CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
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Broad sequencing has dramatically increased the number of putative disease genes (reviewed by Walsh R, et al. Clin Chem. 2017;63[1]:116–128). “Diagnostic sequencing has the potential to become an integral part of the clinical management of patients with inherited cardiac conditions,” the authors wrote. They cautioned, “[E]xpanded testing requires great rigor in the identification of pathogenic variants, with domain-specific knowledge required for variant interpretation.”
Dr. Funke agrees. For many published gene-disease associations, she said, “Claims typically rest on identification of rare variants, usually with little additional supporting data.”
The variety of genes included on HCM panels offered by 45 laboratories surveyed two years ago illustrates the uncertainty about pathogenic genes. Most of the labs in a subset of 14 had 10 to 40 genes on their panels. One had 50; another had 90.
Dr. Funke and colleagues analyzed their data for 766 patients tested with DCM panels from 2007 to 2011. During that time the panel grew from five genes to 46. Clinical sensitivity increased from 10 percent to 37 percent. Inconclusive findings also grew, from about 10 percent to about 60 percent (Pugh TJ, et al. Genet Med. 2014;16[8]:601–608). The investigators noted that “[T]he contributions of individual genes and the pathogenic variant spectrum are still poorly defined.” And they concluded, “Our data illustrate the utility of broad gene panels for genetically and clinically heterogeneous diseases but also highlight challenges as molecular diagnostics moves toward genome-wide testing.”
A variant of uncertain significance finding can affect patients differently, Dr. Funke says. “Depending on how you’re wired, it can be a concern or not. You may assume you have a pathogenic variant or that it signifies nothing.”
Dr. Funke shared a recently reported case in which the finding of a variant of uncertain significance had a powerful adverse clinical impact (Ackerman MJ. Heart Rhythm. 2015;12[11]:2325–2331). The proband died suddenly. The proband’s sister saw a cardiologist but her evaluation was unremarkable. Pan-arrhythmia testing detected a variant of uncertain significance, which the cardiologist concluded must have been the cause of the proband’s death. A prophylactic implantable cardioverter-defibrillator was placed in the sister and in three of her children. Years later it was found that that variant does not affect protein function. When tissue from the proband was finally tested, it was negative.
Dr. Funke
“I am trying to stress that even a physician can misinterpret a VUS,” Dr. Funke says. “That VUS should not have led to the action the physician took.”
To avoid such problems, the Clinical Genome Resource, or ClinGen, is building and centralizing resources to define the clinical relevance of genes and variants (www.clinicalgenome.org). Three questions are crucial: Is this gene associated with a disease? Is this variant causative? Is it actionable?
The ClinGen hypertrophic cardiomyopathy gene curation team is looking at 55 selected genes. Of the first 30 genes curated, only nine had strong or definitive evidence for pathogenicity. Twenty had limited or no evidence. Only two fell in the ambiguous “moderate” category. “Now what I would like to see happen is that labs not even include those genes with limited or weak evidence on panels,” Dr. Funke says.
She is proud of what’s been accomplished over the years through this international effort: “This is what it takes: experts contributing time and expertise to meet a goal in the absence of adequate funding,” she says. Dr. Funke is co-chair of the ClinGen Cardiovascular Clinical Domain Working Group.
She shared another case, one that illustrates how a lack of standards for sequence interpretation can affect patients. A laboratory had interpreted a DCM patient’s variant as likely pathogenic. The patient came to the Laboratory for Molecular Medicine for a second analysis, where the variant was interpreted to be of uncertain significance. Dr. Funke called the other laboratory and resolved the discrepancy. “This is why we need concrete evidence-based standards on which to base our interpretation.”
Since that time, a working group of the American College of Medical Genetics and Genomics and Association for Molecular Pathology published standards and guidelines for interpreting sequence variants (Richards S, et al. Genet Med. 2015;17[5]:405–424). However, “The new criteria are not the final answer,” she said. Three experts from the ClinGen Inherited Cardiomyopathy Expert Panel classified 10 MYH7 variants twice. Using their institutional criteria, concordance was 95 percent. Using the ACMG/AMP criteria, it was only 30 percent. Part of this problem is that the experts were not yet familiar with the new criteria. Another reason: The ACMG/AMP criteria are not specific to cardiomyopathy. “We need to make the criteria tighter with regard to specific diseases,” Dr. Funke says.
To address this need, the ClinGen Cardiovascular Clinical Domain Working Group has adapted and validated the existing variant classification framework for MYH7-associated inherited cardiomyopathies. The recommendations of the expert panel were published online ahead of print on Jan. 4 (Kelly MA, et al. Genet Med. 2018. doi:10.1038/gim.2017.218).
Sharing of sequence data among laboratories is one good way to enhance validation. Much variant-disease association data are public. However, Dr. Funke explains, “Many diagnostic labs have internal data they accumulate over time”—so-called private data. More than 3,000 patients have been studied at her own institution. “From each patient you learn something. Is that variant pathogenic or not?” Unless each laboratory shares its information publicly, though, it all remains in separate silos. “On the other hand,” Dr. Funke said, “once we pool those data, the number of times a variant has been seen increases, which can help clarify its clinical significance. For example, we may cross a threshold where we can confidently move a variant from likely pathogenic to pathogenic.”
One exercise conducted among ClinGen Inherited Cardiomyopathy Expert Panel members illustrates this point. When private data were added to public data, 12 of 60 variants analyzed (48 percent) changed classification. Of those, seven were upgraded from likely pathogenic to pathogenic. Reclassification was accomplished by sharing among “a handful” of laboratories (among them some of the world’s largest cardiomyopathy testing laboratories). As part of their mandate from ClinGen, the NIH-funded working groups are sharing their data in ClinVar.
The Sarcomeric Human Cardiomyopathy Registry consortium, too, observed greater agreement in hypertrophic cardiomyopathy genetic test interpretation with data sharing. “Discordance in variant classification among hypertrophic cardiomyopathy centers is largely attributable to privately held data,” investigators concluded (Furqan A, et al. Circ Cardiovasc Genet. 2017;10[5].doi:10.1161/circgenetics.116.001700). Says Dr. Funke, “We were happy to see they reached the same conclusions we did.”
Dr. Funke presented several suggestions for how to achieve complete harmonization of variant classification. Among them: enhance data submissions, create structured fields for case-level clinical data in public variant databases, and standardize clinical data collection by testing laboratories.
Looking to the next step, Dr. Funke raised the possibility of genetic screening for cardiovascular disease in the general population. “Let’s move forward, but let’s do it carefully and thoughtfully,” she advises. “It is more complicated than people have thought, including myself. We’re finding out we don’t know enough.”
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William Check is a writer in Ft. Lauderdale, Fla.