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Functional Assays Improve Classification of Potential Cystic Fibrosis Risk Variants

NEW YORK (GenomeWeb) – A new analysis of missense variants implicated in the expression of cystic fibrosis (CF) has again highlighted the importance of applying appropriate functional assays to classify such variants and their potential impact in the clinic.

Researchers from Johns Hopkins University focused on four dozen missense variants affecting the CF transmembrane conductance regulator-coding gene (CFTR) that influence CF expressivity. By expressing each of the variants in a human airway cell line lacking endogenous CFTR, they tracked the phenotype in relation to that found in cells with full expressivity of the CF phenotype.

The approach made it possible to find variants previously believed to produce full CF expressivity that showed very low CFTR function, and vice versa. Even so, some surprises did occur. For example, CTFR function remained relatively high in assays of a few variants implicated in high expressivity of the disease phenotype, while other variants linked to partial CF expressivity had little to no effect on CTFR function.

"We show that functional assessment informs variant annotation when full or partial expressivity is present, and that uncertainty of variant effect can be re-interpreted in the context of variable expression of a phenotype," senior author Gary Cutting, a genetic medicine researcher at Johns Hopkins University School of Medicine, and his colleagues wrote in their study, which was published online today in the American Journal of Human Genetics.

With the help of site-directed mutagenesis and PCR verification, the researchers swapped out wild-type CFTR in CF bronchial epithelial cells and systematically replaced it with versions of the gene containing one of 48 CF-associated missense variants — a set that contained 29 variants previously classified as fully penetrant and another 19 variants believed to be partially penetrant.

They went on to evaluate CFTR expression and chloride channel function in these cells with immunoblot assays, PCR-based approaches, RNA sequencing, and/or voltage-current clamp assays, examining the relationship between each variant's anticipated phenotypic expressivity and its actual effects on CFTR function.

They found that CFTR activity was less than 10 percent of that for wild-type CFTR in cell lines containing 21 of the 29 variants classified as having full phenotypic expressivity. Of the remaining eight variants, five showed more modest dips in CFTR gene function, while three maintained CFTR function beyond 25 percent of that described for the wild-type version of the gene.

"Even though [the latter variants] were presumed to be disease causing and were reported as such by clinicians, functional evidence indicates that they are not deleterious," the authors wrote, noting that "all three variants have been reported to occur as part of complex alleles … potentially explaining their presence in individuals with CF."

Meanwhile, the researchers reported that across the 19 variants classified as having partial CF expressivity, CFTR function exceeded 10 percent of wild-type CFTR activity. Nevertheless, a handful of those variants had somewhat muted CFTR activity, falling between 10 percent and 25 percent of wild-type CFTR function.

From these and other results, including comparisons with predictive variant classification algorithms, the authors suggested that "functional testing was informative for the assessment of variants associated with full expressivity and essential for the interpretation of variants found in individuals with partial expressivity of CF."

The team also demonstrated the effectiveness of incorporating functional data into American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) variant annotation criteria to clarify the classification of other potential risk variants for CF, including variants of uncertain significance.

Nevertheless, the authors noted that "correlation between ACMG-AMP and CFTR2 [expert database] annotation is incomplete because the ACMG-AMP guidelines have no defined category for variants causing partial expressivity."