P-105 Genetic variants in the spermatogenic transcription regulators RFX2 and TAF7 enhance the incidence of azoospermia
Authors: Pal S 
Published: June 2022

Abstract

Study question
What are the genetic variants of meiotic regulator genes that are implicated in male infertility among men from West Bengal, India?

Summary answer
TAF7 C16T (MW827584 G &gt; A) and RFX2 562delT (MZ560629delA) new variations may have a major impact on optimal fertility in men from West Bengal.

What is known already
According to published research, roughly 50% of reported infertility cases in India are due to male reproductive abnormalities. Because of the involvement of several gene regulatory networks in the spermatogenesis process, the genetic etiology of idiopathic male infertility remains a mystery. The AZF region of the Y chromosome microdeletion has been discovered as a prevalent cause of male infertility across all ethnicities. Infertility in men has also been linked to sex chromosomal copy number variation and autosomal gene polymorphisms.

Study design, size, duration
This is a case-control study to infer whether the genetic variants are significantly associated in favor of cases or in favor of controls. The study included 160 healthy male controls and 150 azoospermic cases submitted to the Institute of Reproductive Medicine in Kolkata (IRM). All of the subjects agreed to participate in the study and provided blood and sperm samples. The duration of the study is three years. Seminograms were made to filter azoospermic individuals.

Participants/materials, setting, methods
2 ml of venous blood samples were taken from azoospermic men using the venipuncture procedure and stored in EDTA coated vacutainer tubes at -20°C for DNA isolation.QIAamp Blood Mini Kit was used to isolate genomic DNA. For PCR analysis, unique primers for TAF7 and RFX2 functional domains were created. Taq Dye Deoxy Terminator sequencing kit (Applied Biosystems, Foster City, USA) and ABI Prism 377 DNA sequencer were used to analyze PCR results.

Main results and the role of chance
We have found two significant genetic alterations in the coding region of TAF7 and RFX2 which are MW827584 G &gt; A(C16T) and MZ560629delA(562delT) respectively. Both were found to have a strong association to the occurrence of azoospermia. For MW827584 G &gt; A, minor allele ‘A’ has shown elevated risk of azoospermic against the odd 4.684 (95% C.I = 2.900-7.564, Relative risk =2.110, p value = &lt;0.001). Fisher’s exact test was performed to calculate Odds ratios with respective 95% confidence interval (CI). After Bonferroni's correction, a two-tailed P-value of less than 0.02 was considered statistically significant throughout. Only the case sample cohort had the RFX2 deletion MZ560629 delA. Only 9 cases with this unique deletion in heterozygous form were found out of 150 azoospermic patients. This frameshift mutation results in the production of an early termination signal at exon 6, resulting in the creation of a truncated protein. The wild-type RFX2 protein has 729 amino acids but this frameshift mutation results in a 212-amino-acid RFX2 protein that lacks several secondary motifs and domains, rendering the wild-type function useless. In silico analysis of SIFT, PROVEAN, MUTATION TASTER and REGULATION SPOTTER predicted that these genetic variants are disease-causing.

Limitations, reasons for caution
There are some potential limitations to our research. A larger sample size investigation is needed to reconfirm the genetic association study. Moreover, confounding effects of allelic variation of those genetic modifiers may cause differences in the manifestation of fertility state along with the allelic variants of TAF7 and RFX2 genes.

Wider implications of the findings
This diagnostic technique will not only reduce the hazards connected with assisted reproductive technologies (ART), but it will also provide insight into previously unknown areas of infertility and enable clinicians in conducting a more thorough investigation of the patients.

Trial registration number
not applicable