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The four exome test kits examined as part of this study failed to deliver quality results, particularly because they often missed some disease-causing mutations altogether

Human exome sequencing is gaining favor among medical laboratories wanting to use this information for clinical purposes. However, the accuracy of some exome-capture test kits available on the market today has come under question.

A team from the Thomas Jefferson University in Philadelphia surveyed the potential false-negative rate of mutations in 56 disease-causing genes produced by four different commercially available human exome-capture test kits. The researchers found that these test kits failed to deliver quality results, sometimes missing mutations altogether, noted a report published by Medical Daily.

Sequencing Exomes Favored Diagnostic Tool for Its Economy 

This study is of particular interest to pathologists and other diagnostic clinicians because exome sequencing, which analyzes sequences of the protein-coding regions of the human genome (that make up about 1% of the genome’s 3 billion base pairs), is commonly used for diagnosing genetic disorders. Exome sequencing has gained favor because it is significantly less expensive and faster than sequencing a person’s complete genome.

Most lab scientists know that exome sequencing uses exons—segments of DNA and RNA—which contain information needed for coding proteins or peptide sequences. Sequencing the complete exome allows clinicians to check for variations or mutations responsible for potentially life-threatening diseases. Such testing should also report incidental findings for 56 specific genes because they can cause serious, but treatable medical conditions, noted the Medical Daily story.

56 Genes Associated With Nearly 18,000 Disease-causing Variants

A total of 17,774 pathogenic nucleotide variants are annotated in the Human Gene Mutation Database (HGMD) for these 56 disease-causing genes, noted a report published by Genetic Engineering News (GEN).

This is one of the reasons why the American College of Medical Genetics and Genomics (ACMG) has recommended that all patients undergoing genetic sequencing be informed of any variations found in these 56 genes, whether or not the test goal was to find other mutations.

Some Exome Tests Fail to Fully Cover Variant Regions

Because of the ACMG recommendation, several companies were inspired to create commercially available human exome-capturing platforms, according to the researchers at Thomas Jefferson University. They concluded that most of these sequencing datasets miss a high proportion of clinically relevant regions of the 56 ACMG, disease-causing genes.

The study analyzed 44 exome datasets from four different exome capture kits and two-sequence platforms. The researchers also examined the exome methods for their ability to detect clinically relevant mutations in the 56 ACMG genes.


Researcher Eric Londin, Ph.D. (pictured), is an Assistant Professor in Thomas Jefferson University’s Computational Medial Center. He recently presented the results a study at the 2014 conference of the European Society of Human Genetics that found four commonly used commercial exome-capture test kits may miss disease-causing mutations because they fail to fully cover variant regions of 56 disease-causing genes. (Photo copyright Thomas Jefferson University)

“At least one gene in each exome method was missing more than 40% of disease-causing genetic variants, and we found that the worst-performing method missed more than 90% of such variants in four of the 56 genes,” stated Eric Londin, Ph.D., Assistant Professor at Thomas Jefferson University Computational Medical Center, Department of Pathology. He was speaking about the study results at the 2014 conference of the European Society of Human Genetics (ESHG) that took place in Milan, Italy, on May 31 through June 3.

Study Raises Concerns About Exome Technology Performance

“Our concern is that when a clinical exome analysis does not report a disease-causing genetic variant, it may be that the location of that variant has not been analyzed rather than the patient’s DNA being free of a disease-causing variant,” he stressed in his ESHG presentation. “Depending on the [exome test] method and laboratory, a significant fraction [more than 10%] of the exome may be untested, and this raises concerns as to how results are being communicated to patients and their families,” Londin added. Therefore, high false-negative rates using existing sequencing kits are quite possible, he pointed out.

After they assessed the coverage of disease-causing variants in datasets associated with each commercially available human exome-capture test kits, the researchers found the quality of trial datasets comparable to other published clinical and research exome datasets. However, the researchers determined that they performed poorly in regard to coverage of variant locations.

Improving the Diagnostic Potential of Exome Sequencing

The researchers suggested that new exome testing kits be developed to reliably cover genetic mutations that cause known diseases. “If adequate performance cannot be obtained across the exome,” commented Londin, “then further use of targeted disease-specific panels should be explored.”

The study also found that exome datasets generated from smaller amounts of sequence data performed far worse than those from larger datasets. Therefore, another possible improvement would be to generate sufficiently large amounts of sequence data to achieve optimum nucleotide coverage, suggested the GEN report.

“Current consensus and regulatory guidelines do not prescribe a minimum data requirement for clinical exome tests,” observed Londin in his presentation. “The result is that, when a causative variant cannot be identified, it does not necessarily imply that the variant is not present, rather that there may be a technical issue with the exome technology used. In other words, a clinical ‘whole exome’ study may not be ‘wholesome’ in coverage,” he explained.

For pathologists, clinical chemists, and other medical laboratory scientists performing molecular and genetic tests, the findings of the study conducted at Thomas Jefferson University are a reminder that the accuracy and reliability of many gene sequencing methods need to be understood and considered when doing testing for clinical purposes.

—By Patricia Kirk

Related Information:

Exome Sequencing Produces False-Negatives; Misses Many Disease-Causing Mutations

Exome Sequencing May Miss Pathogenetic Variants

A comparative analysis of exome capture