Multiplexed Functional Assessment of Genetic Variants in CARD11

Area of Science:

• Genetics
• Immunology
• Molecular Biology

Background:

• Genetic testing identifies numerous variants in disease genes, but understanding their functional impact remains a challenge.
• CARD11 gene variants are linked to distinct immunodeficiencies, with dominant-negative and gain-of-function effects.
• Accurate variant interpretation is crucial for diagnosing and managing genetic disorders.

Purpose of the Study:

• To develop and apply a high-throughput method for functional assessment of CARD11 variants.
• To identify variants causing decreased or increased protein function associated with immunodeficiency.
• To elucidate the mechanism of dominant-negative activity, including exon skipping.

Main Methods:

• Utilized a "cloning-free" saturation genome editing approach in a diploid cell line.
• Simultaneously scored 2,542 variants in the CARD11 gene region for functional impact.
• Analyzed an exon-skipping mechanism contributing to dominant-negative CARD11 function.

Main Results:

• Successfully assessed the function of 2,542 CARD11 variants.
• Identified variants associated with decreased or increased protein function relevant to immunodeficiency.
• Demonstrated high sensitivity (94.6%) and specificity (88.9%) in classifying known clinical variants.
• Provided immediate utility for interpreting seven coding and splicing variants found in clinical settings.

Conclusions:

• The developed genome editing approach is effective for large-scale functional variant assessment.
• This method significantly enhances the diagnostic utility of genetic testing for CARD11-related immunodeficiencies.
• The approach is generalizable for variant interpretation in other clinically actionable genes and cell types.