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Personal Genome Sequencing Identifies Mendelian Mutations

By Kevin Davies

October 22, 2009 | HONOLULU – The only downside of holding a major scientific conference in Hawaii is that, at some point, one has to step inside and attend a talk or two. But those among the more than 4600 registered attendees at the 2009 American Society of Human Genetics (ASHG) convention who ventured indoors were treated to some excellent talks on the opening day. In particular, two groups presented impressive results of next-generation sequencing studies that conclusively show how it is possible to identify previously unknown mutations responsible for Mendelian diseases.

James Lupski (Baylor College of Medicine) is an authority in the area of structural variants underlying genetic disorders (and has been for two decades). In the early 1990s, his team characterized the novel sub-chromosomal duplication that gave rise to a common form of peripheral neuropathy called Charcot-Marie-Tooth (CMT) disease. Since then, mutations in some 40 genes have been shown to give rise to CMT-like diseases. But none of them accounted for one particularly interested patient: Lupski himself.

Earlier this year, Richard Gibbs, director of the Baylor Genome Center, offered to sequence Lupksi’s entire genome in the hopes of finally identifying the mystery mutant gene. (Gibbs and Lupski were part of the team that interpreted the first personal genome delivered by next-gen sequencing, James Watson, in 2007.)  Using the Life Technologies/Applied Biosystems SOLiD platform, Gibbs and colleagues sequenced Lupski’s DNA to 30-fold coverage.

Not surprisingly, the sequencing produced thousands of single-nucleotide polymorphisms (SNPs) considered putative disease-causing mutations. Gibbs applied a series of filters, removing SNPs already catalogued in the database (and thus considered too common to be the basis of a rare genetic disorder) as well as those found in HapMap samples. Lupski detailed how 6 SNPs in his genome were correlated with known behavioral disorders, 32 were cancer associated (Lupski is a cancer survivor), and 47 were implicated in common diseases.

In the end, Lupski and colleagues found different deleterious mutations in his two inherited copies of a gene called SH3TC2. The gene encodes a protein expressed in the membrane of Schwann cells that could have a role in the myelination of nerve fibers.

Miller Time

Recently, University of Washington geneticist Jay Shendure and colleagues published a report in Nature showing that exome sequencing of genes from patients with a known genetic disorder (Freeman-Sheldon Syndrome) could indeed separate the mutation signal from the noise of other variants. Exome sequencing has the advantage of sequencing just 1 percent of the DNA in the whole genome, but the analysis is limited to mutations that affect protein-coding regions.

Next, Shendure and colleagues set out to replicate their success with a Mendelian disorder where the root cause has not been found. The Shendure team used Agilent arrays to enrich the exome sequences and Illumina GA II for sequencing.

The investigators selected Miller Syndrome, a developmental disorder characterized by limb defects, first described by Marvin Miller and colleagues from the same university. Shendure’s team sequenced the exomes from four individuals with the disorder including a pair of siblings. When two filters were applied – removing variants observed in dbSNP and sequenced HapMap samples – the Shendure team was left with putative mutations in just one gene: DHODH (dehydroorotate dehydrogenase). Mutations in the same gene were subsequently found in six other children with the syndrome.

The two affected siblings studied in the original study also had respiratory infections reminiscent of cystic fibrosis. When the researchers applied a computational program to predict deleterious changes, mutations in another gene were implicated: DNAH5. Interestingly, this gene is associated with Kartagener’s syndrome, a disorder that has cystic fibrosis-like characteristics. Shendure’s conclusion is that the siblings in this unfortunate family inherited not one but two recessive Mendelian traits.

Shendure would not be drawn on the cost of exome sequencing per individual, but noted that an exome could be sequenced on two lanes of the current Illumina flow cell, which could soon be reduced to a single lane.

In subsequent discussion, David Valle (Johns Hopkins) pointed out that interpretation of these results was greatly facilitated by the wealth of medical expertise brought to bear on evaluating the biological significance of specific candidate genes. Nevertheless, as genome sequencing costs continue to drop, these studies (and others recently published in the area of cancer) strongly suggest that whole- or even partial genome sequencing can identify causal alleles associated with rare genetic diseases. Doubtless this is only the beginning.


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