Blood samples

Credit: Jeremy L. Grisham

MILAN, ITALY—New research suggests exome sequencing does not always produce high-quality results with regard to subsets of genes.

The American College of Medical Genetics and Genomics (ACMG) recommends physicians inform patients of clinically actionable genetic findings in the course of clinical exome testing.

Specifically, mutations in 56 specific genes with known clinical importance should be reported, even when they are incidental to the patient’s medical condition.

However, an analysis of 44 exome datasets from 4 different testing kits showed that they missed a high proportion of clinically relevant regions in the 56 ACMG genes.

“At least 1 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,” said Eric Londin, PhD, of Thomas Jefferson University in Philadelphia, Pennsylvania.

He and his colleagues presented these findings at the European Society of Human Genetics Conference 2014 (abstract C07.6).

A central question, according to the researchers, is not how often a clinical diagnosis can be made using exome sequencing, but how often it is missed. And this study suggests 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 that the location of that variant has not been analyzed, rather than the patient’s DNA being free of a disease-causing variant,” Dr Londin said.

“Depending on the method and the 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.”

A total of 17,774 disease-causing genetic variants are annotated in the Human Gene Mutation Database for the 56 genes mentioned in the ACMG recommendations. The researchers examined the coverage of the exome datasets for the locations where the 17,774 disease-causing variants can occur.

Although the exome datasets are comparable in quality to other published clinical and research exome data sets, the coverage of the disease-causing locations was very heterogeneous and often poor.

The researchers believe that clinical labs that implement the ACMG reporting guidelines should recognize the substantial possibility of reporting false negative results.

The team said one potential improvement would be to have clinical exome sequencing use methods designed to provide a maximum yield of all clinically relevant genes.

“Many of the currently used exome kits are designed to provide a very broad dataset including genomic features that do not yet have a well-established clinical association,” Dr Londin said.

“There is a need to develop new kits and methods which provide adequate and reliable coverage of genes with known disease associations. If adequate performance cannot be obtained across the exome, then further use of targeted disease-specific panels of genes should be explored.”

The researchers also found that exome datasets generated from low amounts of sequence data (fewer than 6 gigabases) performed much worse than datasets that were generated from higher amounts of sequence data (more than 10 gigabases).

This finding is consistent with previous studies showing that exome methods do not have a linear relationship between sequence-generated and nucleotide coverage. Instead, a minimum threshold of sequencing data needs to be met before optimum nucleotide coverage is obtained.

“Current consensus and regulatory guidelines do not prescribe a minimum data requirement for clinical exome tests,” Dr Londin said. “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. 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.”

Exome sequencing not always accurate, presenter says