Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity  Karen S. Raraigh, Sangwoo T. Han, Emily Davis, Taylor A. Evans, Matthew J. Pellicore, Allison F. McCague, Anya T. Joynt, Zhongzhou Lu, Melis Atalar, Neeraj Sharma, Molly B. Sheridan, Patrick R. Sosnay, Garry R. Cutting  The American Journal of Human Genetics  Volume 102, Issue 6, Pages 1062-1077 (June 2018) DOI: 10.1016/j.ajhg.2018.04.003 Copyright © 2018 American Society of Human Genetics Terms and Conditions

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 Isc 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 Isc 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. The American Journal of Human Genetics 2018 102, 1062-1077DOI: (10.1016/j.ajhg.2018.04.003) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2018 102, 1062-1077DOI: (10.1016/j.ajhg.2018.04.003) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2018 102, 1062-1077DOI: (10.1016/j.ajhg.2018.04.003) Copyright © 2018 American Society of Human Genetics Terms and Conditions