Abstract
Accurate genetic diagnosis is essential for appropriate treatment in cystic fibrosis (CF). Large copy number variants like duplications in the CFTR gene are rare and often classified as variants of uncertain significance (VUSs) due to unknown characteristics of the inserted material, complicating diagnosis and treatment decisions. We identified a previously uncharacterized exon 22 duplication (CFTRdup22) in the CFTR gene in two anamnestically unrelated people with CF, both exhibiting a mild phenotype. Initial classification as a VUS was based on standard genetic testing. We employed a custom next-generation sequencing (NGS) panel to determine the exact breakpoints of the duplication and conducted mRNA sequencing to confirm its effect on splicing. DNA and RNA analyses allowed for precise breakpoint determination, confirming that the duplication was in tandem and the reading frame remained intact. This, as well as a residual CFTRdup22 function of ~30% as measured via intestinal current measurement, is consistent with a clinically milder CF phenotype. Collectively, the precise characterization of the variants' breakpoints, localization and orientation enabled us to reclassify the variant as likely pathogenic. This study highlights the importance of advanced genetic techniques, such as NGS and breakpoint analysis, in accurately identifying CF-causing variants. It underscores the importance of a comprehensive approach and persistence when suspecting a specific genetic condition. This can aid in reclassifying VUSs, providing a definitive diagnosis for the affected family and enabling appropriate therapeutic interventions, including the use of CFTR modulators.