Infertile Men With Non-Obstructive Azoospermia Exhibit Defects in the MSH5 DNA Mismatch Repair Gene

Presentation Number: OR22-3
Date of Presentation: June 16th, 2013

Alex David Ridgeway*, Jason Kovac, Sarmistha Mukherjee, Josephine Addai, Larry L Lipshultz and Dolores J Lamb
Baylor College of Medicine, Houston, TX

Abstract

INTRODUCTION:  Infertility involves a contributing male factor in 50% of cases.  Non-obstructive azoospermia (NOA) results from spermatogenic failure of which the mechanisms involved are incompletely understood.  Given that spermatogenesis requires prolific cellular division and high fidelity DNA replication, repair systems (i.e. Mismatch Repair; MMR) are needed to correct cellular insults or commit the cell to apoptosis if reparation is not possible.  Epigenetic modifications through DNA methylation can cause MMR deficiences.  Given that MSH5 is a key component of the MMR pathway and that MSH5 deficient mice display sterility, we sought to determine a role for MSH5 in NOA men. 

METHODS:  We examined the global DNA methylation status of NOA men (n=26; n=5 controls) using the Infinium HumanMethylation450 BeadChip to identify DNA methylation abnormalities.  Changes in the methylation status of MSH5 lead to further examination of a cohort of 371 NOA men and 66 fertile controls for specific mutations in the MSH5 gene by denatured high performance chromatography.  The functional effects of MMR defects were tested by examining cellular growth in the presence and absence of MMC (mitomycin C) and MNU (N-Nitroso-N-mehylurea).  MSH5 expression and localization was examined using qPCR, immunocytochemistry and western blots.  

RESULTS:  Analysis of DNA methylation identified significantly aberrant DNA methylation within MSH5 in 4 of 26 NOA men. The key regions of the MSH5 gene that encompass important functional domains of the protein, exon 2 and 15, revealed polymorphisms and genetic aberrations.  A variant (C85T) that altered codon 29 of the MSH5 gene was identified.  This resulted in a proline to serine change (P29S) in 11 infertile men and threonine 418 to alanine in 3 NOA men in exon 15.  Testicular fibroblasts from 30 NOA men were resistant to the DNA alkylating agents MMC/MNU suggesting a functionally defective DNA MMR pathway.  Fibroblasts from control men (n=10) exhibited sensitivity and did not proliferate.  Immunocytochemisty, qPCR and western blots identified significantly decreased levels of MSH5 and cellular mislocalization in NOA men.  

CONCLUSIONS:  The DNA MMR pathway in NOA men exhibits defects in MSH5.  These alterations may affect genetic stability and DNA recombination leading to impaired spermatogenesis.

 

Nothing to Disclose: ADR, JK, SM, JA, LLL, DJL