Beyond Sequencing: Integrating MLPA Reveals Hidden Structural PKD2 Variants and Enhances Mutation Detection in a...

Background and Objectives: The genetic architecture of late-onset autosomal dominant polycystic kidney disease (ADPKD) remains incompletely defined. Pathogenic mutations in two genes that encode polycystin proteins, PKD1 and PKD2, prevail among these patients. Detection of PKD1 alterations by a sequencing method is challenging as this is a large gene with high GC content and multiple pseudogenes. Although next-generation sequencing (NGS) has become the cornerstone of molecular diagnosis, its limited availability in several settings and its limited sensitivity for structural variants may create a diagnostic blind spot, particularly for PKD2. Because PKD2-associated ADPKD follows a milder clinical course with delayed progression to end-stage renal disease (ESRD), its contribution to late-onset disease may be systematically underestimated. We hypothesized that a phenotype-driven strategy integrating copy number analysis would uncover clinically relevant PKD2 variants overlooked by DNA sequencing methods. Materials and Methods: Six well-characterized consecutive ADPKD patients that entered KRT (kidney replacement therapy) above 70 years old underwent targeted PKD2 analysis using bidirectional Sanger DNA sequencing of all coding exons and exon-intron boundaries, complemented by multiplex ligation-dependent probe amplification (MLPA) for copy number variant detection. Results: Pathogenic PKD2 variants were identified in 33% of the patients in this limited cohort. These included the recurrent nonsense variant p.Arg872Ter and a previously undescribed multi-exonic deletion encompassing exons 1-9 that was detectable exclusively by MLPA. Additionally a novel variant of unknown significance (VUS) (p.Leu273Gln) was detected. The high diagnostic yield in this phenotypically highly enriched cohort highlights the value of targeted structural variant analysis. Conclusions: Our findings identify structural PKD2 variation as an under-recognized cause of genetically unresolved late-onset ADPKD and demonstrate that phenotype-guided incorporation of copy number analysis can overcome limitations of NGS alone. A combined sequencing-MLPA approach may therefore provide a more complete and clinically informative molecular diagnosis, particularly in carefully selected patients with late-onset ADPKD.