OBJECTIVE: In this study, we aimed to identify sterility-related variants in a Chinese pedigree with male infertility and to reveal the different phenotypes and intracytoplasmic sperm injection (ICSI) outcomes of the affected members.
METHODS: Physical examinations were performed on male patients. G-band karyotype analysis, copy number variation sequencing, and quantitative fluorescent PCR were conducted to detect common chromosomal disorders in the probands. Whole-exome sequencing and Sanger sequencing were applied to identify the pathogenic genes and the protein expression changes caused by the very mutation were identified by Western Blot in vitro.
RESULTS: A novel nonsense mutation (c.908C > G: p.S303*) in the ADGRG2 was identified in all infertile male patients of the pedigree, which was inherited from their mothers. This variant was absent from the human genome databases. This mutation was also unexpectedly found in a male member with normal reproductive capability. Members with the mutation had different genitalia phenotypes, ranging from normal to dilated phenotypes of the vas deferens, spermatic veins and epididymis. There was a truncated ADGRG2 protein in vitro after mutation. Of the three patients\' wives treated with ICSI, only one successfully gave birth.
CONCLUSIONS: Our study is the first to report the c.908C > G: p.S303* mutation in the ADGRG2 in an X-linked azoospermia pedigree and is the first to report normal fertility in a member with this mutation, expanding the mutation spectrum and phenotype spectrum of this gene. In our study, ISCI had a success rate of only one-third in couples including men with azoospermia with this mutation.

Congenital bilateral absence of the vas deferens (CBAVD) is clinically characterized by the absence of the bilateral vas deferens; the main clinical manifestation is infertility, accounting for 1-2% of male infertility cases. CBAVD may be accompanied by congenital abnormalities in the urogenital system and cystic fibrosis (CF)-related clinical manifestations. CBAVD can develop as a mild manifestation of CF or can be isolated. The main pathogenic mechanism of CBAVD is gene mutation, and CBAVD and CF have a common genetic mutation background. CFTR mutation is the main pathogenic cause of CBAVD and CF, and ADGRG2 mutation is the second most common cause. Although lack of the vas deferens in CBAVD patients causes infertility due to the inability to release sperm, the testes of CBAVD patients have spermatogenic function. Therefore, CBAVD patients can achieve fertility through sperm retrieval surgery and assisted reproductive technology (ART). However, gene mutations in CBAVD patients can have an impact on the ART outcome, and there is a risk of passing on gene mutations to offspring. For CBAVD patients and their spouses, performing genetic counseling (which currently refers mainly to CFTR mutation screening) helps to reduce the risks of genetic mutations being passed on to offspring and of offspring having CF with concomitant CBAVD.

OBJECTIVE: Congenital bilateral absence of the vas deferens (CBAVD) is a major cause of obstructive azoospermia and male factor infertility. CBAVD is mainly caused by mutations in the genes encoding CFTR (cystic fibrosis transmembrane conductance regulator) and ADGRG2 (adhesion G protein-coupled receptor G2). This study aimed to describe CFTR and ADGRG2 variations in 46 Chinese CBAVD patients and evaluated sperm retrieval and assisted reproductive technology outcomes.
METHODS: The CFTR and ADGRG2 genes were sequenced and analyzed by whole-exome sequencing (WES), and variations were identified by Sanger sequencing. Bioinformatic analysis was performed. We retrospectively reviewed the outcomes of patients undergoing sperm retrieval surgery and intracytoplasmic sperm injection (ICSI).
RESULTS: In total, 35 of 46 (76.09%) patients carried at least one variation in CFTR, but no copy number variants or ADGRG2 variations were found. In addition to the IVS9-5 T allele, there were 27 CFTR variations, of which 4 variations were novel and predicted to be damaging by bioinformatics. Spermatozoa were successfully retrachieved in 46 patients, and 39 of the patients had their own offspring through ICSI.
CONCLUSIONS: There are no obvious hotspot CFTR mutations in Chinese CBAVD patients besides the IVS9-5 T allele. Therefore, WES might be the best detection method, and genetic counseling should be different from that provided to Caucasian populations. After proper counseling, all patients can undergo sperm retrieval from their epididymis or testis, and most of them can have their own children through ICSI.

OBJECTIVE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients.
METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed.
RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI.
CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.

OBJECTIVE: Congenital aplasia of vas deferens (CAVD) is an atypical form of cystic fibrosis (CF) and causes obstructive azoospermia and male infertility. Compound heterozygous variants of CFTR are the main cause of CAVD. However, most evidence comes from genetic screening of sporadic cases and little is from pedigree analysis. In this study, we performed analysis in a Chinese pedigree with two CAVD patients in order to determine the genetic cause of this familial disorder.
METHODS: In the present study, we performed whole-exome sequencing and co-segregation analysis in a Chinese pedigree involving two patients diagnosed with CAVD.
RESULTS: We identified a rare frameshift variant (NM_000492.3: c.50dupT;p.S18Qfs*27) and a frequent CBAVD-causing variant (IVS9-TG13-5T) in both patients. The frameshift variant introduced a premature termination codon and was not found in any public databases or reported in the literature. Co-segregation analysis confirmed these two variants were in compound heterozygous state. The other male members, who harbored the frameshift variant and benign IVS9-7T allele, did not have any typical clinical manifestations of CF or CAVD.
CONCLUSIONS: Our findings may broaden the mutation spectrum of CFTR in CAVD patients and provide more familial evidence that the combination of a mild variant and a severe variant in trans of CFTR can cause vas deferens malformation.

Congenital bilateral absence of vas deferens (CBAVD), a frequent cause of obstructive azoospermia and male infertility in Chinese, is mainly due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study aim to explore the promoter region of CFTR gene in CBAVD patients and study the mutations by functional analysis, and to discuss the significance of mutation testing in this area. We performed screening analysis on 65 CBAVD patients and 50 controls to detect mutations in the CFTR gene, and studied the functions of promoter mutations using reporter gene constructs, transient transfection techniques and subsequent assessment of transcriptional activity and expression levels. Mutations c.-195C>A and c.-34C>T in the promoter region of the CFTR gene were detected in 4 of our Chinese CBAVD patients, one of which was novel (c.-195C>A) and located in the conservative area, as well as the binding site of SP1 transcription factor through the prediction of bioinformatics analysis. By reverse transcription qPCR assay and luciferase assay, we validated it as a functional disease-causing variant that down-regulates the CFTR gene expression, and this effect was related to the amount of transcription factors. This study was the first to explore the promoter region of the CFTR gene in Chinese, and we believe that mutations in this region are associated with Chinese CBAVD patients. We also suggest a systematic strategy for genotyping Chinese CBAVD couples, which should help in developing reproductive counseling.