Identification of novel variants in SSX1, CPNE1, and SPTBN5 in men with oligoasthenoteratozoospermia using whole-genome sequencing

Reduced sperm count, impaired motility, and abnormal morphology are characteristics of oligoasthenoteratozoospermia (OAT). Genetic factors play an important role in spermatogenic failure, but the molecular basis of OAT is still not fully understood. Whole-genome sequencing (WGS) was performed in 17 infertile men with normal karyotypes and no Y-chromosome microdeletions, including 10 patients with azoospermia and 7 with OAT. Among the OAT group, three patients carrying prioritized rare variants with potential relevance to spermatogenesis were selected for detailed analysis and presentation in the current study. Variants were called using DeepVariant, and they were annotated by Ensembl Variant Effect Predictor (VEP). Variants were prioritized based on low allele frequency (< 1% in gnomAD), predicted functional impact, relevance to spermatogenesis, and consistency with a recessive inheritance model. Candidate variants were validated by Sanger sequencing and inspection of BAM files. Structural protein modeling and characterization of mutation-induced changes in molecular interactions were also performed using SWISS-MODEL and AlphaFold. We identified a hemizygous splice donor variant (c.*4 + 1G > T) in SSX1 and a missense variant (c.1190 A > G) in CPNE1 in one patient with OAT. SSX1 is a testis-expressed gene, while CPNE1 encodes a calcium-dependent phospholipid-binding protein. In another patient, a stop-gained variant (c.790 C > T) in SPTBN5 was identified, which encodes a spectrin beta chain protein. This study identifies rare candidate variants in SSX1, CPNE1, and SPTBN5 that may potentially be associated with the molecular basis of OAT. These findings provide preliminary evidence supporting further investigation of these genes in OAT and support the application of WGS in unresolved male infertility.