Mutations in SOHLH1 gene associate with nonobstructive azoospermia

In a previous study, we found SOHLH1 (spermatogenesis and oogenesis-specific basic helix-loop-helix 1) as the first testis-specific basic helix-loop-helix transcription factor essential for spermatogonial differentiation. SOHLH1 therefore represents an excellent candidate gene for testicular failure such as nonobstructive azoospermia (NOA). We analyzed whether there were mutations in the SOHLH1 gene in 96 Korean patients with NOA. The sequence analysis discovered three novel variations: one intronic variant (c.346-1G>A), and two nonsynonymous exonic variants (c.91T>C and c.529C>A) with known single nucleotide polymorphisms (SNPs), which included six intronic variants, two synonymous, and two nonsynonymous variants. We examined the consequences of mutations in SOHLH1 using in vivo and in vitro assays. Analysis of transcripts from minigenes carrying the c.346-1G>A revealed that splicing site variation leads to the partial deletion at a cryptic splicing site within exon 4. This deletion results in SOHLH1 with a truncated bHLH domain. Transient transfection assay showed that the SOHLH1 mutant with the truncated domain disrupted the transcriptional activity of KIT promoter, whereas two missense mutations harboring either p.Arg37Gln or p.Pro269Ser did not have a significant effect on its transactivation. Our findings indicate that a splice-acceptor site mutation that probably causes a nonfunctional SOHLH1 protein results in nonobstructive azoospermia by the lack of normal spermatogenesis.