L26/P-101 A biallelic splicing variant in WDR78 causes multiple morphological abnormalities of the sperm flagella

Abstract

Does a homozygous splicing variant in WDR78 lead to male infertility with multiple morphological abnormalities of the sperm flagella (MMAF)? A homozygous WDR78 splicing variant disrupts WD40 domains through aberrant splicing and is associated with MMAF and defective sperm flagellar assembly. Multiple morphological abnormalities of the sperm flagella (MMAF) represent a well-defined genetic cause of male infertility, combining asthenozoospermia with severe structural defects of the flagellum. To date, more than fifty genes, mainly encoding axonemal or peri-axonemal proteins, have been implicated. Many of these proteins are essential for dynein arm assembly and flagellar motility. WDR78 encodes a WD-repeat protein involved in inner dynein arm formation and ciliary motility in animal models. However, its contribution to human male infertility has not yet been established A consanguineous North African family with five infertile brothers and a homogeneous MMAF phenotype was investigated. The study combined whole-exome sequencing, in silico variant interpretation and functional validation using a minigene splicing assay. One affected brother underwent detailed semen analysis according to WHO recommendations. Whole-exome sequencing was performed following a negative result using an extended infertility gene panel. The candidate splice variant was analysed with multiple in silico splicing prediction tools. Its functional impact was assessed using a minigene assay in HEK293T cells, followed by RT–PCR, capillary fragment analysis and Sanger sequencing to characterise the resulting transcripts. Whole-exome sequencing identified an homozygous splice-site variant in WDR78 (NM_024763.5:c.2113-2A>G). This variant showed an extremely low allelic frequency in population databases (6.9 × 10−7 in gnomAD) and was absent from the 1000 Genomes Project, supporting its rarity and compatibility with an autosomal recessive mode of inheritance. In silico analyses consistently predicted a severe splicing defect. No other variant susceptible to be responsible of the phenotype was identified. Functional assessment using a minigene assay demonstrated activation of a cryptic splice site within exon 15, resulting predominantly in a single aberrant transcript characterised by a 53-bp deletion. This abnormal transcript introduced a frameshift and a premature stop codon, predicting a truncated WDR78 protein lacking the final 99 amino acids, including two WD40 repeats. These domains are essential for β-propeller formation and for protein–protein interactions required for inner dynein arm assembly. Disruption of these structural features provides a coherent molecular explanation for the severe abnormalities of sperm flagellar structure and motility observed in the affected brothers, all of whom presented a homogeneous MMAF phenotype. This report is restricted to a single consanguineous family, and additional independent cases are needed to confirm the gene–disease association. Functional validation relied on a heterologous cell system, which may not fully reproduce germ cell–specific splicing regulation and protein function. These findings identifies WDR78 as a novel gene responsible for MMAF and male infertility, justify its inclusion in diagnostic gene panels and underscore the contribution of functional assays to the interpretation of splice-site variants detected by exome sequencing. -