Clinical and functional evaluation of non-missense MYH9 variants in MYH9-related disease

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and variable systemic manifestations including nephropathy. While most pathogenic MYH9 variants are missense substitutions causing dominant-negative effects, the pathogenic potential of non-missense variants, particularly those affecting splicing, remains unclear.

MYH9 (NM_002473.6) variants registered as "DM" (disease-causing variant) or "DM?" (possible disease-causing variant) in HGMD® were curated. After excluding missense and non-analyzable variants, ten intronic or single-base deletions were selected. Splicing effects were assessed by minigene assays in HEK293T cells and compared with SpliceAI predictions. Clinical information was reviewed.

Aberrant splicing was confirmed in three variants: c.3838-2A>G, c.5765+2T>A, and c.5765+2T>G. The c.3838-2A>G variant caused in-frame skipping of exon 29, non-truncating variant, whereas the latter two induced inclusion of a cryptic 50-bp exon with premature termination codons in the final exon that can escape nonsense-mediated decay. These spliceogenic variants were associated with MYH9-RD phenotypes producing abnormal proteins that likely exert dominant-negative effects. The remaining seven variants showed no splicing abnormalities and were reported in non-MYH9-RD contexts, suggesting limited evidence for pathogenicity. SpliceAI predictions were concordant with experimental results.

This study provides systematic evidence that some aberrant splicing MYH9 variants can lead to MYH9-RD with dominant-negative pathogenesis. Conversely, several variants previously annotated as "DM?" showed no functional or clinical relevance, arguing against a pathogenic role and supporting their consideration as likely benign or uncertain significance. Integrating in silico prediction with experimental validation improves variant interpretation and has implications for the clinical management of MYH9-RD.