Novel UBE2B-associated polymorphisms in an azoospermic/oligozoospermic population.
OBJECTIVE
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.


CONCLUSIONS
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.
Huang
Ivan
I
Division of Reproductive Endocrinology, 30 North Medical Drive, University of Utah Health Sciences, Salt Lake City, Utah 84106, USA. fertilitydr@hsc.utah.edu
Emery
Benjamin R
BR
Christensen
Greg L
GL
Griffin
Jeanine
J
Peterson
C Matthew
CM
Carrell
Douglas T
DT
D020121:5' Untranslated Regions; D053713:Azoospermia / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics*
D020121:5' Untranslated Regions; D017931:DNA Primers; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes