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).

Table 1: Primers and PCR conditions used for TYK2 genotyping.










  


    
S.no


    
Gene


    
Primers


    
PCR    conditions


      (35    cycles)


    
Amplicon


      Size


  


  


    
1


    
TYK2


      
 


    
F:5'TCACCAGGCACTTGTTGTCC 3'


      
R:5'CGGCTTCCAGCATGTGTATG3'


    
5 min at 95°C, 40 sec at 94°C, 30 sec at 59°C,50 sec at 72°C


      and 10min72°C.


    
385


  


  


    
PCR*: Polymerase Chain    Reaction 


  


















Table 1: Primers and PCR conditions used for TYK2 genotyping.

Figure 1: Restriction digestion of TYK2 polymorphism rs2304256 (C/A) by Bsm I.


    


    

    


    


    Figure 1: Restriction digestion of TYK2 polymorphism rs2304256 (C/A) by Bsm I.

Immunohistochemical Analysis

Immunohistochemical analysis of TYK2 expression was performed on the proliferative phase endometrial tissue sections from endometriosis patients (n=5) and healthy controls (n=4), using the method described by Bhanoori et al. (2008) [4]. After the incubation of sections with rabbit polyclonal antibody against TYK2 (1:1000, Cell signaling technology, USA) and FITC-conjugated goat anti-rabbit secondary antibody (1:1000, Sigma–Aldrich, USA), they were washed and mounted with an anti-fade mounting medium (Vector Lab, USA) then visualized using an Axioplan 2 epifluorescence microscope (Carl Zeiss, Inc., USA). Negative controls were obtained by substituting TYK2 antibody with the non-immune rabbit serum.

Statistical Analysis

Statistical analysis was performed as per the methods described in our previous study [33].

Results

All subjects (n=300) were successfully genotyped. The genotype distributions of individual SNPs, as well as allele system, were all in Hardy-Weinberg equilibrium (P<0.05) in both the cases and controls. The results were analyzed in a blinded fashion.

Genotyping of TYK2 (rs2304256C/A) Polymorphism

Sequence analysis of TYK2rs2304256 non-synonymous SNP is shown in Figure 2. The homozygotes (C/C) and (A/A) manifested as a single peak, whereas heterozygote (C/A) as double peaks. The genotype and allele (P=0.0432) distribution of TYK2 rs2304256 C/A polymorphism showed significant difference between cases and controls according to codominant, dominant and recessive model (P< 0.05; Table2). The frequencies of allele ’A’ and genotype AA in patients with endometriosis were significantly higher than those in controls, suggesting that allele ‘A’ and genotype AA are associated with endometriosis.

Table 2: Genotype and allele frequencies of TYK2 gene polymorphism in endometriosis cases and controls.










  


    
Genotypes/


      Alleles


    
Cases


      (n=150)


    
Controls


      (n    = 150)


    
P-value


    
Odds    ratio


    
95%    CI


  


  


    
 


      
TYK2


    
 


    
 


    
 


    
 


    
 


  


  


    
rs2304256/


      Genotypes


      Codominant model


    
 


    
 


    
 


    
 


    
 


  


  


    
CC


    
57


    
71


    
-


    
Reference


    
Reference


  


  


    
CA


    
80


    
75


    
0.2353


    
1.3287


    
0.8306-2.1256


  


  


    
AA


      
Recessive model


        AA


        CA+CC


      
Dominant model


        CA+AA


        CC


    
13


      
 


      
13


        137


      
 


      
93


        57


    
4


      
 


      
4


        146


      
 


      
79


        71


    
0.01328


      
 


      
0.02461


      
 


      
0.10219


    
4.0482


      
 


      
3.4635


      
 


      
1.4664


    
1.2519-13.0907


      
 


      
1.1025-10.8802


      
 


      
0.9259-2.3225


  


  


    
Alleles


    
 


    
 


    
 


    
 


    
 


  


  


    
C


    
194


    
217


    
 


    
Reference


    
Reference


  


  


    
A


    
106


    
83


    
0.0432


    
0.5901


    
0.3988-0.8731


  


  


    
CI: Confidence Interval. 


  


















Table 2: Genotype and allele frequencies of TYK2 gene polymorphism in endometriosis cases and controls.

Figure 2: Genotyping of TYK2 gene polymorphism rs2304256 by sequence analysis of the PCR amplifi ed product using a forward primer.


    


    

    


    


    Figure 2: Genotyping of TYK2 gene polymorphism rs2304256 by sequence analysis of the PCR amplifi ed product using a forward primer.

Immunohistochemistry of TYK2

Immunostaining of TYK2 was observed in both glandular epithelial and stromal cells of endometrium. In the endometria of women with and without endometriosis, we observed no significant difference in the expression of TYK2 (Figure 3).

Figure 3: Immunohistochemical analysis of TYK 2 expression in phase-matched (proliferative) endometrium from case (A) and control (B).


    


    

    


    


    Figure 3: Immunohistochemical analysis of TYK 2 expression in phase-matched (proliferative) endometrium from case (A) and control (B).

Discussion

Endometriosis is a polygenically inherited and multifactorial disease. A variety of genetic association studies have focused on the association between cytokine (TNF-a, IL-2, IL-4, IL-6, IL-10, or IL-16) gene polymorphisms and endometriosis risk [24,25,36,37,38]. However, the studies on the down-stream signaling molecules like JAKs and STATs are very few. It is evident from the literature that the Th2 immune response is associated with endometriosis. [1]. Interleukin IL13, a typical Th2 cytokine is the key regulator of inflammatory and immune responses, that is central to endometriosis and associated abnormalities [8]. Elevated mRNA and protein levels of IL-13 was reported in the peritoneal fluid of endometriosis patients [35]. The same changes are observed in the ectopic endometrium of affected women which could be responsible for the defective immunosurveillance leading to endometriotic tissue overgrowth [8]. TYK2 is required for mediating the biological function of IL-13 in processes associated with Th2 immune response [5,21]. IL-13/TYK2 signaling in B cell proliferation, Ig E and MHC class II expression and Th1-cytokines inhibition are well demonstrated in endometriosis [29]. An aberration in TYK2 can lead to abnormal STAT6 signaling, gaining resistance to apoptosis and escaping the immune surveillance [6,20]. Therefore, TYK2gene may have the crucial importance in regulating immune and/or inflammatory responses in endometriosis.

In the current study, SNP in TYK2 rs2304256 was examined to ascertain whether the polymorphism is associated with endometriosis susceptibility in women from South Indian. The non-synonymous SNP rs2304256, a C to An alteration in exon 8 of TYK2, induces a change of valine to phenylalanine at position 362 in the JAK-homology 4 region of TYK2 [30]. Li et al showed that the allele ‘A’ disrupts a putative exonic splicing enhancer binding motif affecting the pre-mRNA processing of TYK2; thus, promoting the inclusion of exon 8 in the mRNA, which is essential for TYK2 binding to cytokine receptor [23]. At the rs2304256 locus, we observed that the allele ‘A’ and genotype AA occurred at significantly higher frequency in cases than in controls, indicating that the mutant allele 'A' could be a genetic risk factor for endometriosis. Of note, Peluso et al found no correlation between the SNP rs2304256 and endometriosis risk in the Brazilian population [27], which is inconsistent with the present finding. Possible reasons for this inconsistency could be: first, ethnic variation seen in the polymorphism analyzed. In point of fact, drastic difference was observed between the two populations in the distribution of the 'A' allele. In the Brazilian population, the mutant allele 'A' was found in 26.4 percent of cases and 23.6 percent of controls, whereas in the South Indian population, the frequencies were 35.3 percent and 27.6 lymphopercent in the current study. The second possible reason could be due to the difference in the sample size used in the study. However, larger population studies may require validating the exact role of this mutation in endometriosis.

The expression of TYK2 in the eutopic endometrium of women with and without endometriosis did not differ significantly in our study. Although there are conflicting results on TYK2 expression in various cancers, the majority of research found higher expression in tumor tissues when compared to controls [18,19]. Additionally, a strong correlation between TYK2 over-expression and late-stage tumors was observed, indicating that TYK2 over-expression contributes to the aggressiveness of tumors. However, we found no significant difference in TYK2 expression between eutopic endometria of women with and without endometriosis in the current study. To validate the role of TYK2 expression in the pathophysiology of endometriosis, more research with a bigger sample size is needed.

The minor allelic frequency for the SNP evaluated was compared with the mutation frequency data from populations of different ethnic origins, obtained from HapMap, 1000Genomes, Genome Aggregation Database (GnomAD) and EXAC database (dbSNP) (Supplementary Table 1). In the cases with the polymorphism under study, we observed that the minor allelic frequency was close to the values reported for Asian populations in the 1000 Genome Database. The frequency of 'A' allele is more common in Asians, Europeans, and Americans than it is in Africans for the TYK2 gene polymorphism rs2304256. Since Indians are a part of Asian ethnic group, the allelic frequency for the SNP rs2304256 found in the cases were very close with the Asians represented in 1000 Genomes, however, further confirmation requires studies with large sample sizes.

JAK-STAT signaling mediates immune regulatory processes that are central in endometriosis development and progression. We believe that the combined effect of various JAK-STAT mediator polymorphisms may disrupt immune homeostasis leading to the establishment and maintenance of endometrial cells at ectopic locations, as illustrated previously by our team considering IL6 and STAT6 [2,3]. The Discovery of how these genetic polymorphisms interact on endometriosis development may be a crucial step in understanding the pathophysiology of endometriosis.

In conclusion, our study shows that the TYK2 gene polymorphism rs2304256 is significantly associated with endometriosis in South Indian women. Analysis of this polymorphism might help to identify patients at high risk for endometriosis development. Although further work is necessary to understand the molecular mechanism, our findings may lead to new insights into the disease pathogenesis.

Author Statements

Acknowledgements

This work was supported in part by grants from the SERB (DST), India (Lr. No: SR/FT/LS-188/2009) and OU-DST PURSE Programme-II (DST Sanction No. SR/PURSE Phase 2/32(G)), Department of Science and Technology (DST), India to Prof. Manjula Bhanoori.

Conflict of Interests

The authors declare that there is no conflict of interests.

Ethical Approval

The study was approved by the ethical committee and review board of Centre of Cellular and Molecular biology (CCMB), Hyderabad. In the study all the participants were of South Indian origin (Dravidian linguistic group).

Informed Consent

Informed written consent form was obtained from all subjects prior to participation in this study.

Author’s Contribution

KVV: execution of experiments, analysis and interpretation of data, statistical analysis and drafting of manuscript. MLM: Data collection. MD: acquisition of data. SS: analysis and interpretation of data, drafting of manuscript. MB: conception and design of study, analysis and interpretation of data, statistical analysis, drafting of manuscript. All authors will have seen and agreed to the ‘Author Contribution’ statement.

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Citation: KV Veena, Manolla ML, Deenadayal M, Shivaji S, Bhanoori M. The rs2304256, a Non-Synonymous Polymorphism in Tyrosine Kinase 2 Gene is Associated with the Risk of Endometriosis. Austin J Obstet Gynecol. 2023; 10(2): 1216.