Clinician-Initiated RNA Testing Enables Reclassification of a COL4A5 Intronic Variant in X-Linked Alport Syndrome

Variants of uncertain significance (VUS) identified through genetic testing frequently remain unresolved in routine clinical care, as functional follow-up studies are rarely pursued outside research settings. We describe a multigenerational family with suspected X-linked Alport syndrome in which a practicing pediatric nephrologist initiated transcript-level functional testing to resolve a noncanonical intronic COL4A5 variant identified during clinical evaluation. The proband, a 9-year-old girl, initially presented at age 5 with isolated microscopic hematuria and mild microalbuminuria. Her father had progressed to end-stage kidney disease with sensorineural hearing loss, and a paternal uncle exhibited hematuria and proteinuria with preserved kidney function, illustrating differences in disease severity and stage among family members. Genetic testing identified a shared intronic COL4A5 variant, NG_011977.2(NM_000495.5):c.937-10T>G, initially classified as a VUS. To clarify pathogenicity, the clinician arranged targeted RNA analysis through a clinical diagnostic laboratory. Transcript analysis demonstrated activation of a cryptic splice acceptor site with retention of nine intronic nucleotides, resulting in an in-frame insertion. No normally spliced transcript was detected by RT-PCR analysis. These findings provided strong functional and clinical evidence supporting reclassification to likely pathogenic based on RNA analysis, segregation, computational prediction, and population data, and confirmed the diagnosis of X-linked Alport syndrome. This case highlights how clinical RNA testing can help resolve uncertain intronic variants when phenotype and segregation raise concern for pathogenicity, improving diagnostic certainty and family counseling.