A de novo paradigm for male infertility

Abstract

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10^-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10^-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Fig. 1. Analysis of the intolerance to loss-of-function and missense variation in genes with de novo mutations.

a Violin plot with quantile lines showing pLI scores in all genes in gnomAD (red), all genes affected by rare protein-altering loss-of-function (LoF) de novo mutations (DNMs) in a control population (http://de novo-db.gs.washington.edu/de novo-db/) (green) and in all genes with a rare protein-altering LoF DNM in our trio cohort (blue). Using the permutation-based, nonparametric test defined by Lelieveld et al. a significant enrichment of LoF DNMs in LoF-intolerant genes in patient cohort was detected in comparison to the number of LoF in fertile control cohort (DNM LoF mutations in patients n = 17, median pLI in patients with male infertility = 0.80, DNM LoF mutations in controls n = 21, median pLI in controls = 3.75 × 10⁻⁵, p value = 1.00 × 10⁻⁵, N simulations = 100,000). The black dot indicates median pLI scores. b Violin plot with quantile lines showing the distribution of Z-scores for genes with predicted benign (n = 59) and pathogenic missense DNMs (n = 63) in infertile patients. A significant increase in predicated pathogenic DNMs in missense-intolerant genes was detected compared to benign missense DNM (Two-sided Mann–Whitney U test, p value of 3.44 × 10⁻⁴). (***p value < 0.001).

Fig. 2. Protein–protein interactions predicted for proteins affected by pathogenic de novo mutations.

Significantly larger number of interactions were observed in proteins affected by de novo mutations than expected for a similar sized dataset of randomly selected proteins (PPI enrichment p value = 2.35 × 10⁻²). The central module of the main interaction network (blue dashed circle) contains 5 proteins involved in mRNA splicing (Supplementary Fig. 6).

Fig. 3. Description of control and TOPAZ1 proband testis histology and aberrant acrosome formation.

a, b H&E stainings of (a) control and (b) Proband_060 with pathogenic mutations in TOPAZ1 gene. The epithelium of the seminiferous tubules in the TOPAZ1 proband show reduced numbers of germ cells and an absence of elongating spermatids based on the analysis of 150 seminiferous tubules in control and patient. c, d immunofluorescent labeling of DNA (magenta) and the acrosome (green) in control sections (c) and TOPAZ1 proband sections (d). (c) The arrowhead indicates the acrosome in an early round spermatid and the arrows the acrosome in elongating spermatids. Spreading of the acrosome and nuclear elongation are hallmarks of spermatid maturation. (d) No acrosomal spreading (see arrowheads) or nuclear elongation is observed in the TOPAZ1 proband. The asterisk indicates an example of progressive acrosome accumulation without spreading. Scale bar: 40 µm (a, b) and 5 µm (c, d).