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Exome Sequencing May Enhance Dx Yield of Mitochondrial Disease

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This article was originally published July 1.

NEW YORK (GenomeWeb) – Exome sequencing may be a useful tool in identifying the genetic underpinnings of a mitochondrial disorder subtype, researchers reported today in the Journal of the American Medical Association.

Currently, a number of different academic and commercial laboratories offer exome sequencing as a means to diagnose rare disorders, but the practice is not yet commonplace. The diagnostic success rate is around 25 to 30 percent, with some groups reporting a wider range of diagnostic success depending on the specific disorder. In addition, most experts argue that more evidence is needed to show both clinical utility and economic feasibility before insurance will adopt policies covering the technique.

In the JAMA study, the researchers from Newcastle University in the UK demonstrated that at least for mitochondrial disorders characterized by multiple respiratory chain complex defects, exome sequencing can find a diagnosis in the majority of cases.

Mitochondrial disorders are a common cause of genetic disease but can be hard to diagnose at the molecular level. A specific subtype of those disorders, which are characterized by multiple respiratory chain complex defects, can be especially hard due to the large number of genes that may be involved.

Patrick Chinnery, professor of neurogenetics at Newcastle University and a senior author of the study, told Clinical Sequencing News that exome sequencing is a huge improvement over candidate gene sequencing when trying to diagnose this mitochondrial disorder.

As reported in the JAMA paper, exome sequencing found a definitive diagnosis in 28 out of 53 patients analyzed and a likely diagnosis in an additional four patients, for a combined diagnostic rate of about 60 percent. As Chinnery's group previously reported in Brain in 2011, candidate gene sequencing only found a diagnosis in one out of 52 patients. "It's a huge shift from almost no diagnosis, to the majority," he said.

The group evaluated 53 patients that had been referred to two national centers in the UK and Germany, all of whom had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation.

They used Illumina's TruSeq 62-mb capture kit and the HiSeq 2000 for the sequencing and used an in-house developed bioinformatics pipeline to analyze the results. They confirmed putative pathogenic variants by Sanger sequencing.

The team classified variants into four groups: presumptive pathogenic, possible pathogenic, unknown significance, and unresolved.

They found presumptive pathogenic variants — defined as homozygous or compound heterozygous mutations in genes previously shown to cause multiple respiratory chain complex deficiencies — in 28 patients, or 53 percent of the patients. Five patients had a pathogenic mutation in the RMND1 gene and were also from consanguineous families. One additional patient had a different pathogenic RMND1 mutation. Five patients had a mutation in AARS2, four patients had mutations to MTO1, two patients had EARS2 mutations, two patients had MTFMT mutations, and one patient had a C12orf65 mutation. In addition, single patients with a clinical presentation resembling previously described cases carried homozygous or compound heterozygous mutations in YARS2, PUS1, MGME1, ETHE1, ELAC2, and TK2.

The group found possible disease-causing mutations — defined as homozygous or compound heterozygous mutations in novel genes predicted to cause a mitochondrial translation defect — in four different genes in four patients, or 8 percent.

"There were few recurrent genes," Chinnery said. The team identified almost as many disease-causing or potential disease-causing genes as there were patients in the study, underlining the fact that when looking at such a heterogeneous disease a candidate gene approach would have been expensive and time consuming, he added. "Only by applying the exome sequencing approach do you get to the answer quickly," he said.

Chinnery said the next step is to follow up with patients for whom exome sequencing did not identify a genetic cause to the disease. He said there are several reasons for why exome sequencing did not yield a diagnosis. In some patients, he said, exome sequencing turned up a potential causative gene, but the gene was novel and functional studies have not yet been done.

For instance, in 15 patients the team identified mutations in genes predicted to encode mitochondrial proteins, but because those mutations were in single patients and there is not yet functional data that can link those genes to the disorder, they were called as variants of unknown significance.

In six patients, exome sequencing did not point to any candidate pathogenic mutations. For these patients, Chinnery said, it is possible that the exome capture did not cover the coding region harboring the mutation or it could be that the pathogenic variants fall in noncoding regions. He said that these patients will be entered into the rare disease arm of the UK's 100,000 Genomes Project for whole-genome sequencing.

The JAMA study is just one of several recent studies signaling that next-generation sequencing is starting to become incorporated into mainstream medicine. Last month, for instance, a New England Journal of Medicine review provided a how-to for diagnostic whole-genome and exome sequencing.

"These types of studies are absolutely needed," Madhuri Hegde, executive director of Emory Genetics Laboratory, told CSN.

One important aspect the study highlights is that having a well-defined clinical phenotype can help increase the chances of success, Hegde said. She attributed the high diagnostic rate in part to the fact that the researchers were analyzing a very specific subtype of disease. The diagnostic rate is "slightly higher than what clinical labs doing exome sequencing report because this is a selected population. All the patients had biochemical evidence," she said.

Chinnery agreed that having a well-defined clinical phenotype is important to increase the success of exome sequencing and said that there is evidence that exome sequencing can have a diagnostic success rate closer to 40 to 50 percent in well-defined cases.

In addition, he added, while the study shows that, at least for this specific disorder, exome sequencing may yield a dramatic improvement in diagnostic rate, further studies need to be done to demonstrate cost-effectiveness. "My feeling is that this is going to result in a huge cost and time savings," he said. "Many patients with mitochondrial disorders take years, even decades, before they receive a diagnosis," he said.

Hegde added that studies such as this one may also help make the case for reimbursement. "We need more data showing diagnostic success," she said. "But then the next step will be to demonstrate how exome sequencing impacts treatment."

EGL offers diagnostic exome sequencing and Hegde said that already her lab has found that a diagnosis can impact patient management. "In general, physicians are excited about the power of this technology, but are looking for guidance about whether to order an exome or not," she said.

She added that although there are still many hurdles to overcome with regards to interpretation and reimbursement for testing, the more studies that are published demonstrating the utility of the approach, the more exome sequencing will become integrated into clinical practice.

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