Application of Genomic Studies in Uncovering Sperm Defects Mechanisms

Background: It is estimated that about 40 % of male infertil-ity cases are idiopathic. While asthenozoospermia is the most frequent condition observed in infertile male, very few genes in relation to this phenotype have been introduced. The main reason for this in many cases is the large number of potential candidate genes. It is assumed that more than two thousand genes are involved in spermatogenesis. Therefore, lots of po-tential candidate genes may exist in each case making it very difficult to identify the pathogenic variation behind the condi-tion. However, novel gene and variation identification by using newly available technologies is a beneficial approach to uncov-er the asthenozoospermia pathophysiology potentially leading to new treatments. Accordingly, in the last decade the advent of Next Generation Sequencing (NGS) technology and particu-larly Whole Exome Sequencing (WES) has revolutionized our knowledge of reproductive genetics. This unprecedented suc-cess is due to the nature of this method in which the exons of known human genes are sequenced with adequate coverage making it possible to study various candidate genes simultane-ously. In recent years, we have actively recruited consanguine-ous families with multiple infertile male patients. Routine tests have failed to establish the cause of infertility in all of these patients while generations of consanguineous marriages indi-cated it to be of genetic nature. Therefore, we applied WES to study these families which led to the identification of novel and rare pathogenic variations in genes involved in natural sperm functions.Materials and Methods: To establish the idiopathy of infertil-ity, all patients underwent routine diagnostic procedure including semen analysis, hormonal assays and karyotyping. Con-sequently, high quality DNA was procured from patients and close family members for WES. Rigorous bioinformatics anal-ysis on WES results led to a shortlist of candidate variants that were verified by conventional PCR and Sanger Sequencing. To confirm the pathogenicity of the identified variants, dynamic protein modeling and appropriate experiments were performed in accordance with the nature of each variation. In one case the sperm sample of patients was treated with the analogue of the predicted defective enzyme and in another, ROS evaluation test was performed on sperm samples.Results: Using WES, we managed to identify a pathogenic frameshift variant in ADCY10 which codes for the soluble adenylate cyclase (sAC), the main source of cAMP in sperm tail and an essential enzyme for the regulation of sperm motil-ity. Functional analysis experiment using treatment of patients’ sperm samples with a cAMP analogue significantly raised the percentage of progressively motile spermatozoa and confirmed the pathogenicity of the variation. Moreover, a rare missense variant in GFPT2 was discovered in another studied family and dynamic protein modeling identified this variation to in-flict negative effects upon the resulting protein. This gene is involved in the antioxidant defense system and its inactivity can lead to high levels of Reactive Oxygen Species in sperm with adverse effects on sperm motility.Conclusion: On the whole, using WES we managed to pro-vide the first evidence of ADCY10 and GFPT2 involvement in human asthenozoospermia. The frameshift variant in ADCY10 was particularly considerable as it had already been shown by a mouse model study that this gene was implicated in astheno-zoopspermia.

Keywords:

Male Infertility , Genomics , WES , Familial , Astheno-zoospermia