studies could be the key to uncovering rare variants associated with disease risk, according to researchers from Decode Genetics.
In a study published this week in Nature Genetics, researchers from the company sequenced the genomes of 87 Icelanders and used that data in combination with GWAS to identify a rare variant associated with a high risk of developing sick sinus syndrome, a disease characterized by heart rhythm disorders.
The researchers first performed a GWAS of 792 Icelandic individuals with SSS and 37,592 Icelandic population controls. The analysis revealed a significant association between SSS and three correlated SNPs on chromosome 14q11. Then, to search for additional signals, the team sequenced the whole genomes of seven of the cases, four of whom contained the variant mostly strongly associated with SSS, and 80 controls who had been chip-typed.
They sequenced the individuals to 10-fold coverage on the Illumina Genome Analyzer with 101-base paired-end reads and called 11 million SNPs in total. They then imputed the SNPs into chip-typed cases and controls with phased haplotype information, and tested for disease association. The 14q11 region was the only region that had a significant association with SSS, with the strongest association a missense mutation in the MYH6 gene, which encodes for the alpha heavy chain subunit of cardiac myosin. All four of the cases with the variant contained the missense mutation, but it was absent from all the cases without the variant and also a control with the variant.
The team next used Sanger sequencing to resequence the exon with the mutation in MYH6 in 351 Icelanders — 118 cases and 223 controls. Combining this data with the original sequencing data revealed that those individuals with the missense mutation had an odds ratio of also having SSS of 12.53. The team determined that carrying the mutation results in a 50 percent lifetime risk of being diagnosed with SSS, while for non-carriers, the lifetime risk is 6 percent.
Further testing of the missense mutation found that it is also associated with several other cardiovascular diseases including atrial fibrillation and thoracic aortic aneurysm. Additionally, more common variants in the same gene affect heart rate.
"We have discovered a rare variant in a previously unidentified SSS susceptibility gene that confers high risk of the disease. The same variant has a substantial effect on heart rate in subjects who have not been diagnosed with SSS," the authors wrote.
"It's one of the first studies to really show that the combination of high-density genotyping and next-gen sequencing can find functional variants," said Hakon Hakonarson at the Center for Applied Genomics at the Children's Hospital of Philadelphia, who is using GWAS in combination with next-gen sequencing to study childhood diseases such as autism (IS 10/12/2010).
The authors wrote that the study adds further evidence that complex diseases are caused by both common variants and rare variants with large effects, citing another example in which a GWAS of hypertriglyceridemia identified four genes, which researchers then sequenced, the results of which "suggested an interplay between common and rare variants in the genetic architecture of this specific phenotype" (IS 7/27/2010).
However, Hakonarson said one reason the Decode researchers' method was successful is because the mutation arose due to a founder effect in the Icelandic population studied, and it is unclear whether the same variant or gene would be implicated in other populations, or even whether the same method would work for more complex diseases where the mutation did not arise under a founder effect.
"They have not proven that they will now be able to find a bunch of rare variants across a bunch of other diseases," he said. "If this was done in another population that didn’t have this founder effect, but had another mutation in the gene, it may not have been picked up."
Additionally, while cardiac disorders tend to be complex and heterogeneous, in this case, the disease more closely resembles a Mendelian disease because it is so population-specific, Hakonarson said, and it's unclear whether similar results would be found studying more complex diseases.
The combined method nevertheless is stronger than either GWAS or whole-genome sequencing alone, he said. There are still a lot of errors in whole-genome sequencing, which makes it difficult to pick out a rare variant from a sequencing error, and GWAS is good for identifying common variants, but not rare ones. But doing the GWAS first allows researchers to narrow their search for rare variants in the sequencing data to specific regions or genes.
"As a method, it's obviously the way to go. Having the GWAS data, and having the GWAS data imputed is a very significant value and gives you a more focused approach to sequencing," Hakonarson said. His group at CHOP, for instance, has been using a similar method with "very encouraging results" to study autism and ADHD.
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