Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B.

Caroline Cazin,Selima Fourati Ben Mustapha,Christophe Arnoult,Zine-Eddine Kherraf,Isabelle Lordey,Amin Bouker,Serge Nef,Raoudha Zouari,Corinne Loeuillet,Pierre F Ray,Yasmine Neirijnck,Nicolas Thierry-Mieg,Françoise Kühne,Lydia Wehrli

doi:10.3390/cells11010118

Abstract

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

Highlights

Infertility, or the inability to conceive, remains a prevalent and ongoing global health concern [1]
We investigated by whole exome sequencing (WES) two unrelated infertile patients born from consanguineous parents and displaying idiopathic non-obstructive azoospermia associated with severe hypospermatogenesis
Because the main aim of this study was to validate or invalidate the implication of the identified candidate genes in non-obstructive azoospermia (NOA), we focused our phenotypic analysis on quantitative spermatogenic defects in KO mice, leading to oligozoospermia or azoospermia

Summary

Introduction

Infertility, or the inability to conceive, remains a prevalent and ongoing global health concern [1]. Infertility is currently estimated to affect 9% of reproductive-aged couples worldwide, with approximately half involving a male factor [2,3]. The diagnostic yield of genetic tests in azoospermia varies depending on the etiology of the disorder, with a diagnosis yield of approximately 90% for congenital bilateral absence of vas deferens (obstructive azoospermia) and only 30% due to a spermatogenic defect (NOA) [7]. This last condition is known as non-obstructive azoospermia (NOA) and is considered as the most severe form of male infertility. Nonobstructive azoospermic men showed the worst health status impairment and should be strictly followed-up regardless of their fertility status [8]

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