X-linked ADGRG2 mutation and obstructive azoospermia in a large Pakistani family.
ABSTRACT: We performed whole exome sequencing to identify an unknown genetic cause of azoospermia and male infertility in a large Pakistani family. Three infertile males were subjected to semen analysis, hormone testing, testicular histology, ultrasonography, karyotyping, Y-chromosome microdeletion and CFTR testing. The clinical testing suggested a diagnosis of obstructive azoospermia (OA). To identify the cause, we performed whole exome sequencing (WES) for 2 infertile brothers and 2 fertile family members. For segregation analysis and variant confirmation, we performed Sanger sequencing. WES data analysis of the family revealed segregated variants in 3 candidate genes. We considered novel nonsense variant c.2440C?>?T(p.Arg814*) in X-linked gene ADGRG2 as biologically most plausible. It is predicted to truncate the protein by 204 amino acids (aa) at a key transmembrane domain. Adgrg2-knockout male mice show sperm loss due to obstructive fluid stasis, while ADGRG2 mutations cause OA in the infertile male patients. Our analysis of testicular histology reveals secondary severe reduction of spermatogenesis, consistent with human and knockout mouse phenotypes. The ADGRG2 nonsense mutation is absent in the largest population databases, ExAC and gnomAD. Analysis of the novel nonsense mutation in extended family members confirmed co-segregation of the mutation with OA in all affected males. The likely pathogenic nature of the mutation is supported by its truncation effect on the transmembrane domain and distinctive ultrasound results. The study demonstrates effectiveness of WES in discovering a genetic cause of azoospermia.
Project description:BACKGROUND:Testis-expressed gene 11 (TEX11) is an X-linked gene and essential for meiotic recombination and chromosomal synapsis. TEX11 deficiency causes meiotic arrest and male infertility, and many TEX11 mutations have been found in azoospermic and infertile men. CASE PRESENTATION:This study reported one novel TEX11 mutation (2653G???T, in exon 29, GenBank accession number, NM_031276) in two brothers with azoospermia. This mutation was firstly screened out by whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon 29. Surprisingly, the same exonic missense mutation (W856C) was observed in two brothers but not in their mother. Histological analysis of testicular biopsy from both brothers revealed meiotic arrest and no post-meiotic round spermatids and mature spermatozoa were observed in the seminiferous tubules. TEX11 expression was observed strongly in spermatogonia and weakly in spermatocytes, but not in Sertoli cells and interstitial cells. CONCLUSIONS:We identified one novel TEX11 mutation in two brothers and summarized the literature regarding TEX11 mutations and male infertility. This study and previous literature indicate that TEX11 mutations are closely associated with male infertility, especially azoospermia, although auxiliary clinical analyses are needed to figure out the causes of male infertility.
Project description:In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.
Project description:<h4>Purpose</h4>It is estimated that 40-50% of infertility among human couples is due to male infertility. Azoospermia is estimated to occur in 1% of all men and to be the cause of 10-20% of male infertility. Genetic defects, including single gene effects, maybe cause of azoospermia in 20-30% of affected males. Here, we aim to identify the genetic cause of azoospermia in a man who is also affected by hereditary spastic paraplegia.<h4>Methods</h4>The proband was subjected to whole-exome sequencing, followed by a comprehensive in silico analysis to identify the azoospermia causative gene.<h4>Results</h4>A novel splice site mutation c.375-2A?>?G in SYCE1 that is thought to be the cause of azoospermia was identified. This variant co-segregated with azoospermia status in the family that has three additional affected males.<h4>Conclusion</h4>SYCE1 gene encodes synaptonemal complex (SC) central element 1 protein which contributes to the formation of the synaptonemal complex during meiosis. Syce1 null male and female mice have been shown to be infertile. There have only been two reports on the effects of SYCE1 mutations in humans; it was shown as the cause of primary ovarian failure (POI) in one and as the cause of nonobstructive azoospermia (NOA) in another. We suggest that the mutation 375-2A?>?G, which affects the acceptor splice site within intron 6 of SYCE1, is the likely cause of azoospermia and subsequent infertility in the family studied. The finding constitutes the third report of SYCE1mutations that affect infertility in humans and further supports its contribution to this condition.
Project description:There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility. Using a candidate gene approach based on genetics of male infertility in mice, we resequenced nine autosomal genes from 78 infertile men displaying testicular failure using custom-made next-generation resequencing chips. Analysis of the data revealed several novel heterozygous nonsynonymous SNPs in four of nine sequenced genes in 14 of 78 infertile men. Eight SNPs in SBF1, three SNPs in LIMK2, two SNPs in LIPE, and one SNP in TBPL1 were identified. All of the novel mutations were in a heterozygous configuration, suggesting that they may be de novo mutations with dominant negative properties.
Project description:To investigate the genetic cause of nonobstructive azoospermia (NOA) in a consanguineous Turkish family through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations.Whole-exome sequencing (WES).Research laboratory.Two siblings in a consanguineous family with NOA.Validating all variants passing filter criteria with Sanger sequencing to confirm familial segregation and absence in the control population.Discovery of a mutation that could potentially cause NOA.A novel nonsynonymous mutation in the neuronal PAS-2 domain (NPAS2) was identified in a consanguineous family from Turkey. This mutation in exon 14 (chr2: 101592000 C>G) of NPAS2 is likely a disease-causing mutation as it is predicted to be damaging, it is a novel variant, and it segregates with the disease. Family segregation of the variants showed the presence of the homozygous mutation in the three brothers with NOA and a heterozygous mutation in the mother as well as one brother and one sister who were both fertile. The mutation is not found in the single-nucleotide polymorphism database, the 1000 Genomes Project, the Baylor College of Medicine cohort of 500 Turkish patients (not a population-specific polymorphism), or the matching 50 fertile controls.With the use of WES we identified a novel homozygous mutation in NPAS2 as a likely disease-causing variant in a Turkish family diagnosed with NOA. Our data reinforce the clinical role of WES in the molecular diagnosis of highly heterogeneous genetic diseases for which conventional genetic approaches have previously failed to find a molecular diagnosis.
Project description:Basonuclin (BNC1) is expressed primarily in proliferative keratinocytes and gametogenic cells. However, its roles in spermatogenesis and testicular aging were not clear. Previously we discovered a heterozygous BNC1 truncation mutation in a premature ovarian insufficiency pedigree. In this study, we found that male mice carrying the truncation mutation exhibited progressively fertility loss and testicular premature aging. Genome-wide expression profiling and direct binding studies (by chromatin immunoprecipitation sequencing) with BNC1 in mouse testis identified several spermatogenesis-specific gene promoters targeted by BNC1 including kelch-like family member 10 (Klhl10), testis expressed 14 (Tex14), and spermatogenesis and centriole associated 1 (Spatc1). Moreover, biochemical analysis showed that BNC1 was associated with TATA-box binding protein-associated factor 7 like (TAF7L), a germ cell-specific paralogue of the transcription factor IID subunit TAF7, both in vitro and in testis, suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis. The truncation mutation disabled nuclear translocation of the BNC1/TAF7L complex, thus, disturbing expression of related genes and leading to testicular premature aging. Similarly, expressions of BNC1, TAF7L, Y-box-binding protein 2 (YBX2), outer dense fiber of sperm tails 1 (ODF1), and glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS) were significantly decreased in the testis of men with non-obstructive azoospermia. The present study adds to the understanding of the physiology of male reproductive aging and the mechanism of spermatogenic failure in infertile men.
Project description:Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs?7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701?; rs147021911) and another from Portugal (p.Arg1931?; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.
Project description:BACKGROUND:Non-obstructive azoospermia (NOA), a serious phenotype of male spermatogenesis failure, is a multifactorial disease which is regulated by genetic, epigenetic, and environmental factors. Some gene structural variants have been demonstrated to be related to NOA. Loss-of-function mutations of KISS1R cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) which result in azoospermia at the pre-testicular level. The objective of this research was to investigate genetic variants of KISS1R in NOA patients. METHODS:The entire coding region of 52 spermatogenesis-associated genes (KISS1R included) was sequenced from 200 NOA patients. Mutation screening was performed to identify genetic variations of these genes by targeted exome sequencing. Sequencing data analysis was carried out by a series of bioinformatics tools. Candidate variants confirmation was performed by Sanger sequencing. Functional analysis of candidate variants was evaluated using SIFT and PolyPhen-2. RESULTS:Three heterozygous missense variants in KISS1R were identified in three patients, respectively. No deleterious variations in other candidate genes were found in the three patients. Two of these three variants, p.A211T and p.G186E, had been reported in the ExAC and dbSNP database, respectively, while the other variant p.A301D was novel. These variants were all predicted to be likely pathogenic by in silico analysis. CONCLUSION:Our study revealed three heterozygous missense variants in KISS1R which expanded the mutation spectrum of KISS1R in infertile men with NOA in the northeast of China.
Project description:BACKGROUND:The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS:We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS:We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS:In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).
Project description:BACKGROUND AND PURPOSE:The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. Mutations in USP26 gene have been proposed to be associated with male infertility. Moreover, the importance of the ubiquitin pathway during different stages of mammalian fertilization and even embryo development has been addressed. Some mutations and haplotypes on this gene have been proposed to be associated with male infertility. In this study, five different mutations on USP26 were investigated: 1737 G > A, 1090 C > T, 370-371ins ACA, 494 T > C and 1423 C > T. METHODS:The study included 166 infertile men with non-obstructive azoospermia, 72 male partners of couples who had previously experienced ≥3 clinical first trimester spontaneous abortions and 60 fertile men. Besides family history of reproduction, hormonal evaluation and semen analysis were performed. DNA was extracted from blood samples. PCR-SSCP, PCR-RFLP and PCR Product Cloning methods were used and resumed by sequencing to insure about the mutations. Moreover, USP26 gene expression was studied by Real-Time PCR after RNA extraction followed by cDNA synthesis from 24 testis biopsies in obstructive and non-obstructive azoospermia patients. RESULTS:The results indicate that there is a haplotype between three observed mutations in Iranian population include: 370-371insACA, 1423C > T and 494 T > C. This haplotype was seen in control group as well. Surprisingly, total frequency of mutations in men with history of idiopathic RPL and azoospermic cases were significantly higher than that of in control groups (p < 0.05). Serum testosterone concentrations and testicular volume did not differ in the mutation positive group compared with the non-mutation group. About the USP26 gene expression, there is a significant difference between the expression levels of obstructive azoospermia, complete maturation arrest samples and SCO samples (P < 0.05). CONCLUSIONS:According to our results, the USP26 gene may play an important role in male reproduction. The alterations of this gene may be involved in male infertility and RPL in Iranian population and may negatively affect testicular function.