Novel UBE2B-associated polymorphisms in an azoospermic/oligozoospermic population

Abstract

Aim: To assess whether abnormalities exist in the UBE2B gene in a population of infertile human males, and to establish biologic plausibility of any discovered mutations.

Methods: We carried out polymerase chain reaction (PCR) amplification and sequence analysis of the 5'-untranslated region and six exons of the UBE2B gene, including flanking intronic regions, in a group of fertile and infertile men. Following the identification of a putative promoter region that contained single or dual triplet deletions within a 10-CGG repeat island, we evaluated the binding affinity of these identified polymorphisms as compared to the wild-type sequence to transcription factor SP1 using a DNA-protein gel shift assay.

Results: There was a novel exonic single nucleotide polymorphism (SNP) noted in exon 4 in 5% of infertile men. In silico 3D modeling of the altered protein showed an innocuous isoleucine for valine substitution. There were no mutations noted within any of the other exons. Three novel intronic SNPs were identified within the fertile group, and seven novel intronic SNPs identified in the infertile group. The DNA-protein gel shift assay noted that both single CGG deletion and double CGG deletion bands had approximately twice the binding affinity compared to the wild-type for SP1. The negative control confirmed no non-specific protein binding.

Conclusion: By themselves, a single or double CGG deletion is unlikely to pose biologic significance. However, such deletions in this suspected promoter region are associated with increased binding affinity for SP1, and might represent one of several factors required for alteration of UBE2B gene expression.