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May 31, 2014

Exome Sequencing May Miss Pathogenetic Variants

Exome Sequencing May Miss Pathogenetic Variants

Source: © Gernot Krautberger - Fotolia.com

  • While a patient may unknowingly skirt unseen dangers, like a latter-day Magoo blindly wandering past open manholes, physicians would rather patients be aware of their health risks, or at least have some notion of the degree to which confidence in their wellbeing is justified. A physician, for example, might hesitate before informing a patient of incidental genetic findings from an exome sequencing report.

    Such reports, a new study maintains, often fail to produce high-quality results. In particular, currently available exome sequencing kits tend to miss variants in specific genes, including the 56 genes considered clinically relevant by the American College of Medical Genetics and Genomics (ACMG). According to the ACMG, incidental pathogenic findings among these 56 genes should be reported.

    The ACMG recommendation, reasoned researchers based at Thomas Jefferson University, assumes that exome sequencing returns data of sufficient quality. Data of poor quality, however, might instill false confidence, much like Magoo’s gauzy outlook. While the fictional Magoo invariably escapes the consequences of his blurry vision, or his obstinate refusal to see that he has a problem, real-life patients might suffer harm if medical professionals were to miss chances to identify (and correct) shortcomings in genomic testing.

    To address this issue, the researchers surveyed the potential false-negative rate of mutations in the 56 ACMG genes. They retrospectively analyzed 44 exome datasets from four different exome capture kits and two-sequence platforms. In addition, the researchers examined the exome methods for their ability to detect clinically relevant mutations in the 56 ACMG genes.

    A total of 17,774 pathogenic nucleotide variants are annotated in the Human Gene Mutation Database (HGMD) for the 56 genes, and data were examined for depth of coverage in the exome datasets.

    The researchers presented their findings June 1 at the annual conference of the European Society of Human Genetics, in a paper entitled “Clinical exome sequence performance for reporting secondary genetic findings.” The paper was read by Eric Londin, Ph.D., assistant professor in the Computational Medicine Center, Department of Pathology, Anatomy, and Cellular Biology, Thomas Jefferson University.

    Overall, the four-exome methods had inadequate depth of coverage for accurate base calling ranging from 5.2 to 34.8% of the pathogenic variant positions. “At least one gene in each exome method was missing more than 40% of disease-causing genetic variants,” said Dr. Londin. “And we found that the worst-performing method missed more than 90% of such variants in 4 of the 56 genes.”

    A central question, the researchers asserted, is not how often a clinical diagnosis can be made using exome sequencing, but how often it is missed, and the study shows clearly that there is a high false-negative rate using existing sequencing kits.

    “Our concern is that when a clinical exome analysis does not report a disease-causing genetic variant, it may be rather that the location of that variant has not been analyzed rather than the patient’s DNA being free of a disease-causing variant,” continued Dr. Londin. “Depending on the method and the laboratory, a significant fraction (more than 10%) of the exome may be untested.”

    One potential improvement would be the development of new kits and methods that provide adequate and reliable coverage of genes with known disease associations. “If adequate performance cannot be obtained across the exome,” commented Dr. Londin, “then further use of targeted disease-specific panels of genes should be explored.”

    Another potential improvement would be the generation of sufficiently large amounts of sequence data to achieve optimum nucleotide coverage. “Current consensus and regulatory guidelines do not prescribe a minimum data requirement for clinical exome tests. The result is that when a causative variant cannot be identified, it does not necessarily imply that the variant is not present,” emphasized Dr. Londin. “In other words, a clinical ‘whole exome’ study may not be ‘wholesome’ in coverage.”

    While the study includes suggestions about ways to improve clinical exome sequencing, it has more immediate import. Specifically, it raises concerns about rates of false-positive results. As Dr. Londin concluded, “Patients and their families should be made aware of this problem and of the implications of the genomic findings of clinical exome sequencing in its current state.”


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