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

Abstract

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.

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

Figure 1

CFTR mRNA, Protein Quantity, and Function Are Variable but Correlated (A) Mean and standard deviations of CFTR mRNA transcript quantity relative to that of HPRT1 (n = 3 for each cell line) for ten independent cell lines expressing WT-CFTR. (B) Immunoblot detecting varying quantities of mature (band C) CFTR from whole-cell lysates of ten cell lines expressing WT-CFTR. Controls include cell lines expressing the CF-causing variants F508del, which causes a folding defect (band B only), and G551D (band C); non-transfected CFBE cells (no signal); and HEK293 cells transiently expressing WT-CFTR (band C). Loading controls for protein quantity (Na⁺/K+ ATPase) are shown below. The plot on the right shows the mean and standard deviations of CFTR quantities for each cell line relative to the WT-CFTR 5 cell line assessed from at least three immunoblots. (C) Representative recordings of CFTR function measured by I_sc for ten WT-CFTR cell lines. Forskolin (10 μM) activates CFTR chloride current, and the amount of current inhibited by the CFTR-specific inhibitor inh-172 (10 μM) determines the level of CFTR function. The plot on the right shows the mean and standard deviations for I_sc derived from at least three measurements for the ten WT-CFTR cell lines. (D) Correlations of the quantity of CFTR mRNA with quantity of mature CFTR (left), quantity of mature CFTR with CFTR function (center), and quantity of CFTR mRNA with CFTR function (right) for ten independent cell lines expressing WT-CFTR.

Figure 2

Independently Derived Cell Lines of CFTR Missense Variants Yield Consistent Interpretation (A) Standard curve (dashed line) for 100% WT-CFTR function derived with CFTR mRNA and CFTR function from 24 independent cell lines expressing WT-CFTR. Predicted CFTR function corresponds to 25% (green), 10% (gold), and 1% (red) of WT-CFTR function across the range of mRNA expression observed in WT-CFTR cell lines. (B) Plot of log CFTR function against CFTR mRNA quantity derived from the correlation shown in (A). After normalization for mRNA quantity, CFTR variants expressed in multiple independent cell lines show consistent levels of residual CFTR function. Four variants illustrate the range of CFTR function observed.

Figure 3

Distinct Distributions of the Residual CFTR Function of Variants Associated with Full or Partial Expressivity of CF The majority of variants associated with full expressivity of CF allow less than 10% WT-CFTR function, and the remainder distribute across three higher ranges of function. None of the variants associated with partial expressivity of CF have less than 10% CFTR function.