Whole genome sequencing, which analyzes a person's complete DNA sequence, has shown to provide enough evidence for doctors to diagnose cases of rare genetic disorders. A cheaper method, called exome sequencing, which sequences the protein-coding regions of the genome, has also been available. But a new study, conducted by researchers at Thomas Jefferson University in Philadelphia, finds that currently available exome sequencing may not always deliver quality results, and may even miss disease-causing mutations.

The results were presented at the annual conference of the European Society of Human Genetics (ESHG) on Sunday. 

Exome sequencing uses exons — segments of DNA or RNA — that contain information needed for coding proteins or peptide sequences. So, even though they represent only a fraction of the genome, they nevertheless carry important information, similar to the trailer of a movie. Sequencing the complete exome allows those who diagnose patients to check for any variations or mutations responsible for potentially life-threatening diseases. But during this testing, incidental findings of 56 specific genes must also be reported, because they can also cause serious, but treatable medical conditions.  

The American College of Medical Genetics and Genomics (ACMG) emphasized the importance of alerting patients to such variations even if the goal of the genetic sequencing was to find other mutations. This recommendation has given rise to several human exome-capturing platforms. But according to the researchers, including Dr. Eric Londin, assistant professor at the Computational Medical Center, Department of Pathology, Anatomy, and Cellular Biology, most of these sequencing sets miss a high proportion of clinically relevent regions of the 56 ACMG genes. 

The study analyzed 44 exome datasets from four different testing kits. "At least one gene in each exome method was missing more than 40 percent of disease-causing genetic variants, and we found that the worst-performing method missed more than 90 percent of such variants in four of the 56 genes," Londin said in a press release. In some cases, genetic variants may have been missed because the clinical exome kit didn't have the ability to identify them. "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," Londin added.

"Depending on the method and the laboratory, a significant fraction (more than 10 percent) of the exome may be untested and this raises concerns as to how results are being communicated to patients and their families," Londin said. High false-negative rates using existing sequencing kits is quite possible, the researchers said. 

A total of 17,774 disease-causing genetic variants are annotated in the Human Gene Mutation Database (HGMD) for the 56 genes mentioned in the ACMG recommendations. The researchers looked at the coverage of these variants in the exome datasets. They found that the trial datasets were comparable in quality to other published clinical and research exome data sets, but with regard to coverage of the variant locations, they performed poorly. The researchers feel that genome sequencing laboratories that conform to the ACMG reporting guidelines, should consider the high rate of false-negative results that can occur when using these kits.

The researchers suggested that the exome sequencing kits be manufactured to focus on genetic mutations that cause known diseases. "If adequate performance cannot be obtained across the exome, then further use of targeted disease-specific panels of genes should be explored," Londin said in the release.

The study also found that exome datasets generated from smaller amounts of sequence data performed far worse than those from larger datasets. This finding is consistent with previous studies showing that exome methods do not have a linear relationship between sequence-generated and nucleotide2 coverage. Instead, a minimum threshold of sequencing data needs to be met before optimum coverage is obtained.

"Current consensus and regulatory guidelines do not prescribe a minimum data requirement for clinical exome tests," 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."

Source: Londin E, et al. At The Annual Conference Of The European Society Of Human Genetics. 2014.