Genotypic Analysis Revealed Association of HLA Alleles with Clinical Parameters in Bangladeshi Patients with Rheumatoid Arthritis

This study investigated distribution of HLA alleles (HLADRB1*01, *03, *04, *07, HLA-DQB1*0201, *0301/4) in 34 healthy controls and 57 rheumatoid arthritis (RA) patients in a Bangladeshi population and correlated the genotypic frequencies with clinical parameters. Frequency distribution of HLADRB1*04 (34%) and HLA-DRB1*01 (32%) were the highest followed by HLADQB1*0301/4 (29%) and HLA-DQB1*0201 (26%) in RA patients while HLADRB1*03 (12%) had lowest frequency. Plasma level of anti-CCP and rheumatoid factor antibodies confirmed diagnosis of RA patients that varied significantly between patients and healthy controls. Likewise, plasma levels of C-reactive protein, triglycerides, cholesterol, HDL-cholesterol and activities of AST and ALT exhibited significant variation between the two groups. In contrast, the levels of glucose, total protein, uric acid, LDL-cholesterol and plasma activity of ALP in RA patients had no significant deviations from healthy controls. Relationship between HLA genotype frequency and clinical parameters revealed that the mean levels of anti-CCP and rheumatoid factor antibodies were highest in the patients harboring HLA-DRB1*04 allele. These findings underpin the correlation between HLA genotype with clinical markers of RA which are indicative of disease severity. The positive correlation of these markers with certain HLA genes may be used to identify susceptible individuals who are likely to have RA in Bangladeshi population.


Introduction
Rheumatoid arthritis (RA) is characterized by chronic inflammation caused by autoimmune disease attacking joints that leads to pain, swelling, stiffness and eventual destruction of joints. RA is thought to be influenced by both genetic predisposition and environmental factors. Based on numerous studies focusing on the molecular basis and genetic predisposition to uncover the risk-factors of RA, it has been well surmised that high-risk genetic context combined with epigenetic and environmental factors leads to the manifestation of the RA symptoms [1] [2] [3] [4] [5]. Three factors-genetic-make up, environment and autoimmunity that contributes to the onset and progression of RA, have been termed as the "Bermuda triangle" [1]. In recent years with the advent of new technologies, our understanding of the aberrant immune functions leading to infiltration and inflammation of the synovium has advanced significantly. The exact triggers and their timing that causes the onset of RA remain elusive. The heritable contribution in the form of genetic predisposition in the pathogenesis of RA has been estimated to be about 60% that including human leukocyte antigens (HLA-DR) and non-HLA genes [2] [3]. It was hypothesized that the genetic factor when combined with certain environmental exposure and lifestyle-related factors such as smoking may trigger the onset of RA by inducing the production of anti-cyclic citrullinated peptide antibody (ACCP) [4].
Despite numerous attempts to uncover the comprehensive genetic basis of RA, only HLA-DRB1 allele was found to be linked with risk of RA beyond any reasonable doubt [5]. Different allelic variations of HLA-DRB1 gene have been linked with RA risk and onset, suggesting a possible role of interaction between T-cell receptor (TCR) and HLA-antigen in the underlying pathological process of RA [6]. In an attempt to uncover the underlying mechanism of certain HLA association with RA, Gregerson et al. proposed a hypothesis called "Shared Epitope (SE)" for the association of different HLA-DRB1 alleles with RA. The study showed an RA associated HLA-DRB1 allele encodes a five amino acid long conserved sequence (QRRAA, RRRAA or QKRAA) that resides within the third hyper variable region (HVR3) of the DRb1 chain. These residues form a part of the helical domain of the antigen-binding site and thus are likely to directly affect the antigen presentation process. The HLA alleles that are shown to encode this conserved amino acid sequence include: DRB1*0401, *0404, *0405, *0408, *0101, *0102, *1402, *09 and *1001 [5]. In different population groups representing different ethnicity the frequency of the HLA alleles that are associated with RA vary significantly [5]. For instance, DRB1*0401 and *0404 alleles are frequently associated with RA in Caucasians whereas DRB1*0405 and DRB1*0101 alleles are found to be highly associated with RA in Japanese and in Israeli Jews respectively [5]. However, no such studies have been conducted to untangle the HLA genotyping associated with RA in Bangladesh.
It is apparent that in order to minimize disease progression, early diagnosis is of utmost importance to better manage the RA patients. Unfortunately, the early F. Shaona et al. American Journal of Molecular Biology diagnosis of RA is still challenging. Since autoantibodies are directly linked with the onset of autoimmune disease, rheumatoid factor (RF)-an autoantibody that targets the Fc portion of IgG, has been one of the most important markers for the RA diagnosis. However, the non-specificity RF has been a major challenge as RF levels can also be elevated in patients that are suffering from other diseases e.g., Sjogren's syndrome and type 2 cryoglobulinaemia [6], infection (such as HCV infection) [8] and in elderly persons [7]. Due to limited specificity of RF, around 60% -70% of RA patients are diagnosed as RF positives. Only 11 -20% of RF positive patients suffering from masculoskeletal problems have RA. For these reasons RF factor is considered as a good indicator of disease severity rather than diagnostic value [7]. Another serological marker for RA is anti-cyclic citrullinated peptide (anti-CCP) antibodies that are reactive to the citrulline amino acid and are observed to be elevated in the sera of patients with RA [9]. In contrast to RF, anti-CCP antibody is observed to have increased specificity when it comes to RA diagnosis and has emerged as an alternative maker for RA [10].
Although RF may have lower specificity in RA diagnosis, this particular serological marker has a great value as a predictor of RA severity and thus is considered as prognostic marker for RA [11]. Elevated levels of RF are found to be associated with risk of developing RA [12]. RA patients with elevated RF levels are prone to develop a more aggressive form of the disease. Rheumatoid nodules are commonly observed as a manifestation of the elevated RF in the serum of RA patients [13].
The evidence of HLA association with the risk of developing RA, prompted the question whether HLA polymorphisms have a role in the inflammatory reaction and thus can be used as a risk predictor for the aggressiveness of rheumatoid synovitis. Studies based on retrospective analysis showed that HLA genotyping is indeed associated with aggressiveness of rheumatoid synovitis [6] [14]. Alleles such as HLA-DR B1*01 are found to be enriched in RA patients characterized by RF-negative representing a variant of RA known to slowly progress compared to RF positive variant of RA. In contrast HLA-DRB1*0401 allele was found to be frequently associated with RA positive variant of RA that is considered as a more aggressive form of the disease [14]. These studies have opened a new window of opportunity to evaluate the risk of developing the aggressive form of RA simply by analyzing the HLA genotypes. Other studies, evaluating the utility of HLA genotyping to predict the progression of RA in prospective manner, claimed that HLA-DRB1*04 positive RA patients are susceptible to develop inflammatory activities in the joints at the early stage of the disease that causes subsequent destruction of joints [15]. Although HLA-genotyping holds much promise as a prognostic marker, the precise value of HLA genotyping to predict disease progression is still not clear. In addition, the association of HLA genotyping and the levels of disease progression marker-RF have not been studied previously.
The current study was specifically designed to evaluate the association between HLA genotyping and RA propensity in Bangladeshi population. Moreover, in this study we sought to uncover the possible link between enrichment of certain HLA DRB1 alleles in the RA patients with elevated levels of clinical parameters such as RF and anti-CCP antibody. In summary the current study has the potential to shed light into the plausible association between HLA genotyping with RA progression by stratifying the patients based on their HLA-allele frequency and clinical parameters-RF and anti-CCP antibodies.

Study Design, Enrollment of Study Participants and Sample Collection
A schematic diagram representing the workflow of the study is shown in Figure 1.

Determination of the Level of Anti-Cyclic Citrullinated Peptide and Rheumatoid Factor Antibodies
Anti-cyclic citrullinated peptide (anti-CCP) and RF levels were measured using routine clinical procedures. Briefly, the measurements were carried out by utilizing the immunoassays for the semi-quantitative determination of the IgG class of auto-antibodies specific to cyclic citrullinated peptide (CCP) and RF mediated agglutination in plasma samples using ABBOTT reagent kit.

Measurement of Biochemical Parameters
Plasma levels of c-reactive protein (CRP), total protein, uric acid, cholesterol,

HLA-Genotyping by Polymerase Chain Reaction
White blood cells of blood samples were used to extract genomic DNA by organic extraction procedure as described by Afruza et al. [16]. All DNA samples were subjected to genotyping for HLA-DRB1 and HLA-DQB1 using PCR-SSP  Table 1.

Statistical Analyses
Demographic data obtained from the structured questionnaire and clinical parameters were analysed using SPSS v21.0 (IBM) in which the results were expressed as mean ± SD for continuous variables and % for categorical variables.
To compare the differences between different variables from the control and patients, Student's independent t-test was performed using Statistical Package for Social Sciences (SPSS, v21).

Identification of HLA Alleles Using Gel Electrophoresis
Amplified PCR products representing the distinct HLA alleles were identified according to the band size by gel electrophoresis. For instance, the presence of

Levels of Anti-Cyclic Citrullinated Peptide and Rheumatoid Factor Antibodies in the Plasma of Study Participants
In order to confirm rheumatoid arthritis, the levels of anti-cyclic citrullinated  (RF) (104.6 ± 22.29 U/mL). The patients suffering from rheumatoid arthritis exhibited significantly higher anti-CCP antibody and RF level (≥10 IU/mL) compared to normal individual (Figure 3(a) and Figure 3(b)). The mean of age of the control and the RA groups were 23.5 ± 11.0 and 50.67 ± 14.29, respectively.
The anthropometric and demographic data of the healthy individuals as well as the potential RA patients are given in Table 2.

Uric Acid, CRP, Glucose and Total Protein Levels in the Plasma of Study Participants
The mean levels of plasma uric acid of healthy subjects and RA patients were On the contrary the mean plasma CRP level (3.69 ± 0.59 mg/dL for healthy control and 9.92 ± 1.26 mg/dL for RA patients) and plasma total protein level (7.99 ± 0.26 mg/dL for healthy control and 6.80 ± 0.14 mg/dL for RA patients) varied significantly between control group and RA patients as revealed by t-test. The data for biochemical parameters among the study participants are given in Table 3.

Lipid Profile Analysis of the Study Participants
The mean triglyceride (TG) levels in the plasma of healthy controls and RA patients were 112.2 ± 8.82 and 234.4 ± 16.54 mg/dL, respectively. Their mean plasma cholesterol levels were 116.4 ± 6.98 and 219.7 ± 11.30 mg/dL, respectively. In both cases significant variation was observed between healthy subjects and RA patients. To further evaluate the lipid profile, HDL-and LDL-cholesterol levels were measured. The mean plasma HDL cholesterol in RA patients (36.45 ± 6.5 mg/dL) was significantly lower compared to that of healthy controls (42.58 ± 9.3 mg/dL). Unlike HDL-cholesterol, determination of LDL-cholesterol level did not exhibit any significant difference between healthy controls (125.69 ± 11.6 mg/dL) and RA patients (132.5 ± 5.8 mg/dL). Data have been presented in Table 4.

Activities of Aspartate Aminotransferase, Alanine Aminotransferase and Alkaline Phosphatase in the Plasma of the Healthy Controls and RA Patients
The activity of AST for the healthy individuals and RA patients showed significant deviation upon statistical analysis. The mean value for controls was 6.50 ±  Figure 4(a)). The mean level of ALT in the plasma of healthy controls was 6.809 ± 0.98 U/L, and for patients, the value was 10.46 ± 0.8397 U/L. Statistical analyses revealed a significantly increased ALT level in the RA patients in comparison to that of healthy individuals (Figure 4(b)).
In addition, measurement of Alkaline phosphatase (ALP) levels for controls and RA patients revealed the mean ALP levels for control and RA patients were 86.72 ± 6.56 and 33.67 ± 2.53 U/L, respectively. Interestingly unlike ALT and AST, the ALP levels exhibited the opposite trend as it was observed to be significantly lower in the RA patients compared to the healthy individuals (Figure 4(c)).

among the Study Subjects
To investigate the association of HLA-alleles with RA phenotype, it is important showed an interesting phenomenon. Highest frequency for this HLA allele was observed for United States (Figure 5(b)).

Distribution of Anti Cyclic Citrullinated Peptide and Rheumatoid Factor Antibodies among the Study Subjects
Subsequently we sought to identify the correlation between patient-specific HLA-

Discussion
The current study investigated HLA-allele distribution in 57 rheumatoid arthri- The association of various biochemical parameters, e.g. uric acid, C-reactive protein (CRP), glucose and total protein levels, with RA has been reported. For example patients with progressive RA are considered to be at risk of having high serum uric acid [20], CRP levels [21] and abnormal glucose metabolism [22]. In line with these findings we also observed high CRP levels in RA patients. CRP is an effective marker of systemic inflammation and considered as the potent biomarker for the onset of cardiovascular disease associated with RA [23]. However, no significant elevation for serum uric acid, glucose and total protein levels was observed in RA patients in comparison with normal subjects. The measurement of lipid profiles of the study subjects revealed a positive correlation of dyslipidemia and RA since, the RA patients exhibited significantly higher levels of TG, total cholesterol and lower levels of HDL compared to the healthy counterparts.
Previously it was reported that RA patients have a higher risk of developing pre-mature cardiovascular disease as evident by their lower HDL cholesterol levels [24]. Another study conducted by Alizade et al. demonstrated the association of RF levels with the extent and severity of coronary artery disease [25]. The risk of cardiovascular disease in RA patients also emerges from the inability of RA patients to manage the risk through exercise. Therefore, it is of utmost importance to monitor the lipid profile in RA patients followed by intervention to minimize the long-term risk of developing cardiovascular diseases. The higher levels of AST and ALT in the RA patients than the healthy controls can be attributed to the medication as part of initiating disease-modifying antirheumatic drugs (DMARD) therapy that the patients were receiving through the study period. The hepatocoxic side-effects of rheumatoid arthiritis drugs such as methotrexate (MTX), leflunomide (LEF) have been reported earlier [26]. These drugs, due to their cytotoxic effects, were deemed responsible for the elevated levels of AST and ALT in RA patients who receive these drugs [26]. In contrast to AST and ALT, intriguingly, ALP level in RA patients was found to be lower compared to healthy individuals. Earlier studies have found the higher levels of ALP in RA patients although the underlying etiology has not been well understood [27].
In order to establish a link between HLA genotyping and clinical parameters of rheumatoid arthritis-anti-CCP and RF antibody, six different HLA alleles (HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DQB1*0201 and HLA-DQB1*0301/4) were analyzed among the study subjects. The selection of these alleles was based on the systematic analysis of the allele frequency of different HLA alleles in South-East Asian population. Based on the frequency analysis, we selected these six different HLA alleles that represent South-East Asian population diversity with respect to HLA-alleles. Analyses of the genotypic distribution of HLA genes in the study subjects indicated that the alleles were widely distributed throughout healthy subjects and RA patients. An earlier study reported a possible association of HLA-DRB1*01 allele with rheumatoid arthritis in populations representing a wide variety of ethnicity and geographical locations [28] [29] [30]. Another study showed the association of the propensity of RA with HLA-DRB1*04 in Chinese population [31]. Furthermore, study with RA patients among Syrian population was found to be strongly associated with both HLA-DRB1*04 and HLA-DRB1*01 [32]. However, no such association has been shown for South-East Asian population. North American Rheumatoid Arthritis Consortium, performed a study involving a large cohort of study-subjects of the North American origin, have uncovered a significant association between the susceptibility of RA and HLA-DRB1*04 [33].
In our study the frequency of RA patients harboring HLA-DRB1*04 allele (34%) was found to be double than that of in healthy individuals (17%) indicating the frequency occurrence of this particular allele is relatively higher in RA patients.
Next we asked whether an association could be established between HLA alleles and RA specific clinical parameters. To achieve this, we first divided the study subjects including the healthy controls and RA patients into different groups according to the presence of distinct HLA alleles and then, the levels of anti-CCP and RF antibodies were compared between the groups. No notable pattern was observed with respect to distribution of anti-CCP antibody levels among the different HLA-groups ( Figure 6(a)) though HLA-DRB1*04 positive RA patients had the highest and HLADQB1*0301/4 positive patients had the lowest levels.
Surprisingly, for RF antibody a strong pattern emerges, which indicated that the RA patients carrying HLA-DRB1*04 allele exhibited significantly higher levels of RF antibody compared to the RA patients having other HLA-alleles. This striking finding indicated that HLA-genotyping has the potential not only to estimate the propensity of RA but also can be utilized to RA progression and severity.
Previously, studies have established a potential link between the RF and with disease activity in rheumatoid arthritis [34].
The scarcity of genotypic studies of South-East Asian population, has posed a major challenge in the estimation of the percentage of population who are at risk