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

Area of Science:

• Genetics
• Molecular Biology
• Medical Science

Background:

• Missense DNA variants can have unpredictable effects on protein function, complicating disease pathogenicity interpretation, particularly with variable expressivity.
• Cystic Fibrosis (CF) exhibits variable expressivity, making it challenging to link specific CFTR gene variants to disease severity.
• Accurate interpretation of missense variants is critical for genetic diagnosis and patient management.

Purpose of the Study:

• To evaluate the utility of functional assays in interpreting the pathogenicity of 48 CFTR missense variants associated with variable CF expressivity.
• To compare the performance of functional assays with computational prediction algorithms in assessing variant impact.

Main Methods:

• Assessed CFTR function of missense variants in a native isogenic context using a human airway cell line lacking endogenous CFTR.
• Stably expressed CFTR mutants in the genome of the cell line.
• Compared functional assay results with pathogenicity assignments and predictions from four algorithms (CADD, REVEL, SIFT, PolyPhen-2).

Main Results:

• 13% (6 of 48) of variants believed to cause disease showed no significant impact on CFTR function.
• Functional assays refined pathogenicity assignments, but four computational algorithms failed to distinguish between variants causing severe, moderate, or minimal CFTR function reduction.
• Variants associated with full CF expressivity showed a range of CFTR function, from <10% to >75% of wild-type.

Conclusions:

• Functional assays are essential for accurate interpretation of CFTR missense variants, especially in cases of variable expressivity.
• Current computational prediction tools should be used cautiously for missense variant interpretation due to limitations in differentiating functional impact.
• The study highlights the complexity of genotype-phenotype correlations in cystic fibrosis.