Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

Abstract

Background: The mismatch repair (MMR) pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility.

Methods: We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs) in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5) using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL) assay in 450 cases. Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation techniques were employed to determine the effects of functional variants.

Results: One intronic SNP in MLH1 (rs4647269) and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn) and MSH5 (rs2075789, Pro29Ser) seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2.

Conclusions: Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility.

Figure 1

Box-and-whisker plots of sperm DNA fragmentation for different genotypes. The boxes represent the 25^th and 75^th percentiles; whiskers are lines extending from each end of the box covering the extent of the data on 1.5 × inter-quartile range. The median value is denoted as the line that bisects the boxes. Circles and asterisks represent the outlier values. Significant differences were measured by multiple linear regression.

Figure 2

FRET imaging of MLH1 and PMS2 interaction in live HEK293T cells. Images of CFP-tagged (green) and YFP-tagged (red) constructs when transiently expressed in HEK293T cells. Co-localization of co-expressed constructs is shown as yellow in overlay images. The pseudocoloured images represent FRET signals corrected for any bleed-through using the micro-FRET method (FRETc). A: Co-localization (overlay) and direct interactions (FRETc) between MLH1-CFP + PMS2 (wt)-YFP were detected in the nucleus. B: Cells co-expressing MLH1-CFP + PMS2 S775N-YFP showed good co-localization of fluorescent signals but little detectable FRETc signal in the nucleus.

Figure 3

Interaction studies between hMLH1 and hPMS2 variants. A: Western blot of total protein extracts (50 μg each) from HEK293T cells transfected with pcDNA3.1-MLH1 and either wild-type pcDNA3.1-PMS2 (775Ser) or pcDNA3.1-PMS2 (775Asn) variants. β-actin was used as controls. B: The lysates of cells co-expressing the two plasmid were immunoprecipitated with anti-MLH1 N-20 antibody, and then detected with anti-PMS2 (A16-4) antibody. Western blot signals were quantified employing Quantity-One software.