Association of ESX1 gene variants with non-obstructive azoospermia in Chinese males

Abstract

Genetic factors are one of the most important causes of non-obstructive azoospermia (NOA). ESX1 is an X-linked testis-biased expressed gene, and a potential biomarker for testicular sperm retrieval in NOA patients, yet few systematic studies have investigated its association with NOA. Here, we performed selected exonic sequencing in a large cohort of Chinese males, and four novel missense mutations (including one compound mutation), one novel synonymous mutation of ESX1 unique to NOA patients were identified. We analyzed the effects of ESX1 mutations on cyclin A degradation and cell cycle progression by immunoprecipitation assay and flow cytometry, and found that the compound mutant p.[P365R; L366V] ESX1 compromised the stabilizing effect of ESX1 on polyubiquitinated cyclin A, thereby causing the failure of M phase arrest in cells. Further studies showed that the deleterious effect of the compound mutations on ESX1 protein function was attributed to p.P365R but not p.L366V alteration. The novel ESX1 mutation p.P365R might confer high risk for NOA in Han Chinese population, probably via affecting cell cycle control.

Whole‐genome sequencing identifies new candidate genes for nonobstructive azoospermia

“…They also found similar variants (c.1094C>G and c.1096C>G) to ours, which are located in the proline‐rich repeat region, but more closer to the end of this region 53 . The compound variants compromised the stabilizing effect of ESX1 on cyclin A, thereby causing the failure of M phase arrest in cells 53 . Therefore, we would predict that also our identified variants, which were analogical to the reported variants would destabilize cyclins, thereby affecting the cell cycle.…”

“…Importantly, within the group of NOA patients examined using the WGS method were samples that had been previously tested using WES with no apparent results. In the case of ESX1 gene, a group from China reported for the first time a rare ESX1 gene variant unique to Chinese patients with NOA and no mature sperm could be found in the testicular biopsy of those patients 53 . They also found similar variants (c.1094C>G and c.1096C>G) to ours, which are located in the proline‐rich repeat region, but more closer to the end of this region 53 .…”

“…In this gene, another mutation (c.1094C >G, c.1096C>G) was reported in the Han Chinese population as a high risk factor for NOA, likely by affecting cell cycle control. 53 TEX13A (c.596G>A) in which translocations including this gene were reported as genetic risk factors for azoospermia; 54 KCND1 (c.1396C>G; c.1723T>G)—this gene is involved in volume regulation by murine spermatozoa; 55 KIAA1210 (c.4207G>T; c.1214G>A)—it was suggested that KIAA1210 is important for regulating the dynamic change of chromatin structures during spermiogenesis; 56 DHRSX (g.14068A>C)—it was reported that aberrations in the region including this gene may result in the deficiency of X–Y pairing or recombination, ultimately leading to the spermatogenic failure; 57 ZMYM3 (c.2367C>G)—knockout of Zmym3 was observed in mice with arrested spermatogenesis at meiotic metaphase; 58 FAM47C (c.1826A>G) —this gene was described as a possible cause of severe oligozoospermia; 59 FANCB (c.1769T>C)—this gene is involved in the germ cell development at critical stages 60 ; USP9Y (c.3293C>T) and ZXF (c.465T>A) —these genes were previously reported in NOA patients (details are presented in Table 2 , Tables S2 – S4 ). Another example of a gene that has been previously described in the male infertility context and aberrant DNA methylation is FAM50B ( locus 6p.25.2), 61 which was identified in a patient with postmeiotic arrest (P9L) following homozygous variant c.293G>A (Table 2 , Table S2 ).…”

“…In the case of ESX1 gene, a group from China reported for the first time a rare ESX1 gene variant unique to Chinese patients with NOA and no mature sperm could be found in the testicular biopsy of those patients 53 . They also found similar variants (c.1094C>G and c.1096C>G) to ours, which are located in the proline‐rich repeat region, but more closer to the end of this region 53 . The compound variants compromised the stabilizing effect of ESX1 on cyclin A, thereby causing the failure of M phase arrest in cells 53 .…”

“… 53 The compound variants compromised the stabilizing effect of ESX1 on cyclin A, thereby causing the failure of M phase arrest in cells. 53 Therefore, we would predict that also our identified variants, which were analogical to the reported variants would destabilize cyclins, thereby affecting the cell cycle.…”