Genetic Association of the Functional WDR4 Gene in Male Fertility

Abstract

Infertility is one of the important problems in the modern world. Male infertility is characterized by several clinical manifestations, including low sperm production (oligozoospermia), reduced sperm motility (asthenozoospermia), and abnormal sperm morphology (teratozoospermia). WDR4, known as Wuho, controls fertility in Drosophila. However, it is unclear whether WDR4 is associated with clinical manifestations of male fertility in human. Here, we attempted to determine the physiological functions of WDR4 gene. Two cohorts were applied to address this question. The first cohort was the general population from Taiwan Biobank. Genomic profiles from 68,948 individuals and 87 common physiological traits were applied for phenome-wide association studies (PheWAS). The second cohort comprised patients with male infertility from Wan Fang Hospital, Taipei Medical University. In total, 81 male participants were recruited for the genetic association study. Clinical records including gender, age, total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), total sperm number, sperm motility, and sperm morphology were collected. In the first cohort, results from PheWAS exhibited no associations between WDR4 genetic variants and 87 common physiological traits. In the second cohort, a total of four tagging single-nucleotide polymorphisms (tSNPs) from WDR4 gene (rs2298666, rs465663, rs2248490, and rs3746939) were selected for genotyping. We found that SNP rs465663 solely associated with asthenozoospermia. Functional annotations through the GTEx portal revealed the correlation between TT or TC genotype and low expression of WDR4. Furthermore, we used mouse embryonic fibroblasts cells from mwdr4 heterozygous (+/‒) mice for functional validation by western blotting. Indeed, low expression of WDR4 contributed to ROS-induced DNA fragmentation. In conclusion, our results suggest a critical role of WDR4 gene variant as well as protein expression in asthenozoospermia.

Keywords: WDR4; genetic variants; male fertility; sperm quality.

Figure 1

Graphic view of the genotyped WDR4 gene.

Figure 2

Phenome-wide scan for associations with WDR4 variants. The blue line represents p = 0.05, and the red line represents the significance level for phenome-wide associations after multiple testing corrections (p = 3.65 × 10⁻⁵).

Figure 3

Profile of WDR4 gene expression in various tissues. Blue represents the male gender and red the female gender.

Figure 4

H₂O₂-induced γH2AX expression increased in wdr4 heterozygous (+/−) MEF cells. The MEFs cells were subcultured into 6 cm culture dish 24 h before being treated with 100 μM H₂O₂. The mwdr4, the γH2AX, and the GAPDH protein expression levels were determined by western blot after H₂O₂ treatment for 24 h.