Uncovering Dual Molecular Diagnoses in Families with Complex Phenotypes through Structural and Clinical Study of Novel COL4A6 Variants

Background The relationship between observed clinical phenotypes and underlying genotypes is blended or skewed in multiple molecular diagnoses, complicating a comprehensive molecular genetic diagnosis. Aim We report two families with dual diagnoses, using the deafness-associated gene, COL4A6, to exemplify its contribution to blended, complex clinical presentations. Design This is an observational study within a large, ethnically diverse rare disease cohort, focusing on families with hearing loss and suspected dual diagnoses, followed by functional and structural studies of novel variants. Methods Families were identified through a large rare disease sequencing initiative. Exome or genome sequencing was performed, with follow-up RNA studies for a synonymous COL4A6 variant. Spatial and temporal expression analysis in zebrafish traced col4a6 expression in the otic vesicle and ear from 1 to 5 days post-fertilization. Structural modeling was used to estimate variant impact on protein structure. Results We identified two families affected by multiple genetic disorders. The first family presented a missense COL4A6 variant (NM_033641.4:c.1480G>A p.(Gly494Arg)), accounting for hearing loss, while a likely pathogenic HEXA variant (NM_000520.6:c.902T>G p.(Met301Arg)) explained Tay-Sachs disease features. The second family exhibited a synonymous COL4A6 variant (NM_033641.4:c.1767G>A p.(Pro589=)), leading to partial exon skipping and hearing loss, along with a pathogenic splice-site variant in DYM (NM_001353214.3:c.1125 + 1G>T p.?), causing the Dyggve-Melchior-Clausen disease phenotype. Conclusions Our findings highlight the importance of recognizing dual molecular diagnoses to untangle blended phenotypes, as well as the diagnostic relevance of synonymous variants with predicted splicing effects.