Whole exome sequencing is a research tool that may one day contribute to personalized medicine.
NEW ORLEANS—Whole exome sequencing of families with intracranial aneurysm may allow researchers to identify single nucleotide variants (SNVs) that influence the formation and rupture of aneurysms, according to a preliminary report presented at the 2012 International Stroke Conference.
Joseph P. Broderick, MD, Albert Barnes Voorheis Chair of Neurology at the University of Cincinnati, and colleagues performed whole exome sequencing on seven families with high numbers of members with intracranial aneurysm. “Probably about 10% to 15% of people who have intracranial aneurysm report a first-degree relative who has aneurysm,” said Dr. Broderick.
Whole exome sequencing is an innovative technology that allows researchers to find rare variants with medium to large effects and works by sequencing the portions of the genome that code protein, instead of sequencing the entire genome. Investigators look for “variants in the code that could lead to a protein that … does not work the way it should and that could predispose to development of an aneurysm,” Dr. Broderick told Neurology Reviews. He added that whole exome sequencing is best used to study families with a high concentration of a particular disorder.
Finding Final Variant Candidates
Genetic samples from 32 members of seven families affected by intracranial aneurysm were sent to the Center for Inherited Disease Research at John Hopkins University for sequencing, and initial results showed 142,104 variants that differed from the standard, reference variants.
To identify final variant candidates from the large set of data, the researchers applied quality filters. “Any technology has the potential for artifacts, or errors … you can imagine that the amount of data that’s generated is enormous,” said Dr. Broderick. The quality filters excluded SNVs based on frequency of missing loci, excess heterozygosity or homozygosity, and strand bias. Of the initial 142,104 SNVs, 93,625 remained after application of quality filters.
Biologic filters further narrowed the pool of rare variants. These filters allowed researchers to retain “only the variants that met our biologic hypothesis—[that] rare exonic and amino acid-altering alleles that segregate in a Mendelian fashion may contribute to intracranial aneurysm pathology,” said Dr. Broderick. Thus, synonymous or nonexonic variants were excluded by the filters, as well as variants with an allele frequency greater than 3%. At this point, 31,129 SNVs remained.
The investigators used inheritance filters to analyze the remaining 31,129 SNVs. If at least three affected family members shared the same variant (autosomal dominant; heterozygote) or two variants in the same gene (autosomal recessive; homozygote or compound heterozygote), the researchers retained the variants. The inheritance filters left 871 SNVs, which were then prioritized based on gene ontology pathway of interest, after which 31 SNVs remained.
However, because the research focused on rare variants, more common SNVs could potentially have been overlooked. Dr. Broderick compared the process of re-examining results to panning for gold. “When you’re panning the stream, you may find flecks of gold that you didn’t look at the first time, but they’re just a lot harder to find.”
Gene Ontology Pathways
Rather than one gene causing all aneurysms, a certain group of genes may relate to why a family has aneurysms, said Dr. Broderick. He noted that since collagen is important to the arterial wall, the researchers hypothesized that it was likely to be involved with aneurysms.
A close analysis of one of the affected families revealed two collagen genes—Collagen 5A12 and Collagen 17A1—that met all the filters and segregated with affected status. The families “have interesting expression patterns of these genes in aneurysmal tissue,” said Dr. Broderick, although more research is necessary to determine the specific variants at work.
The investigators plan to genotype the collagen genes of all available family members and to contrast affected with unaffected family members. “We’re just looking at that data now,” said Dr. Broderick.
In addition to collagen, Dr. Broderick and his colleagues may explore other gene ontology pathways that could contribute to aneurysm, such as those relating to inflammation, blood pressure, and vessel development.
A Complex Picture
Determining who will develop an intracranial aneurysm is a complex task, said Dr. Broderick, as aneurysms result from interactions between genetic variants and environmental factors. “It may very well be that these [genetic] variants don’t have their impact unless they’re occurring in the setting of smoking,” he said, noting that the researchers will conduct secondary analyses focusing on smoking, hypertension, and other risk factors.
“Smoking is by far the most important [risk factor],” said Dr. Broderick. “Seventy to eighty percent of people with an aneurysm have been a smoker at some point in their lives.” He stressed that people could benefit from quitting smoking right now, even though the genetic research is not yet complete.