Genetic risk assessment of HLA DRB1 alleles (01, 04, 12 and 15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan

Genetic risk assessment of HLA DRB1 alleles (01, 04, 12 and 15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic risk assessment of HLA DRB1 alleles (*01, *04, *12 and *15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan Zahoor Ilahi, Najeeb Ullah Khan, Hamza Khan, Mikhlid H. Almutairi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4160713/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Rheumatoid arthritis (RA) is a chronic autoimmune condition influenced by a complex interplay of genetic and environmental factors. Specific genetic variations, particularly in the human leukocyte antigen (HLA)-DRB1 gene, have been strongly linked to RA susceptibility. HLA-DRB1 alleles, part of the major histocompatibility complex (MHC) class II gene family, play a significant role in the immune system. Aims: This study aimed to investigate the association between HLA-DRB1*01, *04, *12, and *15 alleles and RA risk within the Pashtun population of Pakistan. Methods: A cohort of 100 RA patients and 80 healthy controls was recruited for this study. DNA was isolated from blood samples using the salting-out method, followed by genotyping using sequence-specific primer polymerase chain reaction (SSP-PCR). The association of the selected alleles with clinicopathological and demographic parameters of RA was assessed using the Medcalc odds ratio calculator and Chi-square test. Results: Allelic and genotypic analysis revealed that HLA-DRB1 *01 and *04 variants exhibited non-significant associations with RA risk (P=0.1421 and 0.8495, respectively). Similarly, mutant genotypes of HLA-DRB1 *01 and *04 showed non-significant associations with RA (P=0.2922 and P=0.8263, respectively). However, the heterozygous CA genotype of HLA-DRB1 *01 demonstrated a significant association with RA (P=0.0004), whereas HLA-DRB1 *04 did not (P=0.9120). Furthermore, HLA-DRB1 *12 showed a significant association with increased RA risk (P=0.0001), while HLA-DRB1 *15 did not (P=0.5519). Additionally, HLA-DRB1*01, *04, and *12 did not show significant associations with age group (P=0.08, P=0.11, P=0.16) and rheumatoid factor (RF) status (P=0.34, P=0.65, and P=0.74), whereas HLA-DRB1 *15 exhibited non-significant associations with age group and RF (P=0.01 each). Moreover, associations of HLA-DRB1 *01, *04, *12, and *15 with gender (P=0.81, P=0.52, P=0.27, and P=0.09, respectively) and anti-citrullinated protein antibody (ACPA) status (P=0.56, P=0.40, P=0.65, and P=0.008, respectively) were not significant, except for HLA-DRB1 *04, which displayed a significant association with ACPA. Conclusion: HLA-DRB1 *12 and heterozygous genotypes of HLA-DRB1 *01 were significantly associated with RA risk in the Pashtun population. However, further analysis utilizing whole exome sequencing with larger datasets is warranted for more precise results. HLA DRB1 gene Alleles Rheumatoid arthritis risk Figures Figure 1 Figure 2 Figure 3 Introduction Rheumatoid arthritis (RA) is a commonly diagnosed systemic chronic inflammatory disorder/condition that affects about 1% of the global population [ 1 ]. It is characterized by polyarthritis, immunologic abnormalities, autoimmune reactions, high comorbidity, and early death. It is also known to cause gradual joint destruction and impairment [ 2 ]. Extracellular matrix degradation, which causes the normal breakdown of cartilage, tendons, bone, and ligaments, is a defining characteristic of RA. Beside this, different constitutional symptoms include fever, asthenia, weight loss, malaise and anorexia [ 3 ]. RA is mostly caused by interaction of genetic and environmental factors [ 4 – 6 ]. Other risk factors include age, gender and environmental exposure i.e., smoking, different air pollutants and infectious agents [ 7 , 8 ]. RA is typically classified into two types seropositive or seronegative. The classification is based on presence or absence of rheumatoid factor and Anti-citrullinated protein antibodies [ 9 – 11 ]. This condition affects approximately 5 to 10 individuals out of every 1,000 in developed nations [ 12 ]. It's worth noting that from 1990 to 2017, RA had a significant impact on people's health worldwide, resulting in a loss of 3.4 million of life [ 13 , 14 ]. In the period 1980–2018, the prevalence of RA was 460 patients per 100,000 people worldwide with a prediction level of 95% (0.07–1.29%) [ 15 , 16 ]. The prevalence of RA is more in northern America and Europe as compared to other areas [ 17 – 19 ]. A study from 2011 state that prevalence of RA is 0.5 to 1% in Pakistan but it would be increasing as there is not much data present from Pakistani population [ 20 , 21 ]. The prevalence is more in women as compared to men in Pakistan [ 22 , 23 ]. Different genetic factors are identified to be associated with RA. Among different genetic factors, HLA DRB1 alleles are investigated in several studies and found to be significantly associated with RA [ 24 , 25 ]. This association has been identified in different populations across the world. The strength of different alleles varies in different population [ 26 – 30 ]. Other genetic factors associated with RA include tumor necrosis factor (TNF), corticotrophin releasing hormone (CRH), CYP 19 (aromatase), IFN-γ and other cytokines. Some study suggests that different auto immune disease such as insulin dependent diabetes, locus on chromosome X may be associated with RA [ 31 ]. The human leukocyte antigen (HLA) system, which correlates to the major histocompatibility complex (MHC) in humans, is critical in the antigen presentation of intracellular and extracellular peptides as well as the control of innate and adaptive immune responses [ 24 , 32 ]. The conventional HLA loci are the class I (HLA-A, -B, -C, -E, -F, and -G) and class II ( HLA-DR , -DQ, -DM, and -DP) molecules known for their roles in antigen presentation to CD8 + and CD4 + T lymphocytes, respectively. They are transcribed by the most variable area in the human genome, a 4-Mb region of human chromosome 6p21 and are classified as MHC. MHC plays major role in immunity as well as risk associated with different auto immune diseases [ 33 ]. The role of HLA DRB1 in susceptibility to RA is estimated to be 50% [ 32 ]. The HLA DRB1 gene comprises of 6 exons with each exon encoding different domain [ 34 ]. The leader peptide is encoded by exon 1, the extracellular domains by exons 2 and 3, the trans-membrane domain by exon 4, and the cytoplasmic tail by exon 5 [ 35 ]. Exon 1 encodes amino acid patterns that make up the first hyper variable region (HVR1); Exon 2 encodes similar epitope motifs and the third region (HVR3). About 2960 alleles have been identified in HLA DRB1 gene out of which only few are identified to have role in RA [ 24 ]. Several HLA DRB1 alleles have been identified as playing a critical role in the severity and susceptibility to rheumatoid arthritis. These alleles possess a sequence motif in the third HVR of the HLA DRB1 gene, referred to "shared epitope (SE)", which is composed of five amino acids at positions 70–74 of the HLA DRB1 chain. Specifically, this SE contains one of three amino acid sequences: RRRAA, QKRAA or QRRAA [ 36 ]. While the DERAA pattern seems to be primarily accountable for the beneficial protective effects [ 37 ]. The SE sequences facilitate the presentation of self-antigens to T cells, which is believed to be a critical factor in the onset of RA [ 38 ]. Current studies suggest that, RA is primarily initiated by the specific binding of citrullinated peptides by HLA-DR molecules that possess the SE sequence. The HLA-DRB1 SE allele is found in 64–82% of RA cases, a significantly higher occurrence compared to their first-degree relatives (53.9–55%) and the healthy individual population (39–52%). Approximately one-fifth of individuals with RA are estimated to lack the SE alleles. The autoimmune reaction in RA is believed to be triggered by the citrullination of self-peptides, which results in changes to their characteristics. This can cause the stimulation of immune responses and the production of specific ACPA, which are observed in about 75% of individuals with RA [ 24 ]. There are contradictory results regarding HLA DRB1 alleles association with RA. Many studies from different populations have proved its association with RA [ 27 , 39 – 42 ]. However, no such study is present from Pakistani KP population. Hence this study will help in determining the association of HLA DRB1 alleles especially HLA DRB1 *01, *04, *12, *15 with RA in Pakistani KP population. Materials and methods Enrollment of patients and healthy controls The theoretical and practical work for current research study was executed at Institute of Biotechnology and Genetic Engineering IBGE (Health lab), The University of Agriculture, Peshawar, Pakistan (August, 2022 to July, 2023). In the current study, data (Demographics, Pathological and blood samples) were collected from 100 RA patients. Patients with other complications were not selected for the current study. Besides this, data was collected from 80 healthy controls. Data had been taken on standard Performa attached, approved by the ethical committee of IBGE. Following a written consent, taken from both cases and healthy individuals, around 2 cubic centimeters (cc) of blood samples were obtained in Ethylene Diamine Tetra Acetic acid tubes (EDTA tubes) following established biosafety procedures during August 2022 to November 2022. The collected samples were stored at -20°C for further analysis. DNA extraction and primer designing The extraction of Genomic DNA from obtained blood samples of both RA cases and healthy individuals were performed using salting out/non-enzymatic method already adopted in our lab [ 43 – 45 ]. Furthermore, sequence specific primers were designed for selected Alleles using NCBI primer Blast software (Table 1 ). Table 1 Specific primers sequences designed for the detection of SNPs genotypes Alleles Product size Primers Sequence 5'-3' HLA DRB1* 01 255bp Forward Reverse Wild Reverse Mutant TTGTGGCAGCTTAAGTTTGAAT CTGCACTGTGAAGCTCTCAC CTGCACTGTGAAGCTCTCCA HLA DRB1 *04 260bp Forward Reverse Wild Reverse Mutant GTTTCTTGGAGCAGGTTAAACA CTGCACTGTGAAGCTCTCAC CTGCACTGTGAAGCTCTCCA HLA DRB1* 12 248bp Forward Reverse AGTACTCTACGGGTGAGTGTT CACTGTGAAGCTCTCCACAG HLA DRB1 *15 197bp Forward Reverse TCCTGTGGCAGCCTAAGAG CCGCGCCTGCTCCAGGAT Amplification control primers 796bp Forward Reverse TGCCAAGTGGAGCACCCAA GCATCTTGCTCTGTGCAGAT Sequence specific primer (SSP) PCR The Sequence specific primer (SSP) PCR technique was performed to check out the allele and genotype frequencies of certain SNPs in HLA DRB1 gene. The heterozygous and homozygous polymorphisms were observed in alleles using forward and reverse primers. To determine the allele and genotype frequencies for certain SNPs, A volume of 10 µL reaction mixtures were made twice (had different sets of forward but common reverse primers) consisting 2 µL (5x) Master mix, 1 µL template DNA, 0.3 µL of each reverse and forward primers and 6.4 µL of PCR water/buffer (Table 2 ). The amplified product was further confirmed through 1.5% gel electrophoresis. Table 2 The optimized conditions of primers for PCR Alleles Denaturation Annealing Tm Extension Cycles HLA DRB1 * 01 95°C/5min 94°C/30sec 57.8°C/30sec(W) 55.9°C/30sec(M) 72°C/1min 72°C/10min 35x HLA DRB1 * 04 95°C/5min 94°C/30sec 60.1°C/30sec(W) 60.1°C/30sec(M) 72°C/1min 72°C/10min 35x HLA DRB1 * 12 95°C/5min 94°C/30sec 54.7°C/30sec 72°C/1min 72°C/10min 35x HLA DRB1 * 15 95°C/5min 94°C/30sec 54°C/30sec 72°C/1min 72°C/10min 35x Internal control 95°C/5min 94°C/30sec 64°C/30sec 72°C/1min 72°C/10min 35x Statistical Analysis The statistical analysis for the collected research data was done using Statistical Package for Social Sciences (SPSS) software. The Chi-square test was employed to identify the demographic, pathological parameters and genetic polymorphism of RA cases. If the probability values are Lower than or equals to 0.05 the analysis was counted as statistically significant. The certain association of HLA DRB1 polymorphisms with RA risk was figured out through assessment of odd ratios and 95% confidence intervals by using Medcalc odd ratios calculator. Results Allelic and genotypic frequencies distribution of HLA DRB1 * 01 and HLA DRB1 * 04 in RA cases and healthy Controls The genotyping of HLA DRB1 *01 and HLA DRB1 *04 were performed using SSP-PCR procedure to check the allelic and genotypic frequencies of RA cases and healthy individuals (Fig. 1 ). Our result indicated that, In HLA DRB1* 01, the frequency of variant A was higher in cases (49%) than healthy individuals (41.25%) and had non-significant association with RA (OR = 1.37, 95% CI = 0.89–2.08, P = 0.1421). Similarly, the frequency of heterozygous CA genotype was found to be higher in patients (74%) than healthy individuals (47.5%) and significantly linked with the high risk of RA (OR = 3.89, 95% CI = 1.83–8.25, P = 0.0004). Moreover, the frequency of homozygous Mutant genotype AA was high in healthy individuals (17.5%) as compared to patients (12%) and non-significantly linked with the RA (OR = 1.71, 95% CI = 0.62–4.67, P = 0.2922). In case of HLA DRB1*04 , the frequency of variant A was almost same in healthy individuals (45%) and cases (44%) and observed to be non-significantly associated with RA (OR = 0.96, 95% CI = 0.63 to 1.45, P = 0.8495). Moreover, the frequency of heterozygous CA genotype was exactly the same in cases and healthy controls (60%) each respectively, indicating non-significant link with RA (OR = 0.96, 95% CI = 0.48 to 1.92, P = 0.9120). Furthermore, the frequency of homozygous mutant AA genotype was high in cases (14%) as compared to controls (12%) but it was non-significantly associated with RA (OR = 0.89, 95% CI = 0.34 to 2.35, P = 0.8263) (Table 3 ). Table 3 Allelic and genotypic frequencies distribution of HLA DRB1 *01 and HLA DRB1 *04 in RA cases and healthy Controls HLA DRB1 *01 Patients N = 100 Healthy Controls N = 80 OR 95% CI P-value C 102(51%) 94(58.75%) Reference A 98(49%) 66(41.25%) 1.37 0.89–2.08 0.1421 Genotypes CC 14(14%) 28(35%) Reference CA 74(74%) 38(47.5%) 3.89 1.83–8.25 0.0004 AA 12(12%) 14(17.5%) 1.71 0.62–4.67 0.2922 HLA DRB1 * 04 C 112(56%) 88(55%) Reference A 88(44%) 72(45%) 0.96 0.63 to 1.45 0.8495 Genotypes CC 26(26%) 20(25%) Reference CA 60(60%) 48(60%) 0.96 0.48 to 1.92 0.9120 AA 14(14%) 12(15%) 0.89 0.34 to 2.35 0.8263 The frequencies distribution of HLA DRB1 * 12 and HLA DRB1 * 15 in RA cases and healthy controls The genotyping of HLA DRB1 *12 and HLA DRB1 *15 were performed using PCR-SSP procedure to check the allelic frequencies of RA cases and healthy controls (Fig. 2 ). Internal control primer was used to confirm the validity of PCR reaction in each tube (Fig. 3 ). The frequency of HLA DRB1 *12 was higher in cases (94%) than healthy controls (67.5%), which indicated high risk and significant association with RA (OR = 7.54 95% CI = 2.92–19.47, P = 0.0001). Furthermore, the frequency of HLA DRB1 *15 was higher in cases (74%) than healthy controls (70%) yielding high risk but non-significant association (OR = 1.22 95% CI = 0.63–2.34, P = 0.5519) (Table 4 ). Table 4 The frequencies distribution of HLA DRB1 *12 and HLA DRB1 *15 in RA cases and healthy controls HLA DRB1 *12 Present Absent OR 95%CI P value Cases N = 100 94(94%) 6(6%) 7.54 2.92 to 19.47 0.0001 Control N = 80 54(67.5%) 26(32.5%) Reference HLA DRB1 *15 Cases N = 100 74(74%) 26(26%) 1.22 0.63 to 2.34 0.5519 Control N = 80 56(70%) 24(30%) Reference Association of selected HLA DRB1 alleles with demographic and clinical parameters of RA cases In this study, the association of selected HLA DRB1 alleles with demographic and clinical parameters of different RA cases was explored. The results of our research indicated non-significant associations of HLA DRB1 * 01, * 04, * 12 with age groups (P = 0.08, P = 0.11, P = 0.16) and rheumatoid factor (p = 0.34, p = 0.65, p = 0.74) while HLA DRB1 * 15 exhibited significant association with both age group and rheumatoid factors (P = 0.01 each). Additionally, all the selected alleles of HLA DRB1 gene showed non-significant associations with gender (P = 0.81, P = 0.52, P = 0.27 and P = 0.09). Similarly, these selected alleles exhibited non-significant association with ACPA (P = 0.56, P = 0.40 and P = 0.0.65) except HLA DRB1 *04, which displayed non-significant association with ACPA (P = 0.008) (Table 5 ). Table 5 The association of HLA DRB1 gene selected alleles with clinical parameters of Rheumatoid Arthritis PARAMETERS HLA DRB1 *01 HLA DRB1 *04 Age Group CC (%) CA (%) AA (%) OR P VALUE CC (%) CA (%) CC (%) OR P VALUE 35 years 06(9.37) 50(78.2) 08(12.5) 2.76 0.08 20(31.25) 34(54.5) 10(14.2) 0.44 0.11 Gender Female 10(13.5) 54(72.9) 10(13.5) 1.16 18(24.32) 44(59.45) 12(16.2) 1.38 Male 04(15.3) 20(76.9) 02(7.69) 0.86 0.81 08(30.77) 16(61.54) 02(7.69) 0.72 0.52 Rheumatoid Factor Positive 0(00) 08(80) 02(20) 3.98 02(20) 06(30) 02(20) 1.45 Negative 14(15.5) 66(73.3) 10(11.1) 0.25 0.34 24(26.67) 54(60) 12(13.3) 0.68 0.65 ACPA Positive 02(10) 18(90) 0(00) 1.59 10(50) 10(50) 0(00) 0.25 Negative 12(15) 56(70) 12(15) 0.63 0.56 16(20) 50(62.5) 14(17.5) 4.00 0.008 PARAMETERS HLA DRB1 *12 HLA DRB1 *15 Age Group WILD (%) MUTANT(%) OR P VALUE WILD (%) MUTANT(%) OR P VALUE 35 years 06(9.37) 58(90.62) 0.12 0.16 22(34.37) 42(65.63) 0.24 0.01 Gender Female 06(8.10) 68(91.89) 0.19 16(11.11) 58(88.88) 2.26 Male 0(00) 26(100) 10.5 0.27 10(34.37) 16(65.63) 0.44 0.09 Rheumatoid Factor Positive 0(00) 10(100) 1.61 06(60) 04(40) 0.19 Negative 06(6.66) 84(93.33) 0.61 0.74 20(22.22) 70(77.78) 5.25 0.01 ACPA Positive 02(10) 18(10) 0.47 06(30) 14(10) 0.78 Negative 04(5) 76(95) 2.11 0.40 20(25) 60(75) 1.28 0.65 Discussion Rheumatoid arthritis (RA) constitutes a chronic inflammatory disorder primarily affecting women, with a female-to-male ratio of approximately 3:1. The characteristic feature of this condition is symmetrical polyarthritis, which gradually results in the deterioration of joints over time [ 46 ]. The worldwide prevalence of RA is approximately 1%, while in Western populations, the incidence ranges from 1–2%. In Pakistan, the prevalence is observed to be around 0.5% [ 47 ]. Approximately 0.55% of the urban population in northern Pakistan is affected by rheumatoid arthritis, whereas in southern Pakistan, the incidence rate is approximately 0.14% [ 48 ]. RA is caused by a combination of genetic and environmental factors. "The presence of specific genes, namely HLA class II genotypes, plays a vital role in the development and progression of RA [ 49 ]. Approximately 60% of the risk for developing RA is due to genetics, and within that the impact of the HLA is expected to be around 11–37%. The most relevant HLA gene variants associated with RA susceptibility are HLA-DRB1 *01, DRB1 *04, DRB1* 12 and DRB1 *15 [ 37 ]. The significant link between specific alleles and rheumatoid arthritis (RA) varies across populations in distinct geographic regions and among diverse ethnic groups. Therefore, it is imperative to examine various alleles of the HLA gene in RA cases from various populations and geographic areas, exploring whether they play a protective role or act as susceptibility factors [ 39 ]. In the current study we analyzed the frequencies of HLA DRB1 *01, HLADRB1 *04, HLADRB1 *12 and HLA DRB *15 polymorphism in 100 RA cases and 80 healthy controls. Our findings suggested that CA genotype of HLA DRB1 *01 was significantly linked with the high risk of RA (OR = 3.89, 95% CI = 1.83 to 8.25, P = 0.0004) while the variant A (OR = 1.37, 95% CI = 0.89–2.08, P = 0.1421) and mutant genotype AA (OR = 1.71, 95% CI = 0.62–4.67, P = 0.2922) was non-significantly linked with RA risk. In case of HLA DRB1 *04, variant A (OR = 0.96, 95% CI = 0.63 to 1.45, P = 0.8495), Heterozygous CA (OR = 0.96, 95% CI = 0.48 to 1.92, P = 0.9120) and mutant AA genotype was observed to be non-significantly associated with RA risk (OR = 0.89, 95% CI = 0.34 to 2.35, P = 0.8263). Furthermore, the HLA DRB1 *12 indicated high risk and significant association with RA (OR = 7.54 95% CI = 2.92–19.47, P = 0.0001). While HLA DRB1 *15 yielded high risk but exhibited non-significant association with RA (OR = 1.22 95% CI = 0.63–2.34, P = 0.5519). Moreover, the selected alleles of HLA DRB1 gene (*01, *04 and *12 ) showed non-significant association with age groups and RF while HLA DRB1 *15 indicated significant association (p = 0.01 each). In terms of gender all the selected alleles displayed non-significant associations. Similarly, the selected alleles HLA DRB1 *01, *12, *15 indicated non- significant association with ACPA. While HLA DRB1 *04 showed significant association (p = 0.008). Our results were consistent with the findings of other study performed in Iran, China, Pakistan and Syria population revealing non-significant association of HLA DRB1 alleles (*01, *04, *15) with RA risk [ 27 , 39 – 42 ]. However, findings of Sudan population regarding HLA DRB1 *04 didn’t aligned with our results [ 50 ]. Additionally, significant association of HLA DRB1 *12 in Korean population aligns with our findings in KP population [ 51 ]. In conclusion, our study revealed the intricate role of HLA DRB1 gene in RA risk within the KP population. While highlighting the importance of this study, there are certain constraints which need to be acknowledged. These constraints include limited sample size which is not enough to fully capture intricate genetic variation within this diverse ethnic group. Furthermore, our study primarily concentrated on limited number of alleles within the extensive HLA DRB1 gene, suggesting the need for further exploration into additional potential polymorphisms. Hence future studies should aim to incorporate a wider and more diverse dataset employing advanced whole-genome sequencing techniques to expose additional genetic factors contributing to RA. Such approach will enable a more thorough comprehension of the genetic makeup associated with RA, thereby potentially advancing the development of more efficient diagnostic and therapeutic approaches. Declarations Ethical statements Ethical approval This study was conducted at Institute of Biotechnology and Genetic Engineering, Peshawar, Pakistan, under NO: IBGE/2023/007. Consent to participate and publication A written consent was taken from all the participants both RA patients and healthy controls after clearly briefing the aims and objectives of the study in local language. Competing interests The authors declared no competing interests Consent to publish All the authors have read and approved the article for publication Availability of data and materials All the necessary data are included in manuscript, related data will be provided on request from corresponding author Funding There is no funding received to conduct the study, the authors contributed for the study Authors' Contributions ZI did the experimental work and wrote the first draft under the supervision of NUK. HK help in data collection, and data analysis. NUK, MHA and IA critically review the final manuscript. Acknowledgements Not applicable References Prandota J (2023) Solving the New Puzzle (s) 2014–2022: Flammer Syndrome Probably Represents a Mild Endophenotype of Raynaud’s Phenomenon Mainly Associated with Ocular Disturbances and Also Due to Latent T. gondii Infection. 3(1):1–52 Harsini S, Rezaei N (2023) Autoimmune diseases. Clinical Immunology. 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Ann Saudi Med 31(2):171–173 Hameed K et al (1997) The association of HLA-DRB genes and the shared epitope with rheumatoid arthritis in Pakistan. Br J Rheumatol 36(11):1184–1188 Mourad J, Monem F (2013) HLA-DRB1 allele association with rheumatoid arthritis susceptibility and severity in Syria. Revista Brasileira de Reumatologia (English Edition), 53(1): p. 47–56 Naqi N et al (2011) HLA DRββ1 alleles in Pakistani patients with rheumatoid arthritis. J Coll Physicians Surg Pakistan 21(12):727–730 Jehanzeb M et al (2023) Association of candidate genes (ALR2, RAGE, and VEGF) polymorphisms with diabetic retinopathy in type 2 diabetic patients of Khyber Pakhtunkhwa. Pakistan 50(1):227–234 Khan Y et al (2023) Significant association of BRCA1 (rs1799950), BRCA2 (rs144848) and TP53 (rs1042522) polymorphism with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa. Pakistan : p. 1–10 Shahzad S et al (2023) Estrogen receptor alpha (ESR1) gene polymorphism (rs2234693 and rs2046210) with breast cancer risk in pashtun population of Khyber Pakhtunkhwa. 50(3):2445–2451 Maranini B et al (2022) Focus on sex and gender: what we need to know in the management of rheumatoid arthritis. J personalized Med 12(3):499 Sultana T et al (2020) Update on the genotyping distribution, epidemiology and associated risk factors in inflammatory disorder in adult subjects: A cross-sectional study. J King Saud University-Science 32(1):1157–1161 Ashiq K et al (2022) Prevalence and Control of the Hypertension in Patients Suffering from Rheumatoid Arthritis. Pakistan Heart J 55(4):421–422 Aho K, Heliövaara M (2004) Risk factors for rheumatoid arthritis. Ann Med 36(4):242–251 Ali AA et al (2023) Association of Human Leukocyte Antigen (HLA) class II (DRB1 and DQB1) alleles and haplotypes with Rheumatoid Arthritis in Sudanese patients. Front Immunol 14:1178546 Joung C et al (2003) Association between HLA-DR B1 and clinical features of adult onset Still's disease in Korea. Clin Exp Rheumatol 21(4):489–492 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4160713","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283413040,"identity":"3a353f1b-545c-446f-b7b7-6746767dfaae","order_by":0,"name":"Zahoor Ilahi","email":"","orcid":"","institution":"The University of Agriculture Peshawar","correspondingAuthor":false,"prefix":"","firstName":"Zahoor","middleName":"","lastName":"Ilahi","suffix":""},{"id":283413041,"identity":"a4c8a7ea-de8e-4945-8853-fd020cafedb8","order_by":1,"name":"Najeeb Ullah Khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHCCBAbGhoQEBgbmAxA+kGJsIE4LWwLRWhigWngMiNNizn7g4YOPO9Ly+Gf3fPzMu4NBju9GAtvDGXi0WPYkJBvOPJNTLHHn7GZp3jMMxpI3EtgNN+DRYnAgIU2at60iseFG7gYggyFxA9AWyQf4tJx/kP77L1DL/Bs5j38DtdQT1nIjIY2ZsS0HaHgOG8iWBAOQFrwOu/EgWbL3TFrixhtpZpZzz0gAPfaw3RCf9w3O5yR++LkjOXHejeTHN97usJHnO5587GEPHi3A6EhAsBkbJEBkG14NDAzsB5C1gCk2AlpGwSgYBaNghAEAP2BcEhNUy8IAAAAASUVORK5CYII=","orcid":"","institution":"The University of Agriculture Peshawar","correspondingAuthor":true,"prefix":"","firstName":"Najeeb","middleName":"Ullah","lastName":"Khan","suffix":""},{"id":283413042,"identity":"cfd3b024-f1de-4783-8bf3-c46f0bc1ef52","order_by":2,"name":"Hamza Khan","email":"","orcid":"","institution":"The University of Agriculture Peshawar","correspondingAuthor":false,"prefix":"","firstName":"Hamza","middleName":"","lastName":"Khan","suffix":""},{"id":283413043,"identity":"59eff2cd-8f9f-4851-8452-d3934f4eae5c","order_by":3,"name":"Mikhlid H. Almutairi","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mikhlid","middleName":"H.","lastName":"Almutairi","suffix":""},{"id":283413044,"identity":"599ce5fd-77ac-4cb8-9dd5-4c3e2c323de5","order_by":4,"name":"Ijaz Ali","email":"","orcid":"","institution":"Gulf University for Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ijaz","middleName":"","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2024-03-25 04:50:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4160713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4160713/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53675426,"identity":"7aab88ea-8209-4d4a-8429-e5d59d2828de","added_by":"auto","created_at":"2024-03-28 18:39:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126328,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative gel picture of \u003cem\u003eHLA DRB1\u003c/em\u003e (*01 and *04): Representative gel picture in RA cases, which was run on 1.5% gel and aligned with a 100bp ladder for comparison W indicates wild and M indicates mutant allele.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4160713/v1/d17f98f96017a9ee3e1c137d.png"},{"id":53675423,"identity":"e6cb580d-5e33-42de-9028-3dec06829532","added_by":"auto","created_at":"2024-03-28 18:39:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125214,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative gel picture of \u003cem\u003eHLA DRB1\u003c/em\u003e (*12 and *15) Allele: Representative gel picture, which was run on 1.5% gel and aligned with a 100bp ladder for comparison\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4160713/v1/00a0d0a1e4485006a9bb0ce2.png"},{"id":53675434,"identity":"5030093b-6e5e-4f9b-bec6-468a45f90a17","added_by":"auto","created_at":"2024-03-28 18:39:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86891,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative gel picture of internal control primer: Representative gel picture of Internal control primer, which was run on 1.5% gel and aligned with a 100bp ladder for comparison.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4160713/v1/8dc767d585e05ce3c04c77cb.png"},{"id":53675764,"identity":"7ec47330-23a2-4aaf-8eab-bc8a0c5eac27","added_by":"auto","created_at":"2024-03-28 18:47:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":827146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4160713/v1/630b00bd-2f30-4fc1-8e97-c74cdcb60f97.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic risk assessment of HLA DRB1 alleles (*01, *04, *12 and *15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is a commonly diagnosed systemic chronic inflammatory disorder/condition that affects about 1% of the global population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is characterized by polyarthritis, immunologic abnormalities, autoimmune reactions, high comorbidity, and early death. It is also known to cause gradual joint destruction and impairment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Extracellular matrix degradation, which causes the normal breakdown of cartilage, tendons, bone, and ligaments, is a defining characteristic of RA. Beside this, different constitutional symptoms include fever, asthenia, weight loss, malaise and anorexia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. RA is mostly caused by interaction of genetic and environmental factors [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Other risk factors include age, gender and environmental exposure i.e., smoking, different air pollutants and infectious agents [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. RA is typically classified into two types seropositive or seronegative. The classification is based on presence or absence of rheumatoid factor and Anti-citrullinated protein antibodies [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis condition affects approximately 5 to 10 individuals out of every 1,000 in developed nations [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It's worth noting that from 1990 to 2017, RA had a significant impact on people's health worldwide, resulting in a loss of 3.4\u0026nbsp;million of life [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the period 1980\u0026ndash;2018, the prevalence of RA was 460 patients per 100,000 people worldwide with a prediction level of 95% (0.07\u0026ndash;1.29%) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The prevalence of RA is more in northern America and Europe as compared to other areas [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A study from 2011 state that prevalence of RA is 0.5 to 1% in Pakistan but it would be increasing as there is not much data present from Pakistani population [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The prevalence is more in women as compared to men in Pakistan [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent genetic factors are identified to be associated with RA. Among different genetic factors, \u003cem\u003eHLA DRB1\u003c/em\u003e alleles are investigated in several studies and found to be significantly associated with RA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This association has been identified in different populations across the world. The strength of different alleles varies in different population [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Other genetic factors associated with RA include tumor necrosis factor (TNF), corticotrophin releasing hormone (CRH), CYP\u003csub\u003e19\u003c/sub\u003e (aromatase), IFN-γ and other cytokines. Some study suggests that different auto immune disease such as insulin dependent diabetes, locus on chromosome X may be associated with RA [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe human leukocyte antigen (HLA) system, which correlates to the major histocompatibility complex (MHC) in humans, is critical in the antigen presentation of intracellular and extracellular peptides as well as the control of innate and adaptive immune responses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The conventional HLA loci are the class I (HLA-A, -B, -C, -E, -F, and -G) and class II (\u003cem\u003eHLA-DR\u003c/em\u003e, -DQ, -DM, and -DP) molecules known for their roles in antigen presentation to CD8\u0026thinsp;+\u0026thinsp;and CD4\u0026thinsp;+\u0026thinsp;T lymphocytes, respectively. They are transcribed by the most variable area in the human genome, a 4-Mb region of human chromosome 6p21 and are classified as MHC. MHC plays major role in immunity as well as risk associated with different auto immune diseases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of \u003cem\u003eHLA DRB1\u003c/em\u003e in susceptibility to RA is estimated to be 50% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The \u003cem\u003eHLA DRB1\u003c/em\u003e gene comprises of 6 exons with each exon encoding different domain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The leader peptide is encoded by exon 1, the extracellular domains by exons 2 and 3, the trans-membrane domain by exon 4, and the cytoplasmic tail by exon 5 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Exon 1 encodes amino acid patterns that make up the first hyper variable region (HVR1); Exon 2 encodes similar epitope motifs and the third region (HVR3). About 2960 alleles have been identified in \u003cem\u003eHLA DRB1\u003c/em\u003e gene out of which only few are identified to have role in RA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Several \u003cem\u003eHLA DRB1\u003c/em\u003e alleles have been identified as playing a critical role in the severity and susceptibility to rheumatoid arthritis. These alleles possess a sequence motif in the third HVR of the \u003cem\u003eHLA DRB1\u003c/em\u003e gene, referred to \"shared epitope (SE)\", which is composed of five amino acids at positions 70\u0026ndash;74 of the \u003cem\u003eHLA DRB1\u003c/em\u003e chain. Specifically, this SE contains one of three amino acid sequences: RRRAA, QKRAA or QRRAA [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. While the DERAA pattern seems to be primarily accountable for the beneficial protective effects [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The SE sequences facilitate the presentation of self-antigens to T cells, which is believed to be a critical factor in the onset of RA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Current studies suggest that, RA is primarily initiated by the specific binding of citrullinated peptides by HLA-DR molecules that possess the SE sequence. The \u003cem\u003eHLA-DRB1\u003c/em\u003e SE allele is found in 64\u0026ndash;82% of RA cases, a significantly higher occurrence compared to their first-degree relatives (53.9\u0026ndash;55%) and the healthy individual population (39\u0026ndash;52%). Approximately one-fifth of individuals with RA are estimated to lack the SE alleles. The autoimmune reaction in RA is believed to be triggered by the citrullination of self-peptides, which results in changes to their characteristics. This can cause the stimulation of immune responses and the production of specific ACPA, which are observed in about 75% of individuals with RA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are contradictory results regarding \u003cem\u003eHLA DRB1\u003c/em\u003e alleles association with RA. Many studies from different populations have proved its association with RA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, no such study is present from Pakistani KP population. Hence this study will help in determining the association of \u003cem\u003eHLA DRB1\u003c/em\u003e alleles especially \u003cem\u003eHLA DRB1\u003c/em\u003e*01, *04, *12, *15 with RA in Pakistani KP population.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEnrollment of patients and healthy controls\u003c/h2\u003e \u003cp\u003eThe theoretical and practical work for current research study was executed at Institute of Biotechnology and Genetic Engineering IBGE (Health lab), The University of Agriculture, Peshawar, Pakistan (August, 2022 to July, 2023). In the current study, data (Demographics, Pathological and blood samples) were collected from 100 RA patients. Patients with other complications were not selected for the current study. Besides this, data was collected from 80 healthy controls. Data had been taken on standard Performa attached, approved by the ethical committee of IBGE. Following a written consent, taken from both cases and healthy individuals, around 2 cubic centimeters (cc) of blood samples were obtained in Ethylene Diamine Tetra Acetic acid tubes (EDTA tubes) following established biosafety procedures during August 2022 to November 2022. The collected samples were stored at -20\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and primer designing\u003c/h2\u003e \u003cp\u003eThe extraction of Genomic DNA from obtained blood samples of both RA cases and healthy individuals were performed using salting out/non-enzymatic method already adopted in our lab [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, sequence specific primers were designed for selected Alleles using NCBI primer Blast software (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecific primers sequences designed for the detection of SNPs genotypes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlleles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProduct size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSequence 5'-3'\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1*\u003c/em\u003e 01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e255bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse Wild\u003c/p\u003e \u003cp\u003eReverse Mutant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTTGTGGCAGCTTAAGTTTGAAT\u003c/p\u003e \u003cp\u003eCTGCACTGTGAAGCTCTCAC\u003c/p\u003e \u003cp\u003eCTGCACTGTGAAGCTCTCCA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e260bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward Reverse Wild Reverse Mutant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGTTTCTTGGAGCAGGTTAAACA\u003c/p\u003e \u003cp\u003eCTGCACTGTGAAGCTCTCAC\u003c/p\u003e \u003cp\u003eCTGCACTGTGAAGCTCTCCA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1*\u003c/em\u003e 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e248bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAGTACTCTACGGGTGAGTGTT\u003c/p\u003e \u003cp\u003eCACTGTGAAGCTCTCCACAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e197bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTCCTGTGGCAGCCTAAGAG\u003c/p\u003e \u003cp\u003eCCGCGCCTGCTCCAGGAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmplification control primers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e796bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTGCCAAGTGGAGCACCCAA\u003c/p\u003e \u003cp\u003eGCATCTTGCTCTGTGCAGAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSequence specific primer (SSP) PCR\u003c/h2\u003e \u003cp\u003eThe Sequence specific primer (SSP) PCR technique was performed to check out the allele and genotype frequencies of certain SNPs in \u003cem\u003eHLA DRB1\u003c/em\u003e gene. The heterozygous and homozygous polymorphisms were observed in alleles using forward and reverse primers. To determine the allele and genotype frequencies for certain SNPs, A volume of 10 \u0026micro;L reaction mixtures were made twice (had different sets of forward but common reverse primers) consisting 2 \u0026micro;L (5x) Master mix, 1 \u0026micro;L template DNA, 0.3 \u0026micro;L of each reverse and forward primers and 6.4 \u0026micro;L of PCR water/buffer (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The amplified product was further confirmed through 1.5% gel electrophoresis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe optimized conditions of primers for PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlleles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDenaturation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnnealing Tm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExtension\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCycles\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C/5min 94\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.8\u0026deg;C/30sec(W) 55.9\u0026deg;C/30sec(M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026deg;C/1min 72\u0026deg;C/10min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C/5min 94\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.1\u0026deg;C/30sec(W) 60.1\u0026deg;C/30sec(M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026deg;C/1min 72\u0026deg;C/10min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C/5min 94\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.7\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026deg;C/1min 72\u0026deg;C/10min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C/5min 94\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026deg;C/1min 72\u0026deg;C/10min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInternal control\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C/5min 94\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u0026deg;C/30sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026deg;C/1min 72\u0026deg;C/10min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis for the collected research data was done using Statistical Package for Social Sciences (SPSS) software. The Chi-square test was employed to identify the demographic, pathological parameters and genetic polymorphism of RA cases. If the probability values are Lower than or equals to 0.05 the analysis was counted as statistically significant. The certain association of \u003cem\u003eHLA DRB1\u003c/em\u003e polymorphisms with RA risk was figured out through assessment of odd ratios and 95% confidence intervals by using Medcalc odd ratios calculator.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAllelic and genotypic frequencies distribution of\u003c/b\u003e \u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e01 and\u003c/b\u003e \u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e04 in RA cases and healthy Controls\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genotyping of \u003cem\u003eHLA DRB1\u003c/em\u003e*01 and \u003cem\u003eHLA DRB1\u003c/em\u003e*04 were performed using SSP-PCR procedure to check the allelic and genotypic frequencies of RA cases and healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our result indicated that, In \u003cem\u003eHLA DRB1*\u003c/em\u003e01, the frequency of variant A was higher in cases (49%) than healthy individuals (41.25%) and had non-significant association with RA (OR\u0026thinsp;=\u0026thinsp;1.37, 95% CI\u0026thinsp;=\u0026thinsp;0.89\u0026ndash;2.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1421). Similarly, the frequency of heterozygous CA genotype was found to be higher in patients (74%) than healthy individuals (47.5%) and significantly linked with the high risk of RA (OR\u0026thinsp;=\u0026thinsp;3.89, 95% CI\u0026thinsp;=\u0026thinsp;1.83\u0026ndash;8.25, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004). Moreover, the frequency of homozygous Mutant genotype AA was high in healthy individuals (17.5%) as compared to patients (12%) and non-significantly linked with the RA (OR\u0026thinsp;=\u0026thinsp;1.71, 95% CI\u0026thinsp;=\u0026thinsp;0.62\u0026ndash;4.67, P\u0026thinsp;=\u0026thinsp;0.2922). In case of \u003cem\u003eHLA DRB1*04\u003c/em\u003e, the frequency of variant A was almost same in healthy individuals (45%) and cases (44%) and observed to be non-significantly associated with RA (OR\u0026thinsp;=\u0026thinsp;0.96, 95% CI\u0026thinsp;=\u0026thinsp;0.63 to 1.45, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8495). Moreover, the frequency of heterozygous CA genotype was exactly the same in cases and healthy controls (60%) each respectively, indicating non-significant link with RA (OR\u0026thinsp;=\u0026thinsp;0.96, 95% CI\u0026thinsp;=\u0026thinsp;0.48 to 1.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9120). Furthermore, the frequency of homozygous mutant AA genotype was high in cases (14%) as compared to controls (12%) but it was non-significantly associated with RA (OR\u0026thinsp;=\u0026thinsp;0.89, 95% CI\u0026thinsp;=\u0026thinsp;0.34 to 2.35, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8263) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAllelic and genotypic frequencies distribution of \u003cem\u003eHLA DRB1\u003c/em\u003e*01 and \u003cem\u003eHLA DRB1\u003c/em\u003e*04 in RA cases and healthy Controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatients\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHealthy Controls N\u0026thinsp;=\u0026thinsp;80\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102(51%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94(58.75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98(49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66(41.25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.89\u0026ndash;2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1421\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenotypes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28(35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74(74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38(47.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.83\u0026ndash;8.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14(17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u0026ndash;4.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2922\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e112(56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88(55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88(44%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72(45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.63 to 1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8495\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenotypes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26(26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60(60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48(60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.48 to 1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(14%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12(15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34 to 2.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe frequencies distribution of\u003c/b\u003e \u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e12 and\u003c/b\u003e \u003cb\u003eHLA DRB1\u003c/b\u003e*\u003cb\u003e15 in RA cases and healthy controls\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genotyping of \u003cem\u003eHLA DRB1\u003c/em\u003e*12 and \u003cem\u003eHLA DRB1\u003c/em\u003e*15 were performed using PCR-SSP procedure to check the allelic frequencies of RA cases and healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Internal control primer was used to confirm the validity of PCR reaction in each tube (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The frequency of \u003cem\u003eHLA DRB1\u003c/em\u003e*12 was higher in cases (94%) than healthy controls (67.5%), which indicated high risk and significant association with RA (OR\u0026thinsp;=\u0026thinsp;7.54 95% CI\u0026thinsp;=\u0026thinsp;2.92\u0026ndash;19.47, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0001). Furthermore, the frequency of \u003cem\u003eHLA DRB1\u003c/em\u003e*15 was higher in cases (74%) than healthy controls (70%) yielding high risk but non-significant association (OR\u0026thinsp;=\u0026thinsp;1.22 95% CI\u0026thinsp;=\u0026thinsp;0.63\u0026ndash;2.34, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5519) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe frequencies distribution of \u003cem\u003eHLA DRB1\u003c/em\u003e*12 and \u003cem\u003eHLA DRB1\u003c/em\u003e*15 in RA cases and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95%CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCases N\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94(94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6(6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.92 to 19.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl N\u0026thinsp;=\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54(67.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26(32.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e\u003cb\u003e*15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCases N\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74(74%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26(26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.63 to 2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5519\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl N\u0026thinsp;=\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56(70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24(30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation of selected\u003c/b\u003e \u003cb\u003eHLA DRB1\u003c/b\u003e \u003cb\u003ealleles with demographic and clinical parameters of RA cases\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, the association of selected \u003cem\u003eHLA DRB1\u003c/em\u003e alleles with demographic and clinical parameters of different RA cases was explored. The results of our research indicated non-significant associations of \u003cem\u003eHLA DRB1\u003c/em\u003e\u003cb\u003e*\u003c/b\u003e01, \u003cb\u003e*\u003c/b\u003e04, \u003cb\u003e*\u003c/b\u003e12 with age groups (P\u0026thinsp;=\u0026thinsp;0.08, P\u0026thinsp;=\u0026thinsp;0.11, P\u0026thinsp;=\u0026thinsp;0.16) and rheumatoid factor (p\u0026thinsp;=\u0026thinsp;0.34, p\u0026thinsp;=\u0026thinsp;0.65, p\u0026thinsp;=\u0026thinsp;0.74) while \u003cem\u003eHLA DRB1\u003c/em\u003e\u003cb\u003e*\u003c/b\u003e15 exhibited significant association with both age group and rheumatoid factors (P\u0026thinsp;=\u0026thinsp;0.01 each). Additionally, all the selected alleles of \u003cem\u003eHLA DRB1\u003c/em\u003e gene showed non-significant associations with gender (P\u0026thinsp;=\u0026thinsp;0.81, P\u0026thinsp;=\u0026thinsp;0.52, P\u0026thinsp;=\u0026thinsp;0.27 and P\u0026thinsp;=\u0026thinsp;0.09). Similarly, these selected alleles exhibited non-significant association with ACPA (P\u0026thinsp;=\u0026thinsp;0.56, P\u0026thinsp;=\u0026thinsp;0.40 and P\u0026thinsp;=\u0026thinsp;0.0.65) except \u003cem\u003eHLA DRB1\u003c/em\u003e*04, which displayed non-significant association with ACPA (P\u0026thinsp;=\u0026thinsp;0.008) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe association of \u003cem\u003eHLA DRB1\u003c/em\u003e gene selected alleles with clinical parameters of Rheumatoid Arthritis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePARAMETERS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c14\" namest=\"c9\"\u003e \u003cp\u003e\u003cem\u003eHLA DRB1\u003c/em\u003e*04\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge Group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eCA (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAA (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP VALUE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCC (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eCA (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eCC (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eP VALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;35 years\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(22.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e24(66.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e04(11.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e06(12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e26(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e04(62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;35 years\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e06(9.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e50(78.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e08(12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20(31.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e34(54.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10(14.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10(13.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e54(72.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e10(13.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e18(24.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e44(59.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e12(16.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e04(15.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e20(76.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e02(7.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e08(30.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e16(61.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e02(7.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRheumatoid Factor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e08(80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e02(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e02(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e06(30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e02(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14(15.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e66(73.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e10(11.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24(26.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e54(60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e12(13.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eACPA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e02(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e18(90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0(00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10(50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e10(50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0(00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e56(70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e12(15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e50(62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14(17.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePARAMETERS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e\u003cb\u003e*12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c14\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003eHLA DRB1\u003c/b\u003e\u003cb\u003e*15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge Group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eWILD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMUTANT(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP VALUE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eWILD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eMUTANT(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003cp\u003eVALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;35 years\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0(0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e36(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e8.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e04(11.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e32(88.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e4.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;35 years\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e06(9.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e58(90.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e22(34.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e42(65.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e06(8.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e68(91.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e16(11.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e58(88.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0(00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e26(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e10(34.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e16(65.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRheumatoid Factor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0(00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e10(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e06(60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e04(40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e06(6.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e84(93.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e20(22.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e70(77.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eACPA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e02(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e18(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e06(30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e14(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNegative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e04(5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e76(95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e20(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e60(75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRheumatoid arthritis (RA) constitutes a chronic inflammatory disorder primarily affecting women, with a female-to-male ratio of approximately 3:1. The characteristic feature of this condition is symmetrical polyarthritis, which gradually results in the deterioration of joints over time [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The worldwide prevalence of RA is approximately 1%, while in Western populations, the incidence ranges from 1\u0026ndash;2%. In Pakistan, the prevalence is observed to be around 0.5% [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Approximately 0.55% of the urban population in northern Pakistan is affected by rheumatoid arthritis, whereas in southern Pakistan, the incidence rate is approximately 0.14% [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. RA is caused by a combination of genetic and environmental factors. \"The presence of specific genes, namely HLA class II genotypes, plays a vital role in the development and progression of RA [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Approximately 60% of the risk for developing RA is due to genetics, and within that the impact of the HLA is expected to be around 11\u0026ndash;37%. The most relevant HLA gene variants associated with RA susceptibility are \u003cem\u003eHLA-DRB1\u003c/em\u003e*01, \u003cem\u003eDRB1\u003c/em\u003e*04, \u003cem\u003eDRB1*\u003c/em\u003e12 and \u003cem\u003eDRB1\u003c/em\u003e*15 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The significant link between specific alleles and rheumatoid arthritis (RA) varies across populations in distinct geographic regions and among diverse ethnic groups. Therefore, it is imperative to examine various alleles of the HLA gene in RA cases from various populations and geographic areas, exploring whether they play a protective role or act as susceptibility factors [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study we analyzed the frequencies of \u003cem\u003eHLA DRB1\u003c/em\u003e*01, \u003cem\u003eHLADRB1\u003c/em\u003e*04, \u003cem\u003eHLADRB1\u003c/em\u003e*12 and \u003cem\u003eHLA DRB\u003c/em\u003e*15 polymorphism in 100 RA cases and 80 healthy controls. Our findings suggested that CA genotype of \u003cem\u003eHLA DRB1\u003c/em\u003e*01 was significantly linked with the high risk of RA (OR\u0026thinsp;=\u0026thinsp;3.89, 95% CI\u0026thinsp;=\u0026thinsp;1.83 to 8.25, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004) while the variant A (OR\u0026thinsp;=\u0026thinsp;1.37, 95% CI\u0026thinsp;=\u0026thinsp;0.89\u0026ndash;2.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1421) and mutant genotype AA (OR\u0026thinsp;=\u0026thinsp;1.71, 95% CI\u0026thinsp;=\u0026thinsp;0.62\u0026ndash;4.67, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2922) was non-significantly linked with RA risk. In case of \u003cem\u003eHLA DRB1\u003c/em\u003e*04, variant A (OR\u0026thinsp;=\u0026thinsp;0.96, 95% CI\u0026thinsp;=\u0026thinsp;0.63 to 1.45, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8495), Heterozygous CA (OR\u0026thinsp;=\u0026thinsp;0.96, 95% CI\u0026thinsp;=\u0026thinsp;0.48 to 1.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9120) and mutant AA genotype was observed to be non-significantly associated with RA risk (OR\u0026thinsp;=\u0026thinsp;0.89, 95% CI\u0026thinsp;=\u0026thinsp;0.34 to 2.35, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8263). Furthermore, the \u003cem\u003eHLA DRB1\u003c/em\u003e*12 indicated high risk and significant association with RA (OR\u0026thinsp;=\u0026thinsp;7.54 95% CI\u0026thinsp;=\u0026thinsp;2.92\u0026ndash;19.47, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0001). While \u003cem\u003eHLA DRB1\u003c/em\u003e*15 yielded high risk but exhibited non-significant association with RA (OR\u0026thinsp;=\u0026thinsp;1.22 95% CI\u0026thinsp;=\u0026thinsp;0.63\u0026ndash;2.34, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.5519). Moreover, the selected alleles of \u003cem\u003eHLA DRB1\u003c/em\u003e gene (*01, *04 and *12\u003cem\u003e)\u003c/em\u003e showed non-significant association with age groups and RF while \u003cem\u003eHLA DRB1\u003c/em\u003e*15 indicated significant association (p\u0026thinsp;=\u0026thinsp;0.01 each). In terms of gender all the selected alleles displayed non-significant associations. Similarly, the selected alleles \u003cem\u003eHLA DRB1\u003c/em\u003e *01, *12, *15 indicated non- significant association with ACPA. While \u003cem\u003eHLA DRB1\u003c/em\u003e *04 showed significant association (p\u0026thinsp;=\u0026thinsp;0.008). Our results were consistent with the findings of other study performed in Iran, China, Pakistan and Syria population revealing non-significant association of \u003cem\u003eHLA DRB1\u003c/em\u003e alleles (*01, *04, *15) with RA risk [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, findings of Sudan population regarding \u003cem\u003eHLA DRB1\u003c/em\u003e*04 didn\u0026rsquo;t aligned with our results [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, significant association of \u003cem\u003eHLA DRB1\u003c/em\u003e*12 in Korean population aligns with our findings in KP population [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our study revealed the intricate role of \u003cem\u003eHLA DRB1\u003c/em\u003e gene in RA risk within the KP population. While highlighting the importance of this study, there are certain constraints which need to be acknowledged. These constraints include limited sample size which is not enough to fully capture intricate genetic variation within this diverse ethnic group. Furthermore, our study primarily concentrated on limited number of alleles within the extensive \u003cem\u003eHLA DRB1\u003c/em\u003e gene, suggesting the need for further exploration into additional potential polymorphisms. Hence future studies should aim to incorporate a wider and more diverse dataset employing advanced whole-genome sequencing techniques to expose additional genetic factors contributing to RA. Such approach will enable a more thorough comprehension of the genetic makeup associated with RA, thereby potentially advancing the development of more efficient diagnostic and therapeutic approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted at Institute of Biotechnology and Genetic Engineering, Peshawar, Pakistan, under NO: IBGE/2023/007.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA written consent was taken from all the participants both RA patients and healthy controls after clearly briefing the aims and objectives of the study in local language.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have read and approved the article for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the necessary data are included in manuscript, related data will be provided on request from corresponding author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding received to conduct the study, the authors contributed for the study \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZI did the experimental work and wrote the first draft under the supervision of NUK. HK help in data collection, and data analysis. NUK, MHA and IA critically review the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrandota J (2023) Solving the New Puzzle (s) 2014\u0026ndash;2022: Flammer Syndrome Probably Represents a Mild Endophenotype of Raynaud\u0026rsquo;s Phenomenon Mainly Associated with Ocular Disturbances and Also Due to Latent T. gondii Infection. 3(1):1\u0026ndash;52\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarsini S, Rezaei N (2023) Autoimmune diseases. Clinical Immunology. Elsevier, pp 123\u0026ndash;244\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrassi W et al (1998) \u003cem\u003eThe clinical features of rheumatoid arthritis.\u003c/em\u003e 27: p. 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Clin Exp Rheumatol 21(4):489\u0026ndash;492\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HLA DRB1 gene, Alleles, Rheumatoid arthritis risk","lastPublishedDoi":"10.21203/rs.3.rs-4160713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4160713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRheumatoid arthritis (RA) is a chronic autoimmune condition influenced by a complex interplay of genetic and environmental factors. Specific genetic variations, particularly in the human leukocyte antigen (HLA)-DRB1 gene, have been strongly linked to RA susceptibility. HLA-DRB1 alleles, part of the major histocompatibility complex (MHC) class II gene family, play a significant role in the immune system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAims: \u003c/strong\u003eThis study aimed to investigate the association between HLA-DRB1*01, *04, *12, and *15 alleles and RA risk within the Pashtun population of Pakistan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A cohort of 100 RA patients and 80 healthy controls was recruited for this study. DNA was isolated from blood samples using the salting-out method, followed by genotyping using sequence-specific primer polymerase chain reaction (SSP-PCR). The association of the selected alleles with clinicopathological and demographic parameters of RA was assessed using the Medcalc odds ratio calculator and Chi-square test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Allelic and genotypic analysis revealed that HLA-DRB1 *01 and *04 variants exhibited non-significant associations with RA risk (P=0.1421 and 0.8495, respectively). Similarly, mutant genotypes of HLA-DRB1 *01 and *04 showed non-significant associations with RA (P=0.2922 and P=0.8263, respectively). However, the heterozygous CA genotype of HLA-DRB1 *01 demonstrated a significant association with RA (P=0.0004), whereas HLA-DRB1 *04 did not (P=0.9120). Furthermore, HLA-DRB1 *12 showed a significant association with increased RA risk (P=0.0001), while HLA-DRB1 *15 did not (P=0.5519). Additionally, HLA-DRB1*01, *04, and *12 did not show significant associations with age group (P=0.08, P=0.11, P=0.16) and rheumatoid factor (RF) status (P=0.34, P=0.65, and P=0.74), whereas HLA-DRB1 *15 exhibited non-significant associations with age group and RF (P=0.01 each). Moreover, associations of HLA-DRB1 *01, *04, *12, and *15 with gender (P=0.81, P=0.52, P=0.27, and P=0.09, respectively) and anti-citrullinated protein antibody (ACPA) status (P=0.56, P=0.40, P=0.65, and P=0.008, respectively) were not significant, except for HLA-DRB1 *04, which displayed a significant association with ACPA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e HLA-DRB1 *12\u003cem\u003e \u003c/em\u003eand heterozygous genotypes of HLA-DRB1 *01 were significantly associated with RA risk in the Pashtun population. However, further analysis utilizing whole exome sequencing with larger datasets is warranted for more precise results.\u003c/p\u003e","manuscriptTitle":"Genetic risk assessment of HLA DRB1 alleles (01, 04, 12 and 15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 18:38:52","doi":"10.21203/rs.3.rs-4160713/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d21721b-3197-4f93-847e-7080c75da00b","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-04T01:14:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-28 18:38:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4160713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4160713","identity":"rs-4160713","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}