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Am J Hum Genet. 2018 Jun 7;102(6):1062-1077. doi: 10.1016/j.ajhg.2018.04.003. Epub 2018 May 24.

Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity.

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McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:


Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.


CFTR function; CFTR mRNA; CFTR protein; functional testing; heterologous expression system; variable expressivity; variant annotation

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