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Ambry Researchers Report Consequences of RNA-Based Variant Reclassification at ACMG

SEATTLE (GenomeWeb) – At the American College of Medical Genetics and Genomics annual meeting here yesterday, Ambry Genetics researchers reported on the effects of re-analyzing hereditary cancer variants with the help of supplemental RNA genetic testing data — including the consequences for patient and/or family members' medical management in some cases.

During a company presentation at the conference, Rachid Karam, director of the Ambry Translational Genomics lab, noted that RNA-based testing has shown promise for not only classifying variants of uncertain significance (VUS), but also for upgrading or downgrading variant classifications in ways that directly affect patient care.

Based on the variant classification improvements Ambry investigators are seeing in the research context, the company is gearing up to offer RNA genetic testing in the healthcare setting, according to Ambry representative Brigette Tippin Davis, senior vice president of research and development.

In an email message, Davis noted that researchers at Ambry's Translational Genomics lab have been doing RNA genetic testing on a research basis for more than two years and "have seen success in improving our variant classification."

"We have completed the clinical validation," she added. "We look forward to making this available to healthcare providers and their patients this year."

For the hereditary cancer gene variant study that Karam reported, Ambry investigators collaborated with researchers from the Dana Farber Cancer Institute, Cedars-Sinai Medical Center, and elsewhere to look at the "feasibility and impact" of variant reclassification with RNA genetic testing data — work that was also highlighted in a poster at this year's ACMG conference.

The first arm of that analysis focused on 64 suspected splice site variants — previously found with DNA testing — in 13 genes linked to hereditary breast and ovarian cancer (HBOC), Lynch syndrome, or hereditary diffuse gastric cancer (HDGC). Based on the DNA evidence alone, some 90 percent of the splice variants in HBOC genes were considered VUS, as were three-quarters of the HDGC gene variants and all of the variants in Lynch syndrome genes.

However, those classifications changed considerably with the inclusion of RNA genetic testing data. Just 10 percent of the HBOC gene variants were classified as VUS after incorporating RNA data, for example, while 50 percent of the variants appeared benign and 40 percent were classified as likely pathogenic.

The researchers reported similar classification improvements for the variants in HDGC and mismatch repair genes involved in Lynch syndrome, where RNA genetic testing data revealed variants that were both more benign and more pathogenic than previously appreciated.

When the team surveyed healthcare providers to find out if, and how, RNA-based variant reclassification changed medical management for Lynch syndrome, HBOC, or HDGC patients or their family members, it saw pronounced effects for patients with variants reclassified from VUS to likely pathogenic.

Medical management for family members changed, too. When variants previously deemed VUS were reclassified to likely pathogenic, for example, they were typically directed to cascade testing, while RNA test data that shifted a patient's VUS to benign appeared to coincide with decreased screening and follow-up for family members.

Many more individuals could potentially benefit from this type of variant reclassification, according to that team's analysis of 307,812 individuals who previously received genetic testing on 18 hereditary cancer genes. Of the tens of thousands of variants identified in this group, they noted, more than 2,500 variants were predicted to alter splicing — a set that included both VUS and likely pathogenic variants.

All told, the researchers estimated that roughly one in 50 of those receiving DNA tests for hereditary cancer conditions might benefit from RNA-based variant classification.

Karam noted that there are several RNA methods that can be used to quantify transcripts, assess allele-specific expression, and come up with other forms of messenger RNA data. For the research presented at ACMG, the team relied on a high-throughput, massively parallel, RNA sequencing assay they developed called CloneSeq that involves sequencing cloned transcripts produced from RT-PCR products. 

Karam and co-authors described CloneSeq in a Frontiers in Oncology study published last year, where they used CloneSeq to assess germline BRCA1 and BRCA2 variants in samples from hereditary breast and ovarian cancer (HBOC) patients. That analysis led to a likely pathogen exon skipping variant in BRCA1 that was present in a three-generation, HBOC-affected family.

Likewise, Karam and his colleagues have used RNA data to delve into other forms of variation that are difficult to assess with DNA information alone, including tandem duplications. 

In a study that is currently in press and available online in the journal Gastroenterology, the researchers focused on tandem duplications detected in a clinical cohort of 185,943 individuals with a personal or family history of cancer, using RNA genetic testing to drill down on heterozygous MSH2 duplication VUS in five individuals.

Other teams have come up with their own strategies for characterizing and reclassifying unknown variants or VUS with RNA sequencing assays. For example, Madhuri Hegde and her colleagues developed transcriptome and targeted RNA gene transcripts assays for neuromuscular conditions when Hegde was executive director and chief scientific officer for the Emory Genomics Laboratory.

The 2018 American Society for Human Genetics annual meeting included a session on the use of RNA sequencing for boosting sequence interpretation, which included presenters from the Undiagnosed Diseases Network, Brigham and Women's Hospital, and other groups. In that session Hegde and co-authors from Emory, the Georgia Institute of Technology, and Jain Foundation outlined the use of RNA sequencing and monocyte-assays to boost diagnoses in limb girdle muscular dystrophy type 2 or Miyoshi myopathy cases assessed by gene panel sequencing.

"While variant detection provides the exact molecular diagnosis and associated genotype," authors of that poster wrote, "we show that wherever possible a functional assessment such as [a] monocyte assay and RNA-seq in the clinical setting is required for true [genotype-to-phenotype] assessment to implement personalized medicine." 

Hegde is currently vice president and chief scientific officer of PerkinElmer's Global Laboratory Services, which is expecting to soon launch its own RNA-based test derived from approaches Hegde has been developing.