The rs2304256, a Non-Synonymous Polymorphism in Tyrosine Kinase 2 Gene is Associated with the Risk of Endometriosis

Research Article

Austin J Obstet Gynecol. 2023; 10(2): 1216.

The rs2304256, a Non-Synonymous Polymorphism in Tyrosine Kinase 2 Gene is Associated with the Risk of Endometriosis

KV Veena¹, Madhavi Latha Manolla¹, Mamata Deenadayal², Sisinthy Shivaji3,4 and Manjula Bhanoori¹*

1Department of Biochemistry, Osmania University, India

2Infertility Institute and Research Centre (IIRC), India

3Centre for Cellular and Molecular Biology (CCMB), India

4Presently at: Director of Research, Brien Holden Eye Research Centre, L V Prasad Eye Institute, India

*Corresponding author: Manjula BhanooriProfessor, Department of Biochemistry, Osmania University, 500007, Hyderabad, India Email: bhanoorim@yahoo.co.in bhanoori@osmania.ac.in

Received: February 02, 2023 Accepted: March 27, 2023 Published: April 03, 2023

Abstract

The objective of the study was to investigate whether the TYK2 gene influences the risk of developing endometriosis in South Indian women. The non-synonymous SNP, rs2304256, in exon8 of the TYK2 gene was tested for association in a case–control study of 150 affected women and 150 women with no evidence of disease. The genotype frequencies of the polymorphism were compared using polymerase chain reaction and restriction fragment length polymorphism. Immunohistochemistry was used to analyze the distribution and expression of TYK2 in the endometrium of women with and without endometriosis. According to codominant, dominant, and recessive genotype models, statistically significant differences were observed in the genotype distribution and allele frequency (P=0.0432) between the cases and controls. The distribution and expression of TYK2 did not vary in the endometrium of cases and controls. In the present study, we could establish an association between the TYK2 rs2304256 non-synonymous polymorphism with the risk of endometriosis in South Indian women, indicating that this polymorphism may lead to significant disease susceptibility.

Keywords: Endometriosis; Tyrosine kinase 2; Polymorphism; South Indian women

Abbreviations: CI: Confidence Interval; Χ²: Chi Square; D': Disequilibrium Coefficient; LD: Linkage Disequilibrium; HWE: Hardy–Weinberg Equilibrium; OR: Odds Ratio; TYK2: Tyrosine Kinase 2; IL: Interleukin; STAT: Signal Transducer and Activator of Transcription; PCR: Polymerase Chain Reaction; SNPs: Single Nucleotide Polymorphisms

Introduction

Endometriosis is a chronic, endocrine gynecologic disease characterized by the implantation of functional endometrial tissue at ectopic positions. It is observed mainly in the pelvic area including the ovaries, peritoneal surfaces and ligaments including bowel and bladder [10]. It affects 10-15% of women in their reproductive age and is responsible for dysmenorrhea, dyspareunia, infertility and chronic pelvic pain. Retrograde menstrual invasion and implantation of endometrial stromal cells into the peritoneum are the widely accepted explanations for this condition [17]. Although retrograde menstruation is common in 70-90% of women, the much lower endometriosis prevalence suggests that there must be other variables that may contribute to endometriosis pathogenesis. We previously looked at the correlation between genetic variants in multiple candidate genes and endometriosis risk in Indian women [4,13-16,32,33], which suggested that the condition is polygenic and multifactorial [31].

A complex network of cytokines mediates the immunomodulatory mechanisms leading to pathogenesis of endometriosis [39]. An altered secretion of Th1 and Th2 specific cytokines have been implicated in the pathogenesis of the disease. In the peritoneal fluid of affected women, there has been a shift in the balance of Th1/Th2 cytokines toward the Th2 response, contributing to the derangement of immunologic defense mechanism [28]. Elevated levels of Th2-specific cytokines such as interleukin IL-4, IL-5, IL-10 and IL-13 impairs T-cell cytotoxicity, enhancing the endometrial cell implantation and growth at the extra uterine sites [11]. Tyrosine Kinase 2 (TYK2) is located on chromosome 19p13.2 [9] and is implicated in signaling from Th2 cells, for example, through IL-10 and IL-13 receptors activating STAT3 and STAT6 signaling pathway [21,34]. TYK2 is widely studied in the pathogenesis of several tumors because of its critical role in tumor immunosurveillance [20].

Earlier, few genetic association studies have been conducted on TYK2 locus to study its impact on several autoimmune diseases, however, the identification has been duplicated in a large number of recent analyses and TYK2 is now considered to be a molecular marker in a variety of autoimmune and inflammatory diseases [12]. A non-synonymous SNP of TYK2rs2304256, is widely studied for its association with diseases like systemic lupus erythematosus, Crohn’s disease, rheumatoid arthritis, type 1 diabetes mellitus, ulcerative colitis, etc. [7,22,26]. This study was undertaken to investigate the association of TYK2 rs2304256 polymorphism with the risk of endometriosis in South Indian women.

Materials and Methods

Study Population

The case-control study was carried out on three hundred women in their reproductive age, recruited at the Infertility Institute and Research Center (IIRC), Hyderabad, India. The study subjects were obtained as described earlier [33].

Tissue collection

The proliferative phase endometrial tissues were collected and fixed as per the method described by Bhanoori et al (2008) [4].

DNA Extraction

Salting out procedure was used to extract the genomic DNA from one milliliter of anticoagulated whole blood.

Molecular Analysis of TYK2

Genotyping of TYK2 gene polymorphism was carried out by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) and the results confirmed by Sanger sequencing method as described earlier [16]. The primers and PCR conditions are summarized in (Table 1). The PCR products of TYK2 gene (385 bp) was digested with restriction enzyme (BsmI at 65°C) for 3 h and the DNA fragments were electrophoresed through a 2% agarose gel, stained with ethidium bromide. For the TYK2BsmI C/A SNP, the A allele was represented by DNA band of size 251 and 134bp, the C allele was represented by DNA bands of sizes 385 bp; whereas, the heterozygotes displayed a combination of both alleles (385, 251 and 134bp) (Figure 1).