XRCC5 VNTR, XRCC6 -61C>G, and XRCC7 6721G>T Gene Polymorphisms Associated with Male Infertility Risk: Evidences from Case-Control and In Silico Studies

Abstract

We evaluate the association between genetic polymorphisms of XRCC5 VNTR, XRCC6 -61C>G, and XRCC7 6721G>T with male infertility susceptibility. A total of 392 men including 178 infertile males (102 idiopathic azoospermia and 76 severe oligozoospermia) and 214 healthy controls were recruited. XRCC6 -61C>G and XRCC7 6721G>T genotyping was performed by PCR-RFLP whereas XRCC5 VNTR was performed by PCR. The 2R allele and 2R allele carriers of XRCC5 VNTR polymorphism significantly decreased risk of male infertility. The mutant GG genotypes and carriers of the CG and GG genotypes of XRCC6 -61C>G showed increased risk for the male infertility. Furthermore, the G allele of the XRCC6 -61C>G was correlated with increased susceptibility to male infertility. Likewise, the T allele of the XRCC7 6721G>T polymorphism was associated with increased susceptibility to male infertility in azoospermia. In silico analysis predicted that the presence of tandem repeats in XRCC5 gene prompter can be sequence to bind to more nuclear factors. Also, rs2267437 (C>G) variant was located in a well-conserved region in XRCC6 promoter and this variation might lead to differential allelic expression. The XRCC7 6721G>T gene polymorphism occurred in an acceptor-splicing site, but this polymorphism has no severe modification on XRCC7 mRNA splicing. Our results indicate the association of XRCC5 VNTR, XRCC6 -61C>G, and XRCC7 6721G>T gene polymorphisms with male infertility in Iranian men.

Figure 1

Schematic and PCR products of the XRCC5 VNTR: (a) schematic diagram of the XRCC5 gene showing the position of the VNTR polymorphism along with various alleles; (b) PCR products containing XRCC5 VNTR with various genotypes.

Figure 2

Schematic diagram and results of the PCR-RFLP products of XRCC6 and XRCC7: (a) schematic diagram of XRCC6 -61C>G, which showed BanI restriction enzyme site; (a′) polymorphism of PCR-RFLP products of the XRCC6 -61C>G gene in 1% agarose gel electrophoresis. Lane M: 50 bp DNA ladder; lane 1: CC homozygous genotype; lane 2: CG heterozygous genotype; lane 3: GG homozygous genotype; (b) schematic diagram of XRCC7 6721G>T which showed PvuII restriction enzyme site; (b′) polymorphism of PCR-RFLP products of the XRCC7 6721G>T gene in 1% agarose gel electrophoresis, lane M: 50 bp DNA ladder; lane 1: GG homozygous genotype; lane 2: GT heterozygous genotype; lane 3: TT homozygous genotype.

Figure 3

EPD report and schematic diagram of the different alleles of XRCC5 VNTR: (a) EPD report of the TATA-box situations and schematic diagram of VNTR (rs6147172) in the XRCC5 gene promoter; (b) schematic diagram of the different alleles of XRCC5 VNTR 0R, 1R, 2R, and 3R containing variable numbers of nuclear transcription factor Sp1.

Figure 4

Potential binding sites for transcription factors of 1R, 2R, and 3R tandem repeat elements of the XRCC5 VNTR: XBP-1 = X-box binding protein 1; TFII-I = transcription factor II-I; STAT4 = signal transducer and activator of transcription 4; c-Ets-1 = c-E-twenty-six-1; E2F-1 = E2F transcription factor 1; GR-beta = glucocorticoid receptor-beta; GCF = GC factor; NF-kappaB = nuclear factor-kappaB; RelA = v-rel avian reticuloendotheliosis viral oncogene homolog A.

Figure 5

The prediction of core promoter elements, transcription factor binding sites, and sequence conservation of the XRCC6 -61C>G variant in the XRCC6 gene promoter: (a) prediction of core promoter elements via ElemeNT tool; (b) prediction of potential transcription factor binding sites; (c) illustration of sequence conservation of the DNA sequence around rs2267437 (C>G) locus.

Figure 6

Enhancers and silencers of the XRCC7 6721G>T DNA sequence flanking motif and its sequence conservation: screening of the DNA sequence flankings of XRCC7 6721G>T for enhancer and silencer motif via SpliceAid 2 tool (a and a′); illustration of sequence conservation of the XRCC7 6721 across multiple mammalian species (b).