Investigating de novo mutations in severe idiopathic male infertility

Abstract

Infertility affects 1 in 6 couples worldwide and in approximately 50 % of these cases are attributed to a male factor. Whilst causes such as Klinefelter’s and Y chromosome microdeletions have been well established, the genetic causes behind severe spermatogenic failure are largely unknown, with around 40 % of all male infertility cases remaining idiopathic. De novo mutations (DNMs) are known to play a prominent role in many sporadic disorders with reduced fitness. These mutations, however, are vastly understudied in the field of male infertility due to the difficulty of obtaining parental samples. The foundation of this thesis is formed around the hypothesis that DNMs play a vital role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, next generation sequencing (NGS) techniques were utilised to sequence the DNA of males with idiopathic azoospermia or oligozoospermia and their unaffected parents. An initial study was conducted, utilising a targeted 54-gene panel NGS assay to identify DNMs in 75 patient parent trios. However, this proved to be too narrow a gene pool with no possibly causative DNMs identified across the cohort. A further whole exome sequencing (WES) study was then performed in a cohort of 185 males suffering from idiopathic male infertility and their parents. In total, 145 rare protein-altering de novo SNVs were identified. Following a systematic analysis assessing mutational impact and protein function, 29 DNMs were classified as possibly causative. A significant enrichment of loss-of-function DNMs in loss-of-function-intolerant genes (p-value = 1.00×10−5 ) was seen in infertile men compared to controls. A significant increase was also identified in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01×10−4 ). One such gene containing a pathogenic missense DNM, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. A follow-up study in a cohort of 2,506 infertile males identified six rare heterozygous pathogenic missense mutations affecting RBM5, whilst no such mutations were present in a cohort of 5,784 fertile men (p-value = 0.03). Results from a minigene splicing assay suggested that mutations in RBM5 may potentially disrupt its function as a pre-MRNA splicing factor. The work presented in this thesis provides evidence for the role of de novo mutations in severe idiopathic male infertility, highlights several potential candidate genes causing infertility and suggests potential biological processes disturbed by these genes.