Understanding Mendelian Diseases
Exome sequencing has rapidly become one of the main tools for studying the genetic causes of Mendelian disease because academic groups with access to only one or two next-generation sequencing (NGS) systems can use this approach to study the exomes of hundreds of patients with Mendelian diseases per year. Since November 2009, exome sequencing has led to the identification of over 30 new genes in Mendelian diseases.
Exome sequencing involves an initial enrichment of the targeted DNA regions by hybridization with probes followed by NGS. Data is analyzed to pick out the functional variation and to identify novel mutations associated with rare and common disorders.
In an article published August 2009 in Nature Genetics, a team of scientists headed by Jay Shendure, M.D., Ph.D., assistant professor of genome sciences at the University of Washington, demonstrated that targeted capture and massively parallel sequencing could be a cost-effective, reproducible, and robust strategy to identify variants causing protein-coding changes in individual human genomes.
Using this approach they determined 307 megabases across the exomes of 12 individuals. Freeman-Sheldon syndrome, a rare, inherited disorder, was used as a proof-of-concept to show that candidate genes for monogenic disorders can be identified by exome sequencing of a small number of unrelated, affected individuals.
Although the underlying genetic defect behind the disease was already known, the technique zeroed in on the exact gene responsible for the disease, demonstrating that it was feasible to sort out the genetic signal from more than 300 million bases of DNA.
Using the same strategy, Michael Bamshad, M.D., a professor in the department of pediatrics and adjunct professor of genome sciences at the University of Washington, published a paper in November 2009 reporting on the gene underlying the uncharacterized Mendelian disorder Miller syndrome.
For four affected individuals in three independent kindreds, Sarah Ng, the paper’s first author, and her colleagues captured and sequenced coding regions to a mean coverage of 40x and sufficient depth to call variants at about 97% of each targeted exome.
Filtering against public SNP databases and eight HapMap exomes for genes with two previously unknown variants in each of the four individuals identified a single candidate gene, DHODH. This gene encodes an enzyme required in the pyrimidine de novo biosynthesis needed for DNA and RNA synthesis.
Sanger sequencing confirmed the presence of DHODH mutations in three additional families with Miller syndrome. No similar mutations were found in 100 unaffected individuals.
The authors said they had demonstrated that exome sequencing of a small number of affected family members or affected unrelated individuals provides a powerful, efficient, and cost-effective strategy for markedly reducing the pool of candidate genes for rare monogenic disorders and may even identify the responsible gene(s) specifically.
The approach, they noted, is likely to become a standard tool for the discovery of genes underlying rare monogenic diseases and to provide important guidance for developing an analytical framework for finding rare variants influencing risk of common disease.