Introduction

Rheumatoid arthritis [RA (OMIM: 180300)] or susceptibility to rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints. RA is characterized by joint swelling, tenderness, and destruction of synovial joints leading to severe disability and premature mortality [1, 2]. Elevation of the acute-phase reactants erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) indicates the maintenance of activity in patients with RA. The precise aetiology of RA has not yet been established. There is no known specific therapy for RA, but treatment can slow the progression of the disease, indicating the importance for earlier diagnosis and effective disease-suppressing therapy to prevent or minimize the occurrence of the symptoms of RA.

The prevalence of RA in Caucasians (North America and Europe) is 0.5–1% of the general population [2,3,4], but the prevalence varies between 3 and 8% depending on the environmental and genetic risk factors (2–3 times more common in females than in males). Approximately 80% of RA patients develop the disease between 35 and 50 years. The mortality rate of these patients is at least twice as high as in the healthy population.

Stastny [5] first established that RA was associated with HLA-DRw4, while subsequent studies confirmed an association with the HLA-DRB1 gene [6] and the strongest association with HLA-DRB1*04 [7]. This association is observed in many populations; however, in different ethnic groups, a connection has been established between other variants of the HLA-DRB1 gene locus and RA, which may occur together with HLA-DRB1*04 or separately. A particular subset of HLA-DRB1 gene variants (alleles at DRB1*01, *04, and *10 groups), called shared epitope (SE) alleles, is the most important genetic contributor to the risk of developing RA. The genetic variants that are negatively linked to the development of RA are DRB1*01, DRB1*07, and DRB1*13, which are considered to have a protective role in increasing RA.

The HLA-DRB1 gene is part of a family of genes called the human leukocyte antigen (HLA) complex. The HLA complex is the human version of the major histocompatibility complex (MHC). Researchers have identified at least 2479 different versions of the HLA-DRB1 gene (the Immuno Polymorphism Database (IPD)—the international ImMunoGeneTics (IMGT); IPD-IMGT/HLA Release 3.36.0, http://www.ebi.ac.uk/imgt/hla/).

The aims of this study were to compare inflammation biomarkers values, the ESR and CRP among the other haematological and biochemical parameters, between patients with RA and a healthy control group of females of the Public Institution Health Centre of Sarajevo Canton (Bosnia and Herzegovina) and estimate the frequency of the HLA-DRB1 gene variants, the possible contributions of the predisposing and protective gene variants for the risk of developing RA in females using a low-resolution (two-digit) polymerase chain reaction with sequence-specific primers (PCR-SSP) technique.

Patients and methods

Study groups

The peripheral blood samples were collected from 83 subjects with a diagnosis of RA and 164 healthy donors of the Hospital Novo Sarajevo (the Public Institution Health Centre of Sarajevo Canton, Bosnia and Herzegovina) without a diagnosis of RA and inflammation who came for a medical check-up between April 2015 and June 2018. A total of 98.8% of the participants were females. When collecting samples, ethical principles of research were considered. Data relating to the age and sex were reported by donors, and all the participants included in this analysis signed written informed consent. The study was approved by the Research Ethics Committee of the Public Institution Health Centre of Sarajevo Canton, Bosnia and Herzegovina (01-08-5080-2/16 from 25.08.2016).

Haematological and biochemical analyses

A cross-sectional study was performed on 80 RA patients and 82 healthy control subjects. All haematological and biochemical analyses were carried out in the Department of Laboratory Diagnostics of the Hospital Novo Sarajevo. The haematology tests included the CBC (complete blood count) with measurements of the red blood cells (RBC), the white blood cells (WBC) and the platelets (PLT) counts, hemoglobin (HGB), and hematocrit (HCT). We additionally investigated the erythrocyte constants mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width (RDW), and red cell distribution width standard deviation (RDW-SD) along with the platelet constant mean platelet volume (MPV). The differential blood count (DBC) included the number of neutrophils (NE), eosinophils (EO), basophils (BA), monocytes (MO), and lymphocytes (LY). The ESR and CRP levels were also analysed. Moreover, the neutrophil and lymphocyte ratio (NLR) and the platelets and lymphocyte ratio (PLR) were determined.

The samples of peripheral blood were taken in accordance with good laboratory practice standards. For general haematological examination, 3 mL of blood was collected in a vacuum tube containing anticoagulant ethylenediaminetetraacetic acid (EDTA). Haematological parameters were measured on the automated Beckman Coulter DxH 800 Haematology Analyzer. For ESR, 1.8 mL of blood was collected into a vacuum tube with sodium citrate and placed in a 150 mm graduated pipette. The samples for analysis of CRP were collected in a vacuum tube, centrifuged for 10 min at 3500 rpm, and separated from the serum. The CRP was measured by a standard CRP test on the Roche/Hitachi Cobas C311 Chemistry Analyzer (Boehringer Mannheim, Germany).

HLA genotyping

The molecular analysis was carried out on a group of 83 patients with a diagnosis of RA (n = 83; 2n = 166) and 82 control subjects (n = 82; 2n = 164) in the Laboratory of Genetics in the Department of Biology (Faculty of Science of the University Sarajevo, Bosnia and Herzegovina). The control group included healthy subjects who did not have a diagnosis of RA.

Genomic deoxyribonucleic acid (DNA), previously isolated from whole blood collected in EDTA using the salting out procedure [8], was subjected to PCR amplification. For the amplification of the HLA-DRB1 gene locus, the HLA-Ready Gene Class II test system (HLA-Ready Gene DR, Inno-Train, Germany) was used according to the manufacturer’s instructions. This system enables low-resolution typing of HLA-DRB1, DRB3, DRB4, and DRB5 gene loci.

Each sample was genotyped by a set of 24 PCRs. PCR samples in which the primer binds to its specific target have a specific amplification following PCR, while samples without this primer-specific binding do not. The positive control amplicon (HGH, human growth hormone) in each reaction tube is present in two different sizes (430 bp and 800 bp), with the exception of the first tube containing a negative control (NC).

The DNA samples (approximately 50 ng/µL of concentration) were added to the master mix containing distilled water, Ready PCR (deoxyribonucleotide triphosphates—dNTPs, PCR buffer, cresol red and glycerine) and Taq polymerase (5 U/µL, Axi-Taq DNA Polymerase, Inno-Train, Germany). The program has been validated with a thermocycler Eppendorf Mastercycler gradient (Hamburg, Germany). The PCRs were carried out in 10 µL volumes. Samples were first denatured at 96 °C for 2 min, followed by 10 cycles of 96 °C for 15 s, 65 °C for 60 s, 20 cycles of 96 °C for 15 s, 61 °C for 50 s, and 72 °C for 30 s. Evaluation of the results was performed by 2% agarose gel electrophoresis in Tris/Borate/EDTA (TBE) buffer at 70 V for 15–20 min. The bands were visualized under ultraviolet light (UV) by intercalating ethidium bromide (EtBr, 0.7 µg/µL). Interpretation of the results was performed using kit manufacturer’s data sheets.

Statistical analysis

Results were expressed as median and range of values (minimum and maximum), the number, mean ± standard deviation (mean ± SD), decimal or percentage. Kolmogorov–Smirnov test for results distribution and Student’s t test were performed to determine the statistically significant differences in haematological and biochemical biomarkers between the patients with RA and a healthy control group of females using the NCSS 2019 Statistical Software version 19.0.2 with a significance level of 0.05. The correlation analysis between observed parameters and ESR or CRP between groups was performed using Pearson’s coefficient of correlation r and 95% confidence intervals (95% CI). Results with values ≤ 0.05 were considered statistically significant.

The OpenEpi computer program version 3.01 (Open Source Epidemiologic Statistics for Public Health) was used to evaluate the statistical significance of the differences in the frequency of genetic variants between the group of RA patients and the control group by Fisher’s exact test and 2 × 2 contingency tables. A two-tailed or one-tailed P value less than 0.05 was considered significant in all tests. Statistical significance was improved by the Mantel–Haenszel Chi-squared (χ2) test because of the small sample size. The strength of the association between the presence of a particular genetic variant within the HLA-DRB1 gene locus and the occurrence of the disease was estimated by calculating the odds ratio (OR) and 95% CI for the OR value. An OR < 1 indicates protection, whereas an OR > 1 indicates an increased risk.

The software package PowerMarker, version 3.25, was used to analyse parameters for polymorphism estimation within the HLA-DRB1 gene locus, heterozygosity (H), gene diversity and polymorphic information content (PIC), as well as to assess whether the population was in Hardy–Weinberg equilibrium (HWE) among the patients with RA and control group subjects.

Results

The haematological and biochemical analyses

The mean age of the control group subjects was 46.50 ± 8.52 years (between 30 and 59 years of age), and the mean age of females with RA was 52.51 ± 7.26 years (between 34 and 60 years of age).

All values of the haematological–biochemical parameters of the control group were within the normal range for females (Table 1). The values of haematological parameters in patients with RA were also normal except for ESR and CRP. The values for ESR were elevated in RA patients (median 21, range 1–88 and average 23.94 ± 16.54) in comparison with the normal rate and with the control (median 5, range 2–9 and average 5.02 ± 1.92). Similarly, the CRP in RA patients (median 3, range 1–70 and average 9.01 ± 13.44) was considerably higher than normal CRP values and higher that in the control group (median 2, range 0–4 and average 1.98 ± 1.03).

Table 1 Frequencies of the haematological and biochemical parameters and their distribution in the control group and the group of females with RA using Kolmogorov–Smirnov test
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To determine the statistical significance of the differences in haematological–biochemical parameters between patients with RA and a healthy control group of females, we used the Kolmogorov–Smirnov test. The values of WBC (P = 0.0256), MCV (P = 0.0212), MCH (P = 0.0045), RDW (P < 0.0001), RDW-SD (P < 0.0001), MO (P = 0.0048), and EO (P = 0.0225) were significantly increased in RA patients (Table 1). Similarly, the values of the ESR (P < 0.0001) and CRP (P < 0.0001) were statistically increased in RA patients compared to those in the control group. According to Student’s t test, the relative risk related to ESR (95% CI 15.30–22.54; t = 10.31; P < 0.0001) was 2.7 times higher than that related to CRP (95% CI 4.09–9.97; t = 4.72; P < 0.0001). The values of NE and BA were also increased in RA patients but not significantly. The values of RBC (P = 0.0216) and HCT (P = 0.0123) were significantly reduced in patients with RA compared with the control, while the values of HGB, MCHC, PLT, MPV, and LY were reduced but not significantly. The NLR (median 1.86, range 0.26–5.92, and average 2.12 ± 1.17) and the PLR (median 107.56, range 50.28–253.64, and average 120.75 ± 48.23) were increased in patients with RA compared with healthy controls, but these differences were not statistically significant (Table 1).

Using the Pearson’s coefficient of correlation, no statistically significant correlation was found between the analysed haematological parameters and ESR or CRP in the control group. However, in patients with RA, a statistically significant negative correlation between ESR values and HGB (P = 0.04) was found, but there was a positive association between ESR and RDW (P = 0.0013), RDW-SD (P = 0.0001), PLT (P = 0.0043), or NE (P = 0.0055) (Table 2). There was a statistically significant positive correlation between CRP values on one side and WBC (P = 0.009), RDW-SD (P = 0.0351), PLT (P = 0.0041) or NE (P = 0.0001) values on the other side. The correlation between ESR and CRP in patients with RA was also established. With the ESR value increased, the CRP value of CRP (P < 0.0001) also increased (Table 2).

Table 2 Comparison of haematological parameters with ESR and CRP in the group of RA females using Pearson’s correlation coefficient (r)
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The frequency of allele groups and genotypes of the HLA-DRB1 gene locus in patients with RA and control females

The mean age of the control group subjects was 40.52 ± 14.06 years (range 21–68 years), and the age of patients with RA was 52.89 ± 7.63 years (age range between 34 and 77).

Among the subjects of the control group, 13 different allelic groups of the HLA-DRB1 gene locus (DRB1*01, *03, *04, *07, *08, *09, *10, *11, *12, *13, *14, *15, and *16) were present all 13. The most common allelic groups among the control subjects were DRB1*15 (15.2%), *01 (14.6%), and *16 (12.8%), followed by *07 and *13 (11%) and *11 (9.8%) allelic groups. The other allele groups included *04, *03, *14, *08, *10, *12, and *09 are less represented among subjects in the control group (Table 3). The most prevalent genotypes in the control subjects were DRB1*01/*15 (8.5%), followed by DRB1*01/*04, DRB1*11/*16, DRB1*13/*15, and DRB1*07/*16 (6.1%), DRB1*01/*07 (4.9%), DRB1*07/*13, DRB1*03/*16, and DRB1*15/*15 (3.7%). The remaining 34 different genotypes had a frequency under 2.5% (Table 4).

Table 3 Comparative analysis of the frequency of allelic groups of the HLA-DRB1 gene locus between patients with RA and control group of females
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Table 4 Comparative analysis of the frequency of genotypes of the HLA-DRB1 gene locus between patients with RA and the control group of females
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Among patients with RA, of the 13 allele groups of the HLA-DRB1 gene, the most common were DRB1*04 (15.7%), *03, and *15 (12.6%) after that DRB1*01 (11.4%), *11, and *16 (10.2%). The other allele groups were *07, *13, *14, *08, *10, *12, and *09 (Table 3). The most frequent genotypes in RA patients were DRB1*01/*07 and DRB1*03/*04 with 6% DRB1*01/*14, DRB1*11/*15, and DRB1*04/*04 with 4.8%, respectively. The genotypes DRB1*07/*13, DRB1*03/*11, DRB1*03/*15, DRB1*01/*08, DRB1*07/*15, DRB1*13/*16, DRB1*15/*16, DRB1*03/*03, DRB1*04/*15, and DRB1*04/*16 had frequencies of 3.6%, respectively. The remaining 24 different genotypes had frequencies under 2.5% (Table 4).

The analyses of allelic groups of HLA-DRB3, DRB4, and DRB5 genes (Table 5) showed that the DRB3 gene variants were most common in the control group (39.5%) as well as in RA patients (45.3%).

Table 5 Frequencies of HLA-DRB3, HLA-DRB4 and HLA-DRB5 gene variants and the association of HLA-DRB haplotypes with susceptibility to RA
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The HLA-DRB1 gene locus is highly polymorphic and can be observed in the control group and in the group of patients with RA. It shows a high degree of heterozygosity (0.93 and 0.89), gene diversity (0.89 and 0.89), and PIC (0.88 and 0.88). We observed 43 different genotypes of the DRB1 gene locus in the control group and 39 in the RA patient group. The control group was in HWE, and the group of RA patients was not (P = 0.0285).

The frequency of HLA-DRB1 gene variants in patients with RA in comparison with control female subjects

The distribution of allelic groups of the HLA-DRB1 gene locus in RA patients was compared with controls (Table 3). The frequency of the DRB1*04 (P = 0.0177) and *03 (P = 0.0416) allele groups in RA patients was significantly higher than in the control group. The frequencies of DRB1*08 and *11 allele groups were slightly higher in RA patients than in the control group, but were not statistically significant. In contrast, the frequencies of allele groups DRB1*01, *07, *10, *13, *14, *15, and *16 were higher in the control group than in RA patients but not significantly.

The frequencies of the DRB1*04/*04 (P = 0.0367) and DRB1*03/*04 (P = 0.05) genotypes were significantly higher among RA patients than in the control group (Table 4). A significantly increased frequency of DRB1*01/*15 (P = 0.0074) and DRB1*07/*16 genotypes (P = 0.0471) was found in control subjects compared with RA patients. These genotypes have not been observed in patients with RA (DRB1*01/*15) or were reduced (DRB1*07/*16).

Homozygous genotypes DRB1*04/*04, DRB1*03/*03, and DRB1*16/*16 were present in RA patients, but not detected in the control group, whereas homozygous genotypes DRB1*01/*01 and DRB1*11/*11 were present in the control group, but were not detected in RA patients.

The frequency of allelic groups of the HLA-DRB5 gene was significantly increased in the control group when compared with RA patients (P = 0.0331). The frequencies of allelic groups of the DRB3 and DRB4 gene loci were higher among RA patients than in the control group but not significantly (Table 5). Furthermore, we examined haplotypes of the HLA-DRB genes (DRB1 alone and DRB1-DRB3/4/5 haplotypes). The occurrence of DRB3, DRB4, and DRB5 is correlated with two-digit alleles of DRB1. We estimated 10 haplotypes (Table 5). The frequencies of the DRB1*03-DRB3* (P = 0.0443) and DRB1*04-DRB4* (P = 0.0259) haplotypes were significantly higher among RA patients than in the control group.

In this study, we found there was significantly higher expression of alleles of the HLA-DRB1*03 (P = 0.0061), *04 (P = 0.0001), *08 (P = 0.0270), *10 (P = 0.0254), *11 (P = 0.0024), and *14 (P = 0.0100) groups among RA patients with ESR values above 10 mm/h (Table 6). The HLA-DRB1*04, *09 and *14 variants were expressed more in RA females with CRP values above 5 mg/L but not significantly. RA patients with normal CRP values had significantly higher frequency of DRB1*03 (P = 0.0008), *13 (P = 0.0165), and *15 alleles (P = 0.0008).

Table 6 Frequencies of HLA-DRB1 gene variants in the group of RA females with normal and increased values of ESR and CRP
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Discussion

The cause of RA is still unknown. An important role for the development of RA belongs to the immune complexes that occur in the injured cells of the synovia and the inflamed blood vessels. To date, no specific biochemical or immunological marker has been found for RA. Some laboratory tests serve only to screen the activity and therapeutic effect in RA.

In our study, we showed that the values of the ESR (P < 0.0001) and CRP (P < 0.0001) were significantly increased in RA patients compared to those in the control group and was largely above the upper normal limit. ESR and CRP were also increased in RA patients when compared with control in the research accomplished by Sokolovic et al. [9]. We found a strong association between ESR and CRP in patients with RA (P < 0.0001), but the relative risk related to ESR was more than twofold (2.7 times) than that related to CRP. The results of this study indicate that ESR and CRP were the most sensitive indicators of disease in patients with RA. The persistence of a high serum CRP concentration is usually a severe prognostic sign. The classification criteria for RA, including ESR and CRP measurements, allow early aggressive treatment of RA [10].

Multiple factors, including HLA-DRB1 gene variants, influence the susceptibility to RA. In this study, we showed that the alleles of the HLA-DRB1*04 (P = 0.0177) and HLA-DRB1*03 (P = 0.0416) groups were the most frequent in RA patients and, therefore, represent a risk factor for the development of this disease. In RA, the HLA supports an odds ratio (OR) of ~ 2.8, whereas most non-HLA loci have an OR in the range of 1.1–1.4 [11]. The role of allele groups HLA-DRB1*08, *09, *10, *11, *12, and *14 in the development of RA was not completely defined in this study based on HLA typing. Furthermore, the frequencies of the DRB1*04/*04 (P = 0.0367) and DRB1*03/*04 (P = 0.05) genotypes were significantly higher among RA patients than in the control group and, therefore, represent risk genotypes for the occurrence of RA. However, Fejzic and co-workers [12] found that alleles of HLA-DRB1*01 and *04 group as well as DRB1*01/*13 genotype were the most frequent in RA patients of the Federation of Bosnia and Herzegovina. Homozygous genotype DRB1*04/*04 has not been observed in the control group of females along with DRB1*03/*03 and DRB1*16/*16 genotypes. Correspondingly, the strong genetic associations between the HLA-DRB1*04-DRB4* (P = 0.0259) or HLA-DRB1*03-DRB3* (P = 0.0443) haplotypes and a risk for the development of RA have been established in females.

On the other hand, the most frequent variants in the control group of females were HLA-DRB1*15, *01, *16, *07, and *13 and possibly have a protective role in the formation of RA. However, the differences observed between the control group and patients suffering from this disease were not statistically significant in this research study. Nevertheless, the DRB1*01/*15 (P = 0.0074) and DRB1*07/*16 (P = 0.0471) genotypes can be considered protective factors for RA, i.e., prevent the development of the disease. These genotypes were not observed in patients with RA, or their number was reduced. The frequency of allelic groups of the HLA-DRB5 gene was significantly increased in the control group when compared with RA patients (P = 0.0331). According to our results, the HLA-DRB5 gene represents a protective factor for RA.

In addition, we observed a significantly increased frequency of alleles of the HLA-DRB1*03, *04, *08, *10, *11, and *14 groups in RA patients with ESR values above 10 mm/h and decreased frequency of DRB1*03, *13, and *15 alleles in RA patients with CRP values above 5 mg/L, suggesting that certain HLA-DRB1 gene variants could be associate with milder or severe symptoms of disease.

The results of the H, gene diversity and PIC obtained in this study indicate that the HLA-DRB1 gene locus was highly polymorphic in the control group and in patients with RA. We observed a greater number of homozygous females in the group of patients with RA (~ 11%) than in healthy controls (~ 7% homozygotes).

HLA-DRB1 is the most important gene locus for RA susceptibility, particular for HLA-DRB1 gene variants at the *01, *04, and *10 groups, which encode a conserved five amino acid sequence motif (QRRAA/RRRAA/QKRAA) at 70–74 in the third hypervariable region (HVR3) of the DRβ1 chain that are described as the shared epitope (SE). The SE-coding alleles include members of the HLA-DRB1*04 group (*04:01, *04:04, *04:05, *04:08, and *04:210), HLA-DRB1*01:01 or *01:02, HLA-DRB1*14:02, and HLA-DRB1*10:01. The protective HLA alleles (HLA-DRB1*01:03, *04:02, *08:02, *11:02, *11:03, *13:01, *13:02, and *13:04) have, instead of the SE motif, a different, but shared sequence at the same location in the beta chain of HLA-DR molecules, consisting of the amino acid residues DERAA.

In different ethnic groups, RA-associated HLA-DRB1 alleles differ: HLA-DRB1*04:01, *04:04, and *04:08 are the predominant RA-associated alleles in Caucasians [13], *01:01 and *01:02 in Israeli Jews [14] and *01:01, *04:01, *04:04, and *04:05 in Latin Americans [15]. Shared epitope (SE) alleles and DRB1*09:01 were significantly associated with RA susceptibility in the Japanese population [16]. In addition, the alleles of the HLA-DRB1*04 group dominated in RA populations of Finland [17], the England, the Netherlands [18], and Slovakia [19]. The allele groups DRB1*04 and *01 are equally represented in the British and the Spanish population [20], while DRB1*01 and *10 dominated in the Mediterranean populations [21, 22].

In contrast, there are other HLA-DRB1 gene variants that are negatively associated with RA and, therefore, have a protective role. The allele group HLA-DRB1*13 was found to be significantly lower in RA patients compared to the control group in many studies [17, 18, 21], but in the control group of Mexican Americans, the HLA-DRB1*08 allele group is statistically significantly more frequent and is considered a protective allelic group [23]. Alleles of the DRB1*07 and *13 groups showed protective effects in a Slovak population [19].

The presence of specific HLA-DRB1 gene variants increases the risk of developing RA, while other variants provide protection against disease. Therefore, HLA typing could be helpful in prediction of RA development and establishing and confirming a definitive diagnosis of autoimmune diseases in some subjects. Because there is not specific therapy for RA, treatment could be slow down the progression of the disease indicating the importance for earlier diagnosis. A strong association ESR and CRP with RA could be used to establish definitive diagnosis and introduce of early treatment of RA to prevent the occurrence of RA symptoms.