COLUMBUS, Ohio — A few strategies can be applied to try to resolve variants of unknown significance, according to panelists at the National Society of Genetic Counselors annual meeting.
Sequencing and genetic analysis of patient samples can pick up variants that haven't been seen before as well as those whose impact on disease isn't clear. In 2015, the American College of Medical Genetics and Genomics, in association with the Association for Molecular Pathology and the College of American Pathologists, developed a system to classify variants into one of five categories based on the evidence supporting them. Variants of unknown or uncertain significance, though, have limited evidence and the guidelines said they shouldn't be used in clinical decision making.
But additional evidence can reclassify VUS as benign, likely benign, pathogenic, or likely pathogenic. In a session on bioinformatics tools at the NSGC meeting in Columbus, panelists said RNA sequencing, functional studies, and re-analyzing patient samples at a later date can move VUS from one column to another.
With new resources, Invitae senior VP Steve Lincoln, a session speaker, noted that more VUS are being reclassified.
In some cases, the Mayo Clinic's Eric Klee, another speaker, said that RNA sequencing is being used to supplement genomic analysis. It can, for instance, uncover instances of exon skipping, expressed gene fusions, expression loss, and splicing.
Klee recounted that for one child genetic analysis identified a compound heterozygous mutation in C2CD3 that involved both a pathogenic variant and a VUS. Mutations in that gene had been linked to Oral Facial Digital Syndrome XIV, a condition whose phenotype overlapped with that of the patient. RNA-seq revealed reduced expression of the gene as well as evidence of exon skipping events at two points that led to out-of-frame protein products. They reclassified the compound heterozygous mutation as pathogenic or likely pathogenic.
Klee added that functional work at the protein level, involving protein modeling, far UV spectra analysis, and western blot analysis, could also push VUS into other categories.
As an example, he described an instance where he and his team identified a rare, de novo heterozygous mutation in GABBR2 in a patient with intellectual disability and hypotonia. Previously, Klee noted that the gene had only been linked to smoking and nicotine dependence.
But around the time they found that mutation, a paper was published that identified other mutations in GABBR2 in two patients with seizures as well as intellectual disability and hypotonia. Through protein modeling, they found that these mutations fell within transmembrane domains and could be locking the protein in an active state, a suspicion they confirmed through patch-clamp studies and modeling in zebrafish.
Functional studies, though, take "a lot of effort," Klee said.
Another way to resolve VUS is to reanalyze, reannotate, or reinterpret patient data.
One case Klee presented involved a five-year-old boy with seizures, developmental delay, and balance issues, but who had a normal MRI and was negative for fragile X and Willi-Prader syndromes. Exome sequencing uncovered two VUS, one in NRCAM and one in SLC29A2, though neither were associated with the boy's symptoms.
However, reanalysis of the exome results revealed another mutation, this one in BRPF1. Two studies this year linked mutations in that gene to developmental delay, eyelid droop, and more, leading his team to call it likely pathogenic in their case.
Similarly, another exome sequencing case involving a nine-year-old girl with intellectual disability, motor and speech delay, and more initially pointed to a de novo deletion in ZNF148 that hadn't before been linked to symptoms like hers. Later reannotation, though, found that a new paper had provided that connection.
"As we move forward, I think this is a key part of testing, to go back and look at existing data," Klee said.