Association of TYK 2 rs 34536443 polymorphism with Susceptibility to Systemic Lupus Erythematous in the Iranian Population

*Corresponding Author: Mahdi Mahmoudi, Ph.D., Rheumatology Research Center, Shariati Hospital, Kargar Ave., Tehran, Iran. Email:mahmoudim@tums.ac.ir, Tel/Fax: (+98) 218-822-0067, PO-BOX: 1411713137, Received: 09 June 2018; Accepted: 26 August 2018 Introduction Systemic lupus erythematous (SLE) is a prototypic systemic autoimmune disease that is characterized by the production of multiple autoantibodies against self-antigens owing to a breakdown in self-tolerance [1]. One important characteristic of SLE is the high female to male ratio. The pathogenesis of SLE is complex and recent evidence suggests that its etiology encompasses both genetic risk factors and environmental factors [2]. Although the exact pathogenesis of SLE remains unclear, increased understanding of the underlying genetic background of SLE can help improve the prognosis for SLE. Several studies have pointed to the significance of the type I interferon (IFN) pathway in SLE pathogenesis [3-6]. SLE patients have been shown to have increased serum IFN-α levels; thus, the production of type I IFN may be important to the etiology of SLE [7]. The ligation of type I IFN with its receptor activates the IFN-α receptor (IFNAR), Janus kinases (JAK) 1 and tyrosine kinase (TYK) 2 and signal transcription factors such as the signal transducer and activator of transcription (STAT) [8]. TYK2 (MIM 176941; gene ID 7297), a large multidomain kinase, is the first member of JAK family, spans 27.9 kb nucleotides and comprises 25 exons. It is located on chromosome 19p13.2 in humans and on chromosome 9 in mice [9]. The role of TYK2 gene in the development of autoimmune and inflammatory diseases such as Crohn’s disease (CD) [10], endometriosis-related infertility (ERI) [11], multiple sclerosis (MS) [12], rheumatoid arthritis (RA) [13, 14], systemic sclerosis (SSc) [13], Behçet’s disease (BD) [15] and SLE [2, 9, 16-21] has been demonstrated. Several variants of TYK2 have been investigated [22]. One rare variant of the TYK2 gene, rs34536443 single nucleotide polymorphism (SNP) causes the substitution of G to a C nucleotide in TYK2, resulting in a Pro1104Ala (P1104A) variation in the TYK2 protein. It has been suggested that this alteration may cause a conformational change that may affect the folding and function of TYK2 protein [23, 24]. The associSystemic lupus erythematous (SLE) is a multifactorial autoimmune disorder which affects many organs and displays various symptoms. Genetic components contribute to the incidence and development of SLE. A rare functional variant within the tyrosine kinase 2 (TYK2) gene (rs34536443) is a common genetic candidate for several autoimmune diseases, including SLE. This case control study was performed to investigate the possible association of TYK2 single nucleotide polymorphism (SNP) with a predisposition for and clinical features of SLE in the Iranian population. Genotyping was conducted on 600 patients with SLE and 600 sex-, ageand ethnicity-matched control subjects from the Iranian population. Patient and control samples were genotyped for one SNP (rs34536443) by applying allelic discrimination real-time PCR. Statistical analysis of the allele distribution revealed no significant association (OR = 0.67, CI: 0.38-1.17, P value = 0.163) between the rs34536443 C allele and susceptibility to SLE. The CC genotype was not detected in either the patients or controls. Moreover, the CG genotypes showed no significant association with the risk of SLE (OR = 0.66, CI: 0.37-1.72, P value = 0.15). These findings suggest that TYK2 rs34536443 is not associated with SLE susceptibility in the Iranian population. Further investigation is required to examine the mechanisms by which polymorphisms in this gene lead to SLE development.


Introduction
Systemic lupus erythematous (SLE) is a prototypic systemic autoimmune disease that is characterized by the production of multiple autoantibodies against self-antigens owing to a breakdown in self-tolerance [1].One important characteristic of SLE is the high female to male ratio.The pathogenesis of SLE is complex and recent evidence suggests that its etiology encompasses both genetic risk factors and environmental factors [2].Although the exact pathogenesis of SLE remains unclear, increased understanding of the underlying genetic background of SLE can help improve the prognosis for SLE.
Several studies have pointed to the significance of the type I interferon (IFN) pathway in SLE pathogenesis [3][4][5][6].SLE patients have been shown to have increased serum IFN-α levels; thus, the production of type I IFN may be important to the etiology of SLE [7].The ligation of type I IFN with its receptor activates the IFN-α receptor (IF-NAR), Janus kinases (JAK) 1 and tyrosine kinase (TYK) 2 and signal transcription factors such as the signal transducer and activator of transcription (STAT) [8].TYK2 (MIM 176941; gene ID 7297), a large multidomain kinase, is the first member of JAK family, spans 27.9 kb nucleotides and comprises 25 exons.It is located on chromosome 19p13.2 in humans and on chromosome 9 in mice [9].
The role of TYK2 gene in the development of autoimmune and inflammatory diseases such as Crohn's disease (CD) [10], endometriosis-related infertility (ERI) [11], multiple sclerosis (MS) [12], rheumatoid arthritis (RA) [13,14], systemic sclerosis (SSc) [13], Behçet's disease (BD) [15] and SLE [2,9,[16][17][18][19][20][21] has been demonstrated.Several variants of TYK2 have been investigated [22].One rare variant of the TYK2 gene, rs34536443 single nucleotide polymorphism (SNP) causes the substitution of G to a C nucleotide in TYK2, resulting in a Pro1104Ala (P1104A) variation in the TYK2 protein.It has been suggested that this alteration may cause a conformational change that may affect the folding and function of TYK2 protein [23,24].The associ-Systemic lupus erythematous (SLE) is a multifactorial autoimmune disorder which affects many organs and displays various symptoms.Genetic components contribute to the incidence and development of SLE.A rare functional variant within the tyrosine kinase 2 (TYK2) gene (rs34536443) is a common genetic candidate for several autoimmune diseases, including SLE.This case control study was performed to investigate the possible association of TYK2 single nucleotide polymorphism (SNP) with a predisposition for and clinical features of SLE in the Iranian population.Genotyping was conducted on 600 patients with SLE and 600 sex-, age-and ethnicity-matched control subjects from the Iranian population.Patient and control samples were genotyped for one SNP (rs34536443) by applying allelic discrimination real-time PCR.Statistical analysis of the allele distribution revealed no significant association (OR = 0.67, CI: 0.38-1.17,P value = 0.163) between the rs34536443 C allele and susceptibility to SLE.The CC genotype was not detected in either the patients or controls.Moreover, the CG genotypes showed no significant association with the risk of SLE (OR = 0.66, CI: 0.37-1.72,P value = 0.15).These findings suggest that TYK2 rs34536443 is not associated with SLE susceptibility in the Iranian population.Further investigation is required to examine the mechanisms by which polymorphisms in this gene lead to SLE development.
Because of the involvement of TYK2 in the pathogenesis of some autoimmune diseases, the current study was undertaken to investigate the functional impact of one TYK2 disease-associated variant, SNP rs34536443, in SLE patients.The association of this polymorphism and clinical manifestations of SLE also were investigated.It is hoped that the results obtained from this study enhance understanding of the pathogenesis and therapeutic strategies for systemic autoimmune diseases, especially SLE.

Population and sample preparation
In the present case-control study, SLE patients who met American College of Rheumatology [30] criteria were enlisted from cases referring to the rheumatology clinic of Shariati Hospital in Tehran, Iran.Healthy subjects were matched with the case group with respect to age, sex, and ethnicity.This study was approved by the local ethical committee of Tehran University of Medical Sciences.Before blood sampling, written informed consent was obtained from each participant.Genotyping was conducted on the 600 SLE subjects.Table 1 shows the clinical data of 235 SLE patients (18 men with a mean age of 37.6 ± 13.6 years and 217 women with a mean age of 39.1 ± 11.4 years).The control group comprised 600 healthy matched controls with no clinical evidence or history of any type of autoimmune disease.

Sampling and genotyping
About 5 ml of whole blood in EDTA anticoagulant were gathered by means of venipuncture under sterile conditions from all participants.Extraction of genomic DNA was performed on peripheral blood leukocytes using a standardized phenol-chloroform technique [31].The purity of the isolated genomic DNA was quantified spectrophotometrically using Nanodrop 2000c (Thermo Fisher Scientific; USA) and showed that the A260/A280 ratio extended from 1.7 to 1.9, indicating good quality DNA.The samples were stored at -20°C until analysis.For DNA genotyping, approximately 10 ng of the genomic DNA was used.Genotyping of TYK2 (rs34536443) was performed by the MGB TaqMan allelic discrimination method (Applied Biosystems; USA).All DNA samples were genotyped for rs34536443 by StepOnePlus real-time PCR (Applied Biosystems) according to manufacturer instructions.

Statistical analysis
Pearson's chi-square test was applied to assess the association of alleles and genotypes with SLE as well as to evaluate the relations of genotypes with the clinical data of the patients.All P values were two-tailed.The odds ratio (OR) and 95% confidence interval (CI) were also estimated for each allele and genotype.The Benjamini-Hochberg correction method was applied to control for a false discovery rate (FDR) in multiple comparisons [32].All the statistical analyses were carried out using SPSS for Windows (version 22.0; IBM; USA).SHEsis online software was used for genotype analysis, linkage disequilibrium and the Hardy-Weinberg equilibrium [33].

Results
One SNP (rs34536443) in 600 SLE individuals and 600 unrelated healthy controls was genotyped.Table 1 presents the clinical specifications of 235 SLE patients.

Allele frequency
In the NCBI SNP tool, the G allele is used as the reference allele; thus, the frequency of the C allele was reported (https://www.ncbi.nlm.nih.gov/snp/?term=rs34536443).As shown in Table 2, the frequency of the C allele for rs34536443 SNP was 1.70% in the SLE group, which is same as what was observed in the control group (2.61%).No significant difference was detected in the allelic distribution of rs34536443 variant between the SLE and control groups (OR = 0.67, CI: 0.38-1.17,P value = 0.163).

Genotype frequency
At the genotypic level, the GG genotype was assumed as the reference genotype.The heterozygous genotype rs34536443 CG was observed in 3.40% of the SLE group and 5.22% of controls, which was not a significant difference (OR = 0.66, CI: 0.37-1.72,P value = 0.15).The CC genotype could not be detected in either group.

Association between genotypes and clinical features
We investigated whether or not the rs34536443 genotypes in the TYK2 gene were associated with specific clinical features of SLE patients.Table 3 lists the results.None of the clinical data was significantly different between subjects for the genotypes of GG and CG (none of the subjects were genotyped as CC for rs34536443 SNP).

Discussion
As TYK2 appears to be a genetic determinant of autoimmune and inflammatory diseases, the present study hypothesized that TYK2 polymorphism might be involved in the pathogenesis of SLE in the Iranian population.The genotype and allele frequencies of rs34536443 SNP in TYK2 in patients with SLE compared with healthy controls was investigated in relations to the risk of SLE.To our knowledge, this study is the first attempt undertaken to detect this possible association in SLE susceptibility in the Iranian population.SLE is a complex, systemic autoimmune disease for which a spectrum of modifying and susceptibility genes are responsible for disease initiation and perpetuation.It is widely accepted that several cytokines are involved in the pathogenesis of autoimmune and inflammatory diseases.Numerous studies have suggested a role of cytokines such as IFN-α in SLE development and has been well documented that IFN-α is a pleiotropic cytokine involved in the immunopathogenesis and development of SLE [34][35][36].In SLE, IFN-α stimulates a signaling pathway and encoding genes involves molecules on this pathway that may be associated with SLE risk [37].
IFNAR, which lacks intrinsic kinase domains, consists of the transmembrane subunits IFNAR1 (α-subunit) and IFNAR2 (β-subunit).TYK2 encodes a proximal tyrosine kinase on the IFN-α signaling pathway and, during antigen stimulation of dendritic cells, induces T-helper 1 differentiation [38].Upon the IFN-α-IFNAR interface, TYK2 interacts with IFNAR1, which results in TYK2 phosphorylation and activation [39].Activation of TYK2 results in IFNAR phosphorylation and JAK1 activation, which in turn leads to dimerization and activation of the STAT transcription factors.Subsequently, phosphorylated STAT proteins form homo/heterodimers and localize to the nucleus, where they regulate transcription of several genes, including IFN [40].
TYK2 is considered as an interesting candidate for association studies with regard to genetic susceptibility to autoimmune and inflammatory diseases.The association between TYK2 gene polymorphism and autoimmune and inflammatory diseases have been examined in many studies, but the results have been inconsistent [12,16,19,25,26,41,43].Some studies have reported a strong linkage of TYK2 haplotypes or individual SNPs with SLE risk [2,9,16,41].The rs34563443 variant of TYK2 alters a conserved proline (major allele) to alanine (protective minor allele) at position 1104 in the tyrosine kinase domain of

P value GG CG CC
Faezi et al.Association of TYK2 rs34536443 polymorphism with... the TYK2 protein, seemingly conferring a functionally less-efficient variant of the TYK2 protein [23].Moreover, TYK2 function in primary T-cells, fibroblasts and B-cells is affected by the P1104A variant, leading to dysregulated signaling processing of pro-inflammatory cytokines [44].
Peluso et al. reported decreased susceptibility to ERI in Brazilian women due to the C allele of TYK2 rs34536443 polymorphism [11].Zervou et al. found no significant association between the rs34536443 variant and JIA in the Greek population at either the genotype or allelic levels [45].Diogo et al. demonstrated that allele C of rs34536443 protects against inflammatory bowel disease (IBD), RA and SLE [44], while others demonstrated that the allele is a risk factor for psoriasis [46] and JIA [47].Tao et al. (2010) performed a meta-analysis comprising 21,497 cases and 22,647 controls to determine the different variants of TYK2.They demonstrated the association of rs34536443 polymorphism with autoimmune and inflammatory diseases [48].In this study, the C allele of rs34536443 SNP did not effect SLE susceptibility in the Iranian population, suggesting the role of genetic diversity in different populations and the involvement of different genes in the etiopathogenesis of this multigenic disease.
In the SNP database (http://www.ncbi.nlm.nih.gov/snp/), the frequencies of the C and G alleles of rs34536443 were estimated to be 0.01 and 0.99, respectively.The results in-dicated a frequency for the C allele in the control group of 2.61%, which is similar to that of the SNP database.Moreover, the frequency of the C allele in our SLE group was 1.70, which is similar to those of the control group and the SNP database.

Conclusion
In conclusion, the current study demonstrated no influence of rs34563443 polymorphism on SLE risk in the Iranian population.It is worth mentioning that in order to reach a definite conclusion, further studies using larger sample sizes are required to determine the exact mechanism by which polymorphisms in this gene result in SLE development.The information from this and related studies should increase understanding of SLE pathogenesis and the possibility of development of promising therapies for the disease.

Table 2 .
Allele and genotype distribution of TYK2 gene rs34536443 SNP in SLE patients and healthy controls.

Table 3 .
Various clinical features of RA patients in accordance with the frequencies of 34536443 genotypes.