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TLR-8, TNF-α, and ESR-1α Gene Polymorphism Susceptibility in Onset of Arthritis

Published online by Cambridge University Press:  01 January 2024

Maryam Mukhtar ,
Nadeem Sheikh Open the ORCID record for Nadeem Sheikh [Opens in a new window] ,
Andleeb Batool ,
Tayyaba Saleem Open the ORCID record for Tayyaba Saleem [Opens in a new window] ,
Muhammad Babar Khawar Open the ORCID record for Muhammad Babar Khawar [Opens in a new window] ,
Mavra Irfan Open the ORCID record for Mavra Irfan [Opens in a new window] ,
Saira Kainat Suqaina Open the ORCID record for Saira Kainat Suqaina [Opens in a new window]  and
Chiara Mazziotta
Maryam Mukhtar
Affiliation:
Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan Rhumatology Domain, Center of Molecular Medicine, Karolinska Institute, Department of Medicine, Stockholm, Sweden
Nadeem Sheikh*
Affiliation:
Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan
Andleeb Batool
Affiliation:
Department of Zoology, Government College University, Lahore, Pakistan
Tayyaba Saleem
Affiliation:
Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan
Muhammad Babar Khawar
Affiliation:
Applied Molecular Biology and Biomedicine Lab, Department of Zoology, University of Narowal, Narowal, Pakistan
Mavra Irfan
Affiliation:
Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan
Saira Kainat Suqaina
Affiliation:
Cell and Molecular Biology Lab, Institute of Zoology, University of the Punjab, Lahore, Pakistan
*
Correspondence should be addressed to Nadeem Sheikh; nadeem.zool@pu.edu.pk
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Abstract

Arthritis is a genetic disorder characterized by bones and joint degradation assisted by severe pain and inflammation. It is evident by the studies that 0 candidate genes variations play vital role in its development and progression. Therefore, we investigated the genetic variation of TLR-8, TNF, and ESR-1α genes in the Pakistani population. A case-control study comprising 300 RA, 316 OA, and 412 control subjects was conducted. PCR-RFLP and direct sequencing methods were used for determining genetic variations. Analysis was performed by using PLINK and MEGA 6.0 software. Allelic and genetic frequencies of polymorphisms identified on rs3764879 (TLR-8), rs3764880 (TLR-8), rs5744080 (TLR-8), rs1800629 (TNF), rs2228480 (ESR-1α), and rs1451501590 (ESR-1α) were significantly varied among RA, OA, and controls. Novel functional mutations SCV000844945 and SCV000844946 on TLR-8 as well as a non-functional SCV000804801 and functional variation SCV000804802 on ESR-1α were also identified and reported for the first time in the studied population. Multiple site analyses indicated that polymorphisms on TLR-8 and ESR-1α genes were significant risk factors in disease onset to the next generation. In conclusion, TLR-08 and ESR-1α were significant in the onset of arthritis whereas the TNF was not found as a significant risk factor in the onset of RA and OA.

Type
Research Article
Information
Genetics Research , Volume 2022 , 2022 , e27
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
Copyright © 2022 Maryam Mukhtar et al.

1. Introduction

Rheumatoid arthritis (RA) and osteoarthritis (OA) both are bone disorders characterized by the mutual interactions of environment and genetics. However, RA is an autoimmune disorder whereas OA is non-autoimmune but in both diseases bones and joints get affected and degraded one way or another [Reference Mukhtar, Sheikh, Batool, Khawar, Fatima and Mehmood1]. Innate and adaptive immunities enable the immune system to fight against multiple disorders. Innate immunity detects the presence of antigens through pattern recognition receptors (PRRs) recognized specific pathogenic molecules in microbes along with host endogenous ligands and leads to the pro-inflammatory cytokines, costimulatory molecules, and interferon (IFNs) expression [Reference Kumar, Kawai and Akira2,Reference O'Neill and Bowie3]. The PRR was further classified into two major classes, i.e., cytosolic and transmembrane. Toll-like receptors (TLRs) are the first known transmembrane PRR. In humans, total of ten TLRs were classified on cell surfaces (TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, and TLR-10) and in endosomes (TLR-3, TLR-7, TLR-8, and TLR-9) [Reference Beutler4]. TLR-8 functions like TLR-7 as involved in the reorganization of self RNA, within snRNP autoantibodies complexes, viral RNA, and multiple small molecule agonists [Reference Ablasser, Poeck and Anz5–Reference Vollmer, Tluk and Schmitz10]. However, TLR-8 is more likely activated by AU-rich ssRNA and senses it through its secondary structures [Reference Forsbach, Nemorin and Montino6, Reference Sarvestani, Williams and Gantier11]. TLR-8 expressed itself in myeloid DCs, monocytes, and neutrophils [Reference Forsbach, Nemorin and Montino6, Reference Hattermann, Picard, Borgeat, Leclerc, Pouliot and Borgeat12, Reference Janke, Poth and Wimmenauer13]. Human TLR-8 got activated by the endogenous ligands might lead to the onset of many inflammatory diseases such as rheumatoid arthritis RA as well as OA.

Tumor necrosis factor-alpha (TNF-α), a pleiotropic and pro-inflammatory cytokine, is encoded by the TNF-α gene which is allocated on chromosome 6 : 31 [Reference Sezgin, Barlas and Ankarali14]. In RA patients, its overproduction primarily by macrophages in joints causes the establishment of rheumatoid synovitis, formation of pannus tissue as well as the destruction of the joint. Its elevated level results in the increase of synoviocyte proliferation and leads to the activation of a secondary mediators’ cascade, playing a role in inflammatory cell recruitment, joint destruction, and neo-angiogenesis [Reference Camussi and Lupia15]. It along with interleukin-1β (IL-1β) increased impaired cartilage function and stiffness of articular chondrocytes [Reference Chen, Xie, Rajappa, Deng, Fredberg and Yang16, Reference El-Tahan, Ghoneim and El-Mashad17]. In vitro studies indicated that pro-inflammatory TNF-α upregulates mRNA matrix metalloproteases-1 and -3 chondrocyte which leads to the pathogenic of OA as a potential risk factor [Reference Chen, Xie, Rajappa, Deng, Fredberg and Yang16–Reference Kunisch, Kinne, Alsalameh and Alsalameh18]. TNF-α mRNA expression in knee OA patients was found to be 1.56 times greater as compared to controls [Reference Sezgin, Barlas and Ankarali14].

Estrogen Receptor 1 (ESR-1) gene encodes for estrogen receptors (ERs), located on chromosome located on 6q25 [Reference Ooi, Inokuchi and Harada19]. The main determinant of bone strength is the cortical bone dimensions, and it is regulated by the estrogen receptor (ER) mediated and mechanical loading (see [Reference Khosla, Melton and Riggs20, Reference Lanyon, Armstrong, Ong, Zaman and Price21]). It has been evident that estrogens function as a protector against bone matrix loss and are mediated by estrogen receptor-alpha (ER-α) [Reference Lindberg, Weihua and Andersson22], [Reference Movrare, Lindberg, Ohlsson, Faergemann and Gustafsson23, Reference Sims, Dupont and Krust24]. It stimulates transcription through activation functions (AFs), i.e., AF-1 located on N-terminal and AF-2 located in the ligands binding domain [Reference Birjesson, Windahl and Lagerquist25]. ER-α activates the genes by the hormone-receptor binding which is then followed by gene activation with estrogen response element (ERE) having promoters. It increased the transcriptional activity from ERE-reporter transfected into osteoblast cell-line [Reference Zaman, Cheng, Jessop, White and Lanyon26]. We have recently found that PADI-4 gene polymorphism was not only involved in the onset of RA but was also found to be a significant risk factor in OA onset [Reference Mukhtar, Sheikh, Batool, Khawar, Fatima and Mehmood1]. Both RA and OA are genetic disorders attributed to the environment and daily lifestyle, but the etiology of both diseases is still a mystery concerning the Pakistani population; therefore, the current study was designed to determine the polymorphism of TLR-8, TNF, and ESR-1α gene and its association with the onset of both RA and OA.

2. Materials and Methods

2.1. Ethical Approval and Sampling

The current study was approved by the ethical committee of the Department of Zoology, University of the Punjab, Lahore, Pakistan, and ethical committees of the government and semi-government hospitals of Punjab province of Pakistan from where samples were collected. All participants were already diagnosed with RA and OA by the physician. Blood samples (3 cc) were collected from each subject in EDTA coated tubes with BD syringes after taking informed written consent from the patient/guardian along with their clinical data. Blood samples were also collected from age and sex-matched healthy control subjects with a negative family history of arthritis. Samples were stored at 4oC before being further processed.

2.2. Genotyping

DNA was extracted from each blood sample of controls as well as patients manually by using a modified organic DNA extraction method [Reference Sambrook and Russell27] and was quantified (conc. ng/μl) and qualified (260/280) by using Nanodrop. Three SNP’s rs3764879, rs3764880, and rs5744080 on TLR-8; two SNP’s rs1800629 and rs361525 on TNF, and four SNP’s rs2234693, rs9340779, rs2228480, and rs1451501590 on ESR-1α were selected on basis of their either direct or indirect involvement in either the pathogenesis or progression of disease. For the amplification of targeted polymorphic sites, specific primers used are presented in Table 1. The polymerase chain reaction (PCR) was conducted in a 25 μl reaction mixture consisting of 10 μl of master mix (Thermo Scientific), 3 μl of forward and reverse primer each, and 12 μl of DNase, RNase-free water. The PCR conditions include initial denaturation at 94oC for 5 mins followed by 35 repeated cycles of denaturation at 94oC for 45 secs, annealing temperature presented in Table 1 for 45 secs, extension at 72oC for 30 secs, and then final extension at 72oC for 10 mins. The products were run on 2% agarose for confirming their product sizes. Genotyping was performed by restriction fragment length polymorphism (RFLP) and direct sequencing. For RFLP, PCR products were digested with their respective enzymes (Table 1) and were run on 2% agarose gel. For the direct sequencing method, PCR products were sequenced by using a sequencer, and sequences were visualized on BioEdit software.

TABLE 1: PCR-RFLP materials and conditions with respective polymorphic sites.

F.P: forward primer; R.P: reverse primer; A.T: annealing temperature; RFLP: restriction fragment length polymorphism; I.T: incubation temperature; T.D.T: thermal deactivation temperature.

2.3. Statistical Analysis

The genetic data for each subject was tabulated and passed through Hardy Weinberg Equilibrium (HWE) Test. Allelic and Genotypic Test was performed. The significance level was determined by the chi-square test and Fisher exact test. Linkage Disequilibrium and Haplotype analysis were performed fanalysesple site analysis. The change in the amino acid sequence was determined by MEGA 6.0 software.

3. Results

The mean age of RA males was 38.62 years and that of RA females was 38.98 years with a mean age of diagnosis in years was 33.06 and 31.56, respectively. Both RA males and females were in the normal BMI range, i.e., 18.75–25.5 kg/m2. The mean age of OA males was 55.27 years and for females was 49.19 years with a mean age of diagnosis in years was 46.97 and 42.73, respectively. In the OA group, obesity, i.e., BMI range of 25.5–30.5 kg/m2 was predominating.

It was observed that allele G on rs3764879 (TLR-8) was prevalent in patients whereas on rs3764880 (TLR-8) allele G got replaced by allele A in patients. On the rs5744080 (TLR-8) polymorphic site, mutant allele T was prevalent among RA and OA individuals as compared to controls. Two intronic novel mutations were also identified of about 50bp G>C and 39bp T>A before the rs3764879 (TLR-8) polymorphic site. The novel SNP’s G>C and T>A identified through sequencing were submitted to ClinVar NCBI for obtaining accession numbers SCV000844945 and SCV000844946, respectively. Allele C replaced allele Ton rs1800629 (TNF) polymorphic site in both cases whereas on rs361525 (TNF) polymorphic site allele C was found to be prevalent among patients as well as controls. No mutation exists on rs2234693 (ESR-1α) and rs9340779 (ESR-1α), i.e., allele G was found to be significant among patients as well as in controls whereas on rs2228480 (ESR-1α) allele G was more prevalent in patients as compared to controls. Similarly, on the rs1451501590 (ESR-1α) site, allele G replaced allele C in patients. Two novel mutations were also identified, i.e., SCV000804801 (23G>A) and SCV000804802 (241T>A).

All SNPs followed HWE (p = 1.00), and it was observed that in RA individuals except for rs3764879 (TLR-8) allelic frequency of rs3764880 (TLR-8), rs5744080 (TLR-8), SCV000844945 (TLR-8), and SCV000844946 (TLR-8), polymorphic sites were not significantly varied in comparison to controls. However, in OA cases, except for SCV000844945 (TLR-8), rest of the SNPs were not significantly associated with the onset of disease at the allelic level. As a result of the genotypic analysis, it was observed that except for rs3764879 (TLR-8), all other SNPs were significantly associated with the onset of RA and except for rs5744080 (TLR-8) all other SNPs were significant risk factors for OA onset. It was observed that rs1800629 (TNF) was not significantly associated with the onset of disease at an allelic level; however, it is significantly associated at the genotypic level. In RA, subjects rs2228480 (ESR-1α), SCV000804801 (ESR-1α), and SCV000804802 (ESR-1α) were significantly associated with the onset of disease at an allelic and genotypic level whereas rs1451501590 (ESR-1α) was not found to be a significant risk factor in disease onset. In OA, subjects rs2228480 (ESR-1α), rs1451501590 (ESR-1α), SCV000804801 (ESR-1α), and SCV000804802 (ESR-1α) were significantly associated with the onset of disease at both allelic and genotypic levels (Table 2).

TABLE 2: Single site analysis of all targeted polymorphic sites in RA as well as OA subjects.

The linkage disequilibrium (L.D) for RA and OA is presented in Figures 1(a), 1(b), 2(a), and 2(b). As a result of L.D, it was observed that in both RA and OA individuals, all SNPs together were significant risk factors with the onset of disease as D’ = 1.000; r2 = 0405 and D’ = 0.660; r2 = 0.350. It was observed that SCV000844945 and SCV000844946 were also significant risk factors for the next generation in the onset of disease. As no mutation was observed at rs2234693 and rs9340779 polymorphic sites, therefore, they were excluded from multiple site analyses. As a result of L.D for RA subjects, it was observed that rs2228480, rs1451501590, SCV000804801, and SCV000804802 together were 100% disease onset risk factors in the next generation as D’ = 1.000 and r2 = 0.360 whereas it also increased the risk of OA onset up to 71% in next-generation D’ = 0.717; r2 = 0.423. The L.D for ESR-1 is presented in Figures 3(a) and 3(b) for RA and Figures 4(a) and 4(b) for OA, respectively.

FIGURE 1: (a, b) Linkage disequilibrium chart of TLR-8 gene polymorphic sites in RA patients and controls.

FIGURE 2: (a, b) Linkage disequilibrium chart of TLR-8 gene polymorphic sites in OA patients and controls.

FIGURE 3: (a, b) Linkage disequilibrium chart of ESR-1α gene polymorphic sites in RA patients and controls.

FIGURE 4: (a, b) Linkage disequilibrium chart of ESR-1α gene polymorphic sites in OA patients and controls.

On the TLR-8 gene, it was shown that all haplotypes were significant (p < 0.01) for the start of disease because their frequency was higher in patients compared to control participants. This was true for both RA and OA. However, the frequency of CACGT and CATGT was higher in controls; therefore, they act as protectants in the onset of disease. ESR-1α haplotype analysis indicated that the frequency of AAGT was higher in controls as compared to patients; therefore, it acts as a protectant in RA and OA onset. The significant haplotype in disease onset is presented in Table 3. Change in amino acid sequences with respective polymorphic sites is presented in Table 4.

TABLE 3: Haplotype analysis of TLR-8 and ESR-1α gene.

TABLE 4: Change in amino sequence by MEGA 6.0 software.

4. Discussion

RA and OA are the two major types of arthritis characterized as multifactorial disorders such as age, BMI, autoimmunity, hormonal, environmental, and genetics which play role in their development and progression. iEssential information has been developed concerning the genome-wide causative mutations and their inheritance through multiple techniques including genome-wide association studies (GWASs), candidate gene association studies Twin, linkage analysis, segregation analysis, and twin studies [Reference Valdes and Spector28]. Genetic association studies in different populations evidenced that disease development and progression risk are complex processes that might interact with innate as well as acquired host responses to inflammatory, biomechanical, hormonal, environmental, and immunological stimuli [Reference Zhang and Jordan29]. Therefore, the current study targeted for the first time to determine the association of TLR-8, TNF, and ESR-1α gene polymorphism with the onset of not only RA but also OA in the Pakistani population.

Currently, the study demonstrated that rs3764879 of TLR-8 was associated with RA onset at an allelic level whereas rs3764880 and rs5744080 were associated at the genotypic level. In OA development, rs3764879 and rs3764880 were significant risk factors genotypically. Two novel functional mutations never reported before, i.e., SCV000844945 and SCV0008449456 were also identified on TLR-8 and found to be more associated with OA development as compared to RA onset. Overall polymorphism on the TLR-8 gene was found to be significant in disease onset. Like current findings, a replicate study reported the linkage of RA onset and rs3764880 polymorphism in the Caucasian Spanish population. In addition, signaling of TLR-8 induced pro-inflammatory cytokines [Reference Ohto, Tanji and Shimizu30]. Polymorphism on rs3764880 leads to a decrease in inflammatory cytokines levels. This polymorphism in the start codon causes TLR-8’s first three amino acid deletions giving rise to the TLR8v2 isoform [Reference Wang, Eng, Lin, Liu, Chang and Lin31]. A study conducted on OA Chinese individuals reported rs5744080 polymorphisms as a significant risk factor in the onset of disease in male subjects [Reference Yang, Lee and Lee32]. It is well documented that variants on the TLR-8 genes were associated with autoimmune and infectious diseases like systemic lupus erythematosus, type 2 diabetes, and tuberculosis [Reference Enevold, Nielsen and Jacobsen33–Reference Xiao, Liu and Lin35]. However, more studies should be conducted to determine its association with RA and OA development.

The current study revealed a strong association of Rs1800629 polymorphic site variation with both RA and OA development. In the pipeline of current findings, the same functional polymorphism rs1800629 with RA as well as OA onset was also reported in the north Indian population, Chinese population, and Egyptian population [Reference Gambhir, Lawrence, Aggarwal, Misra, Mandal and Naik36–Reference You, Li and Li38]. In contrast, no association between rs1800629 and RA was reported in the Brazilian population [Reference Pinheiro, Gomes and Ronda39]. On the other hand, the current study reported no association between rs361525 and disease development. However, contrary to current findings, another study conducted on the Pakistani population reported a positive association between rs361525 and OA development in the Pakistani population [Reference Naqvi, Bibi, Murtaza and Javed40]. Like the findings of the current study, no positive association has been reported in the Chinese, Mexican, and Iraqi populations [Reference Naqvi, Bibi, Murtaza and Javed40–Reference Mahmood, Schmalzing, Dörner, Tony and Muhammad42].

It was declared from the present study that no association exists between rs2234693 and rs9340779 with the onset of both RA as well as OA in the studied population. The current study reported that polymorphism on rs2228480 polymorphic site acts as a significant risk factor in the onset of both RA and OA. It was also revealed that rs1451501590 polymorphism was associated with OA development only. Although the vital role of estrogen in the pathogenesis of RA was indicated by many studies, no significant effects were reported by estrogen receptor agonists on RA symptoms [Reference Dziedziejko, Kurzawski, Safranow, Chlubek and Pawlik43]. Van Vollenhoven [Reference van Vollenhoven, Cifaldi, Ray, Chen and Weisman44] reported a lack of clinical advantages of selective ESR-1α agonist treatment in RA patients. Like current findings, no association was reported between RA and rs2234693 and rs9340779 polymorphic sites in the polish and Japanese populations [Reference Dziedziejko, Kurzawski, Safranow, Chlubek and Pawlik43]. Studies conducted in Caucasian, European, and American populations reported a non-significant association between rs2234693 and 9340779 and RA [Reference Lian, Yoo, Pei, Cho, Cheng and Chun45, Reference Loughlin, Dowling, Mustafa, Smith, Sykes and Chapman46]. In contrast to current findings, strong association of rs2234693 and rs9340779 with OA was reported in Japan, Korean, and Mexican populations [Reference Bergink, van Meurs and Loughlin47, Reference Borgonio-Cuadra, González-Huerta, Duarte-Salazár, de los Ángeles Soria-Bastida, Cortés-González and Miranda-Duarte48]. A meta-analysis conducted by Hu et al [Reference Hu, Costenbader, Gao, Hu, Karlson and Lu49] reported that polymorphism on rs2234693 might reduce the risk of OA risk and the rs9340799 may not be associated with OA risk in the Chinese population. A weak association relationship in rs9340799and OA was reported in Europeans but not Asians whereas rs2234693 was not significantly associated with OA in both populations [Reference Ren, Yang and Yin50]. Another study conducted by Ma et al. [Reference Ma, Wei and Zheng51] reported that the risk of OA incidence decreased with rs9340799 and rs2228480 whereas rs224693 was a strong factor in disease onset. Jin et al. [Reference Jin, Hong and Yang52];reported in their case-control study that the minor allele of rs2228480 was an increased risk of knee OA in his Meta-analysis, indicated the same results. The current study also reported the identification of two significant risk SNPs, i.e., SCV000804801 and SCV000804802. SCV000804802 was a functional novel SNP and caused the replacement of phenylalanine with isoleucine.

ESR-1α polymorphisms altered the estrogen receptor expression in the ESR-1, and ESR-2 genes could affect the expression that leads to immunological consequences. The effect of estrogen was mediated by estrogen receptors located in the multiple immune cells (T cells, B cells, monocytes, and macrophages) as well as in the thymus [Reference Tsokos and Nepom53]. This effect can be achieved by directly manipulating the profile of T-helper cytokine from interleukin (IL)-2, TNF-α, and interferon (IFN)-γ (pro-inflammatory) to IL-4, IL-6, and IL-10, transforming growth factor (TGF)-β (anti-inflammatory) direction [Reference Al-Salem and Al-Awadhi54].

The current study concluded that TLR-8 and ESR-1α gene polymorphism are the significant risk factors in the onset of both RA as well as OA individuals in the Pakistani population. However, larger scale studies in other populations should be conducted to determine novel mutation susceptibility.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

All authors contributed equally to this study.

Acknowledgments

The authors would like to acknowledge the Vice-Chancellor of the University of Punjab Lahore for ethically allowing us to conduct this study as well as the Higher Education Commission, Pakistan, for providing funds for the conduction of the study. Higher Education Commission, Pakistan, has funded the current project.

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Figure 0

TABLE 1: PCR-RFLP materials and conditions with respective polymorphic sites.

Figure 1

TABLE 2: Single site analysis of all targeted polymorphic sites in RA as well as OA subjects.

Figure 2

FIGURE 1: (a, b) Linkage disequilibrium chart of TLR-8 gene polymorphic sites in RA patients and controls.

Figure 3

FIGURE 2: (a, b) Linkage disequilibrium chart of TLR-8 gene polymorphic sites in OA patients and controls.

Figure 4

FIGURE 3: (a, b) Linkage disequilibrium chart of ESR-1α gene polymorphic sites in RA patients and controls.

Figure 5

FIGURE 4: (a, b) Linkage disequilibrium chart of ESR-1α gene polymorphic sites in OA patients and controls.

Figure 6

TABLE 3: Haplotype analysis of TLR-8 and ESR-1α gene.

Figure 7

TABLE 4: Change in amino sequence by MEGA 6.0 software.