[Detection of pathogenic gene mutations in thirteen cases of congenital bilateral absence of vas deferens infertility patients].

OBJECTIVE

To detect the cystic fibrosis transmembrane transduction regulator () gene mutations and congenital bilateral absence of vas deferens (CBAVD) susceptibility gene mutations in patients with CBAVD, and to explore their association with the risk of CBAVD.

METHODS

Whole-exome sequencing and Sanger sequencing validation were conducted on the pathogenic genes , adhesion G protein-coupled receptor G2 (), sodium channel epithelial 1 subunit beta (), carbonic anhydrase 12 (), and solute carrier family 9 member A3 () in thirteen cases of isolated CBAVD patients. The polymorphic loci, intron and flanking sequences of gene were amplified by polymerase chain reaction (PCR) followed by Sanger sequencing. Bioinformatics methods were employed for conservative analysis and deleterious prediction of novel susceptibility gene mutations in CBAVD. Genetic analysis was performed on the pedigree of one out of thirteen patients with CBAVD to evaluate the risk of inheritance in offspring.

RESULTS

Exome sequencing revealed gene exon mutations in only six of the thirteen CBAVD patients, with six missense mutations c.2684G>A(p.Ser895Asn), c.4056G>C(p.Gln1352His), c.2812G>(p.Val938Leu), c.3068T>G(p.Ile1023Arg), c.374T>C(p.Ile125Thr), c.1666A>G(p.Ile556Val)), and one nonsense mutation (c.1657C>T(p.Arg553Ter). Among these six patients, two also had the homozygous p.V470 site, additionally, mutations in gene exon regions were not detected in the remaining seven patients. Within the thirteen CBAVD patients, three carried the homozygous p.V470 polymorphic site, four carried the 5T allele, two carried the TG13 allele, and ten carried the c.-966T>G site. Four CBAVD patients simultaneously carried 2-3 of the aforementioned gene mutation sites. Susceptibility gene mutations in CBAVD among the thirteen patients included one missense mutation c.2312A>G(p.Asn771Ser), two SLC9A3 missense mutations c.2395T>C(p.Cys799Arg), c.493G>A(p.Val165Ile), one missense mutation c.1514G>A(p.Arg505His), and one missense mutation c.1061C>T (p.Ala354Val). Notably, the SLC9A3 gene c.493G>A (p.Val165Ile) mutation site was first identified in CBAVD patients. The five mutations exhibited an extremely low population mutation frequency in the gnomAD database, classifying them as rare mutations. Predictions from Mutation Taster and Polyphen-2 software indicated that the harmfulness level of the SLC9A3 gene c.493G>A (p.Val165Ile) site and the gene c.1514G>A (p.Arg505His) site were disease causing and probably damaging. The genetic analysis of one pedigree revealed that the c.1657C>T (p.Arg553Ter) mutation in the proband was a de novo mutation, as neither the proband's father nor mother carried this mutation. The proband and his spouse conceived a daughter through assisted reproductive technology, and the daughter inherited the proband's pathogenic mutation c.1657C>T (p.Arg553Ter).

CONCLUSION

gene mutations remain the leading cause of CBAVD in Chinese patients; however, the distribution and frequency of mutations differ from data reported in other domestic and international studies, highlighting the need to expand the mutation spectrum in Chinese CBAVD patients. The susceptibility genes , , , and may explain some cases of CBAVD without mutations. Given the lack of specific clinical manifestations in CBAVD patients, it is recommended that clinicians conduct further physical examinations and consider scrotal or transrectal ultrasound before making a defi-nitive diagnosis. It is advisable to employ gene mutation testing in preconception genetic screening to reduce the risk of CBAVD and cystic fibrosis in offspring.