Trio-based whole-exome sequencing and Minigene assay identify a novel MYH11 splice site variant (c.2997+5G>C) in recurrent fetal megacystis

Background Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a severe visceral myopathy attributed to defects in smooth muscle contraction. Although pathogenic variants in genes such as ACTG2 , LMOD1 , MYLK , MYH9 , and MYH11 have been associated with MMIHS, the complete genotypic spectrum remains inadequately characterized. Methods We reported a non-consanguineous Chinese family exhibiting recurrent fetal megacystis. Trio-based whole-exome sequencing (Trio-WES) was performed on the parents and the affected fetus. The functional impact of a novel splice site variant identified in the MYH11 gene was assessed using a Minigene splicing assay in vitro . Results Trio-based whole-exome sequencing identified compound heterozygous variants in the MYH11 gene: a maternally inherited 1.9 Mb deletion encompassing MYH11 at 16q13.11, and a novel paternally inherited splice site variant (c.2997 + 5G > C). Initially classified as a variant of uncertain significance (VUS), the pathogenicity of the c.2997 + 5G > C variant was confirmed through functional validation, which demonstrated aberrant splicing. This confirmation facilitated a definitive prenatal diagnosis. Conclusions This study identifies a novel pathogenic splice site variant in MYH11 associated with MMIHS and provides functional evidence supporting its role in the disease’s etiology. Our findings highlight the critical importance of integrating genomic analyses with functional assays to refine interpretation of VUS, expand the mutational spectrum of MMIHS, and improve genetic counseling for affected families.