Sequencing of FTO and ALKBH5 in men undergoing infertility work-up identifies an infertility-associated variant and two missense mutations

Abstract

Objective: To assess whether men with reduced semen quality exhibit genetic variants in the genes coding for the messenger RNA methylation erasers FTO and ALKBH5.

Design: DNA of men undergoing infertility work-up was extracted and the FTO and ALKBH5 genes were sequenced. Statistical analysis was used to study the correlation between the identified ALKBH5 and FTO variants and sperm quality.

Setting: University hospital infertility clinic.

Patient(s): Semen samples from 77 unselected men that had been referred to Oslo University Hospital for routine semen analysis as part of infertility work-up.

Intervention(s): Not applicable.

Main outcome measure(s): Immunohistochemistry and Western blot were used to confirm the presence of ALKBH5 and FTO in human testis. DNA extraction from samples was followed by Illumina MiSeq amplicon high throughput sequencing and sequence alignment. Variant calling was carried out using GATK's UnifiedGenotyper. Standard semen parameter analysis was performed according to World Health Organization guidelines.

Result(s): We found an FTO genetic variant to be associated with reduced semen quality. We also identified two FTO missense variants, one mutation (p.Cys326Ser) was located in the important linker between the two protein domains; the other mutation (p.Ser256Asn) was situated in a flexible loop able to interact with other molecules.

Conclusion(s): The discovery of two missense mutations with potentially detrimental effect on the functionality of the methylation eraser protein FTO, as well as a genetic variant of the same protein that is associated with altered semen quality could suggest that aberrant demethylation of messenger RNA is a factor involved in reduced male fertility.

Keywords: 6-methyladenine; ALKBH5; FTO; infertility; mRNA methylation; semen quality.