ASSOCIATION BETWEEN HLA-DRB1 ALLELES AND SUSCEPTIBILITY TO RHEUMATOID ARTHRITIS AND TREATMENT RESPONSE

Maha r. Abd el-wahed , abeer a. Shehab, heba a. Selim 3, mona e. Hashem 1 and dina g. Abd el-hamed. 1. Department of Clinical Pathology, Faculty of Medicine, Zagazig University. 2. Department of Clinical Pathology, Faculty of Medicine, Ain Shams University. 3. Departments of Rheumatology, Faculty of Medicine, Zagazig University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The most important genetic factor implicated in predisposition to RA is human leukocyte antigen (HLA) major histocompatability (MHC) genes, HLA-DR4 is the major genetic factor implicated but its relative importance varies across ethnic groups (Okada et al., 2012).
HLA gene products play a key role in antivirus and antitumor defense, They function in regulation of immune response to foreign antigens and discrimination of self from non-self antigens. Alloantigens are taken up by antigen presenting cells which process them and re-express the antigens on the cell surface along with HLA to be recognized by T-cell receptor (Powell et al., 2012).
The extremely high levels of polymorphism and heterozygosity within the MHC genomic region provide the immune system with a selective advantage against the diversity and variability of pathogens . However, the high level of polymorphisms and mutations in the MHC has the added risk of generating autoimmune diseases and other genetic disorders. (Deitiker, Atassi 2015).
There is an association between HLA-DRB1 alleles and RA susceptibility (shared epitope hypothesis) (SE), HLA-DRB1*04:04, 04:01, 01:01, 10 , and 48 sex matched healthy volunteers as a control group. Patients suffering from other rheumatological diseases were excluded from the study. The study protocol was approved by the institutional ethics committee and written informed consent was obtained from all participants The patients were subjected to: i) full clinical history and thorough clinical examination. ii) Estimation of disease activity according to DAS28 Score. iii) routine laboratory investigations, including Complete blood count, Liver and Kidney function tests, rheumatoid factor (RF) and C-reactive protein CRP assay and ESR measurement. iiii) Specific laboratory investigation: All patients and controls were subjected to molecular HLA-DRB1 Typing by Luminex X-Map technology.
HLA typing. Using a Luminex LABScan 100 and LAB Type SSO Class II DRB1 Typing Kit. LAB Type SSO is based on reverse SSO technique, Biotinylated amplicon is chemically denatured and the separated strands are allowed to rehybridize to complmentary DNA probes conjugated to fluorescently coated beads. This is followed by removal of unbound material by wash solution, After wash, Streptavidin conjugated Phycoerythrin (SAPE) is added which binds to any biotinylated hybridized product, this is followed by wash step again to remove unbound 1063 substance, then a flow analyzer LAB scan 100 is used to identify the fluorescent intensity of (PE) on each microsphere. (Deshpande et al., 2010).
EDTA-anticoagulated whole-blood samples were collected from patients and controls and processed as follows: 1. DNA extraction using the spin column technique (QIAamp DNA Blood Mini kits;Qiagen, Hilden, Germany) was performed according to the manufacturer's guidelines; 2. Measuring concentration of extracted DNA was done by Qubet fluorometre by using specifically formulated dye that binds selectively to dsDNA to minimize the effects of contaminants in the sample then detection of target-specific fluorescence, and the target concentration needed to begin amplification reaction was adjusted to 20ng/µl. 3. polymerase chain reaction (PCR) amplification of the HLA-DRB1 gene (exon 2) target (LAB Type SSO class II HLA-DRB1 Typing kit; Hannover, GERMANY) was performed and Presence of amplified product was confirmed by gel electrophoresis through visualization of band at 270 bp under UV transluminator. 4. Denaturation/ Neutralization the amplicons were chemically denatured by denaturation and neutralization buffer to form single stranded DNA. 5. Hybridization: combination of appropriate volume of bead mixture (LAB Type SSO Bead Mix; Hannover, GERMANY) with hybridization buffer according to manufactures guide-lines. 6. Washing: of hybridization bead mixture was done by wash buffer for a total of 3 washes. 7. Labelling: The conjugate Streptavidin conjugated phycoerythrin (SAPE) was added, streptavidin binds to the biotin of the PCR product and Phycoerythrin is a fluorescent dye used for labeling by binding to any biotinylaed hybridized product. 8. Washing: was done again for labeled hybridized product. 9. Injection and reading on Luminex: a suspension of bead mixture with wash buffer was injected on Luminex.

Interpretation of results:-
A flow analyzer, the Luminex LAB Scan 100 identifies the fluorescent intensity of PE (Phycoerythrin) on each microsphere The assignment of the HLA type is based on the reaction pattern compared to pattern associated with the published HLA gene sequence. The microsphere mixture consists of a set of fluorescently labeled microspheres that bear unique sequence-specific oligonucleotide probes for HLA alleles. Each microsphere mixture includes negative and positive control microspheres for substraction of non-specific background.

1064
Patients were classified into 5 groups according to treatment protocol given: Patient's improvement and response to treatment was evaluated by measuring the change in two consequtive DAS scores, one was calculated at the time of the study and the other from previous records. Patients were classified into 3 groups according to degree of improvement; moderate improvement, stable and deteriorated Statistical Analysis:-All data were collected, tabulated and statistically analyzed using SPSS 20.0 for windows (SPSS Inc., Chicago, IL, USA). Quantitative data were expressed as the mean ± SD & median (range), and qualitative data were expressed as absolute frequencies (number) & relative frequencies (percentage). t test was used to compare between two groups of normally distributed variables. The Kruskall Wallus test was used to compare between more than two groups when they were not normally distributed. Percent of categorical variables were compared using Chi-square test and Fisher Exact test when appropriate. All tests were two sided. p-value < 0.05 was considered statistically significant, ≥ 0.05 was considered statistically insignificant.

Results:-
The general Characteristics of the study patients and control group are shown in (table 1). Clinical and laboratory data of our patients are shown in (table 2 and table 3).   The frequencies of HLA-DRB1 alleles in RA patients and normal controls are summarized in (table 4). HLA-DRB1*04 and HLA-DRB1*16 alleles were statistically more frequent in rheumatoid arthritis patients compared to controls, p values (0.01, 0.02, respectively), Odds ratios (2.5, 5.0, respectively), whereas HLA-DRB1*07,*11 and *13 alleles were more frequent in controls than patients (ORs were 0.4, 0.7 and 0.6, respectively) but the difference in each case didn't reach a statistically significant level (p>0.05 for each). The relation between HLA-DRB1 alleles and activity markers of the disease as CRP and RF is shown in (table 5 and 6). HLA-DRB1*03 and HLA-DRB1*16 alleles were found to be statistically more frequent among seronegative patients ( (RF ≤ 15 U/ml) than seropositive group (RF > 15 U/ml) (p values were 0.03, 0.02) respectively, Odds ratios were (0.22 and 0.16) respectively, while no significant association was obtained between any of HLA-DRBI alleles and CRP levels in rheumatoid patients. (P>0.05).   The relation between HLA-DRB1 alleles and disease activity (according to DAS28 Score) is shown in table (7). A statistically significant association was found between HLA-DRB1*04 and DRB*13 alleles and disease activity score , as they were associated with the highest activities among HLA-DRB1 alleles. (p= 0.03). Types of treatment given to RA patients are summarized in table (8). The relation between HLA-DRB1 alleles frequencies and response to treatment is shown in table (9). A statisticaly significant relation was identified between each of the following alleles; HLA-DRBI *01,*04,*11,*15 and *16 and response to treatment (p was <0.05 for each). The best response to treatment was in patients carrying HLA-DRBI*15, followed by DRB1*11 alleles, p values (0.0005, 0.005) respectively, Whereas The worst response was detected in patients carrying HLA-DRBI *04 , DRB1*01, and in those with DRB1*16 alleles (p values 0.001, 0.009 and 0.02) respectively,

Discussion:-
Rheumatoid arthritis (RA) is a chronic autoimmune disease with articular and systemic manifestations, It affects about 0.5-1% of adult population, mostly middle aged females (Thierry et al., 2014).
The pathogenesis of rheumatoid arthritis results from complex interaction between genes and environment leading to breakdown of immune tolerance. (Okada et al., 2012).
The significant association of particular alleles with RA is not consistent in all human populations in different geographical areas or among different ethnic groups, hence the analysis of different alleles of HLA in RA patients of many populations and geographical areas with regard to either having protective role or being a susceptibility factor is necessary (Sandoughi et al., 2011).
This study was carried out on 48 rheumatoid arthritis (RA) patients admitted to Rheumatology department of Zagazig University Hospitals during the period from June 2016 to December 2017, in addition to 48 sex matched healthy 1068 volunteers as a control group.
The aim of the study was to assess Human Leukocyte antigen HLA-DRB1 alleles association with susceptibility to rheumatoid arthritis and relation between certain alleles and response to treatment.
Upon comparing frequency distribution of HLA-DRB1 alleles between patients and controls, a significant difference was demonstrated as regards frequency of HLA-DRB1*04 and HLA-DRB1*16 being higher among patients compared to controls (p= 0.01 and 0.02, respectively), (Odds ratios= 2.5 and 5, respectively) which suggests that HLA-DRB1*04 and*16 could be a specific susceptibility alleles for rheumatoid arthritis.
Rest of alleles had no significant differences between patients' and control groups. However, it is important to note that some alleles in the present study were found to be more frequently prevalent in controls than patients, e.g HLA-DRB1*07,*11 and *13 (ORs were 0.4, 0.7 and 0.6, respectively) despite that the difference in each case didn't reach a significant level (p>0.05 for each). The results of the present study , as mentioned before, has shown a trend of negative association of DRB1*11 and *13 to RA which might point to their significance as protection alleles, however they didn't reach a statistically significant value. The significant finding of a negative association of DRB1*11 and *13 to RA by other studies, as by In a study carried out in Romania, HLA-DRB1*04 has shown to be a susceptibility allele in addition to DRB1*01and*14 (Loredana et al., 2012). In Morocco, HLA-DRB1*04 has also been reported as a susceptibility allele while DRB1*07 as a protective allele (Atouf et al., 2008).
Apart from HLA-DRB1*04, the obvious discrepancy between the results of different studies, including our results, as regards frequency distribution of HLA-DRB1 alleles among RA patients can be attributed to the differences in population race, geographical distribution and ethnic origin.
The association between HLA-DRB1 alleles and activity markers of the disease as CRP and RF was assessed. By comparing HLA-DRB1 allele distribution between seropositive and seronegative patients, we found that HLA-DRB1*03 and HLA-DRB1*16 alleles were found to be statistically more frequent among seronegative patients ( (RF ≤ 15 U/ml) than seropositive group (RF > 15 U/ml) (p values were 0.03, 0.02) respectively, Odds ratios were (0.22 and 0.16) respectively. This might point to a possible protective effect of HLA-DRB1* 03 and HLA-DRB1*16 1069 alleles against high serum RF level.
In the present work the frequency of HLA-DRB1*04 allele was higher in RA patients with high RF levels (28%) than in patients with low levels (14.3%), but the difference didn't reach a statistically significant level ( OR=2.4, P= 0.3), a finding which could be better defined among Egyptians on studying larger number of patients. In other populations, several previous studies reported the association between HLA-DRB1*04 and high RF level such as that carried out by AL-Timimi et al., (2014) who confirmed the association between HLA-DRB*01 and DRB1*04 alleles and RF positive Kurd patients, where 28.9% of their patients carrying DRB1*01 alleles were RF positive compared to 5.6% who were RF negative and 22.3% of patients having DRB1*04 alleles were RF positive compared to 5.6% who were negative (ORs= 5.9 and 4.8, respectively). Similar findings were also reported by On the other hand, Kinikli and associates (2003) reported no association between alleles and seropositivity of RA in Turkish population.
As regards serum CRP, our results showed no statistically significant association between any HLA-DRB1 alleles and CRP level in rheumatoid arthritis patients.
Considering the relation between HLA-DRB1 alleles and disease activity ( as assessed by DAS28 Score), our results showed a statistically significant association between HLA-DRB1*04 and DRB1*13 alleles and disease activity, implying that they were associated with the highest activities among HLA-DRB1 alleles according to DAS28 Score (p= 0.03).
Our results partially match with those described by Soliman et al., (2016) in a study on 40 Egyptian RA patients and 20 controls. They reported a significant association of HLA-DRB1* 04 (P= 0.005). They have reported also the association of HLA-DRB1*01 alleles with disease activity (P=0.002), but not DRB1*13, as was obtained in our study.
Another Egyptian study, in 2009 by Farouk and his companions, has investigated the effect of HLA-DRB1 alleles on outcome of rheumatoid arthritis in 29 Egyptian RA patients and 15 healthy controls. The study has reported that HLA-DRB1*01 and DRB1*04 were significantly more expressed among patients with active disease than among those with inactive disease (measured by SADI score). But, in contrast to our study, they have reported that HLA-DRB1*13 ( in addition to HLA-DRB1*03, *11, *12, and *14) were significantly more expressed among inactive cases.
Meanwhile, Fathi and associates (2008) in a study on 60 Egyptian rheumatoid arthritis patients have reported the significant association of HLA-DRB1 *04 with high activity of the disease, but not DRB1*01 or DRB1*13 alleles.
To further enlighten the possible relation between HLA-DRB1 alleles and RA in the current study, patients were categorized into 4 groups according to level of disease activity, which were determined according to DAS28 Score and included the following groups: remission (DAS < 2.6), low disease activity (2.6< DAS < 3.2) , moderate disease activity (3.2 < DAS < 5.1) and high disease activity ( DAS > 5.1) groups.
Al-Timimi and associates (2014) reported that 20% of their patients (Kurds) with HLA-DRB1*04 were of moderate activity and 20% were of high activity, while 3.3% of patients with HLA-DRB1*13 were of moderate activity and 8% were of high activity.
Summing the above data, it can be noticed that HLA DRB1 *04 allele remains the single allele that has been consistently reported by almost all studies, including the present one, as regards its association to disease susceptibility as well as disease activity, despite the different populations studied by different groups.
In the present work the relation between HLA-DRB1 alleles and response to treatment was assessed, patients were classified into 5 groups according to type of treatment regimen given (Disease modifying antirheumatic drugs DMARDs). 25 patients (52%) were on Methotrexate and Antimalarial Drugs, 10 patients (21%) were on methotrexate and prednisone, 7 patients (15%) were on Salazopyrine and Prednisone, 3 patients (6%) were given Methotrexate only and 3 patients (6%) were on Avera. Response to treatment was evaluated by measuring the change in two consequtive DAS scores, the first from patients records and the second at the time of the study, Patients with decrease in DAS Score from initial value > 1.2 were of good improvement, decrease in DAS < 1.2 but > 0.6 were of moderate improvement, decrease in DAS <0.6 were of no improvement.
In a study performed on patients from Pakistan by Ali et al., (2006), 91 RA patients had been receiving methotrexate for at least 6 months and, clinical response to methotrexate was assessed after 12-24 weeks of treatment, those showing 50% or more reduction in ESR, number of swollen joints and morning stiffness as compared to those criteria at admission were classified as responders while those showing little improvement in these parameters were called non-responders.
They found that HLA-DRB1*03 allele was significantly more common among non-responders (37.2% compared to responders (14.3%) (p = 0.004). They added that HLA-DRB1*15 was present more frequently in the nonresponders (46.5%) than in the responders (40.8%) , however the difference was not significant. No other alleles showed significant association with response to treatment in their study, as they have reported.

Conclusion:-
HLA-DRB1*04 and *16 alleles appear to be susceptibility factors for development of rheumatoid arthritis in our population, patients having high frequencies of HLA-DRB1*04 and *13 were predisposed to high disease activity state. In addition HLA-DRB1* 15, *11, *04, *01 and *16 alleles may be predictors for treatment outcome in rheumatoid arthritis patients.