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Broad Institute and UW Researchers Demonstrate Exome Sequencing to Study Simple Subsets of Complex Disease

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By Monica Heger

As exome sequencing continues to prove itself as a useful method for identifying disease genes in monogenic disorders, preliminary results suggest that the technique can also help understand the causes of more complex diseases.

In a study published last week in the New England Journal of Medicine, researchers from the Broad Institute and Massachusetts General Hospital used exome sequencing to find the disease gene in familial hypolipidemia, a disorder characterized by very low levels of LDL cholesterol. Meanwhile, at the Beyond the Genome conference in Boston last week, Jay Shendure, an assistant professor of genome sciences at the University of Washington, presented preliminary results of an exome sequencing study of sporadic autism. That project is a collaboration between Shendure's lab and Evan Eichler's group, also from the University of Washington. The two studies have implications for better understanding heart disease and autism, both complex diseases.

In the NEJM study, researchers used Agilent's SureSelect in-solution capture method and the Illumina Genome Analyzer to sequence the exome of two siblings with extremely low levels of LDL cholesterol. The siblings had been identified out of a family of 12, where four out of ten siblings had unusually low levels of LDL. The researchers chose the two siblings with the most extreme phenotypes and sequenced their exomes to around 200-fold coverage. They also sequenced 60 control exomes of unrelated individuals with the similar ancestry using the same method.

Sequencing identified more than 18,000 variants in each individual, but after filtering out the variants in public databases and those found in the 60 control exomes, the researchers narrowed this number to just under 300. They then looked for variants that were present in both siblings, and found just one mutated gene —ANGPTL3 — where both siblings had two nonsense mutations.

The team then sequenced the ANGPTL3 gene with Sanger sequencing in 38 family members spanning three generations and found that the two other siblings with hypolipidemia also had both nonsense mutations.

The researchers also identified 13 additional family members who had only one of the mutations — indicating that they segregate independently. Those 13 members had lower levels of LDL than the 21 family members without either mutation, but not as low as the four members with both mutations. They also had normal levels of HDL cholesterol and triglycerides, while the four affected family members had lower than normal levels of HDL and also triglycerides.

"We've identified a new gene that regulates LDL cholesterol and the effect is very strong," said Sekar Kathiresan, senior author of the study. Identifying natural causes of low levels of cholesterol is important because heart attacks are the leading cause of death in the US, and lowering cholesterol is the best way to lower the risk of a heart attack, Kathiresan said. The ANGPTL3 protein is expressed in the liver and circulates in the blood, which makes it "an extremely attractive drug target," he added.

In mouse models, the ANGPTL3 gene had been shown to regulate triglycerides, but this is the first time it has been linked to cholesterol regulation in humans. "What was entirely new and surprising was the strong role this gene has in LDL cholesterol regulation," Kathiresan said.

He added that it's an interesting lesson because it shows how, for some diseases, the mouse model does not transfer to humans. As genomic techniques become more common, it may allow researchers to rely less on animal models for studying disease.

The study was done as part of the National Human Genome Research Institute's Medical Sequencing project. Kathiresan said the team is continuing to use exome sequencing to study extreme phenotypes of cholesterol and triglyceride levels, which also appear to be monogenic. They are using the same sequencing strategy, except now the sequencing is done on Illumina HiSeq 2000 instruments at the Broad Institute.

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Kathiresan's team is also involved in the National Heart, Lung, and Blood Institute's Exome Sequencing project, for which they are studying more complex cardiovascular diseases such as early onset heart attack. They are analyzing about 1,000 samples through collaborations with institutions across the country.

While exome sequencing has been used to find the disease genes in rare, monogenic diseases such as Joubert syndrome (IS 2/9/2010), Miller syndrome (IS 9/29/2009), and Schinzel-Giedion syndrome (IS 6/1/2010), it hasn't been clear whether the approach would work for more complex diseases. Kathiresan said that exome sequencing should still be useful, but would require many more samples to find significant results. "For complex diseases, it will require probably on the order of thousands of exomes to be able to see a genetic signal that overcomes the noise," he said.

Sporadic Autism

A team at the University of Washington, including researchers from both Jay Shendure and Evan Eichler's labs, is also using exome sequencing to study complex diseases. Shendure presented preliminary results from their project studying sporadic sporadic autism — autism thought to be caused by de novo mutations, as opposed to inherited — last week.

Shendure and his team sequenced 60 exomes from 20 trios — a mother, father, and affected child. The group used NimbleGen's in-solution capture and sequenced the exomes on the Illumina GA. Their general strategy was to generate 76-base paired-end reads and to sequence to around 72-fold coverage.

Shendure said the team chose to look at sporadic autism rather than inherited autism because autism is such a complex disease and so little is known about the genetics of it. The researchers determined that looking at sporadic autism might enable them to find the "needle in the haystack," since sporadic cases would be more likely to be caused by de novo mutations. "We elected to pursue a model that might allow us to winnow down, to identify disease genes with a reasonable efficiency."

And indeed, he said that of the thousands of single nucleotide variants identified in exome sequencing, eliminating any that were also found in the parents removed the vast majority of candidate mutations. Further filtering against public databases then brought the number down to just a handful of mutations that the team verified with Sanger sequencing.

The method — sequencing the exomes of an affected child and both parents — appears to be a good model for studying sporadic disease, Shendure said. "Trio exome sequencing works for identifying de novo coding mutations." The technique is "useful in the context of genetically heterogeneous disorders where large-effect alleles are expected," he said.

While the team has identified a number of promising genes, Shendure declined to provide details because the results are still very preliminary and have not yet been published.

However, studies like those of the University of Washington group and the Broad Institute group are starting to demonstrate exome sequencing's utility in more than just rare, Mendelian diseases.

"Things are moving beyond Mendelian disease in the classical sense of the word, but the success of the studies is still predicated on the relative simplicity of the disease families in which they are studied," Shendure said.

For example, while autism is a very complex disease, sporadic autism is a much simpler subset. Similarly, the NEJM study looked at a rare, extreme phenotype that nonetheless could have implications for other more common, genetically complex cardiovascular diseases.

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