The Genetic Variants of HLA-DRB1 Alleles and the Chance of Developing Rheumatoid Arthritis: Systematic Review and meta-analysis

Rheumatoid arthritis (RA) is more common in those who have special human leukocyte antigen (HLA) genetic types. One of the most important genetic risk factors for RA lies in HLADRB1 locus. The purpose of this meta-analysis was to gure out which HLA-DRB1 allele frequencies are associated with a risk of RA per allele and phenotype group. The statistics data was calculated using RevMan version 5.4.1. This meta-analysis includes nine articles involving 3004 RA patients and 2384 healthy controls. In the allele group, three HLA-DRB1 alleles; HLA-DRB1*10 (OR = 1.88, 95%CI = 1.25-2.83, p = 0.002), HLA-DRB1*04 (OR = 2.38, 95%CI = 1.73-3.29, p < 0.00001), and HLA-DRB1*01 frequencies were shown to be considerably higher in RA patients compared to controls, potentially increasing the chance of disease development. Four HLA-DRB1 alleles among the included studies (*03, *07, *11, *13, and *14) which was observed that these are more prevalent in healthy persons than in RA patients, and may contribute as a protective factor against disease onset. Only the DRB1*04 subtypes DRB1*0401 (OR = 1.37, 95 percent CI = 1.05-1.79, p = 0.02) and DRB1*0404 (OR = 1.73, 95% CI = 1.19-12.53, p = 0.004) show a signicant association with the risk of RA in our pooled effect. Moreover, the ndings demonstrated a signicant relationship between HLA-DRB1 and the risk of disease in various ethnic groups. 5.4.1. The Mantel-Haenszel technique was used to analyze the effect of HLA-DRB1 alleles in RA. We used odds ratio (OR) and 95 percent condence interval (CI) to measure the effect of HLA-DRB1 alleles in RA. The forest plot was also shown, along with an explanation of the total pooled P-value. The diamond at the bottom of the forest the outcome of combining and averaging studies. The of the diamond are the 95 % and they be interpreted the way as any other individual studies on the


Introduction
The most frequent type of autoimmune arthritis is rheumatoid arthritis (RA). It is caused by the immune system attacking healthy body tissue, means that when the body's defense mechanism is not functioning properly [1]. Although the disease affects people of all ethnic groups around the world, its prevalence varies by race, ranging from 0.1 percent in native Africans [2] to 5% in Pima Indians [3]. However, it is unknown what causes this diseases, Nevertheless, major histocompatibility complex (MHC), also known as Human leukocyte antigen (HLA) in human, is one of the most important genetic variables in RA. One of the genetic loci linked to RA variations is the MHC class II regions. The most important genetic risk factor for the onset of RA is found in the MHC class II region [4,5].
RA has been linked to more than 30 risk alleles. The HLA-DRB1 gene in the MHC II region is the gene most strongly linked to RA, which is thought to account for up to one-third of the genetic risk to RA [6]. Most individuals with RA have preferred HLA-DRB1 alleles with a shared epitope [7,8]. Amino acid sequences in the DRβ chain at locations 70 to 74 were retained in RA-associated DRB1 alleles as either QRRAA, or RRRAA, which is the shared epitope (SE) [7]. The SE-RA link appears to be complicated and variable among populations [9]. Differences in clinical and laboratory markers between DR4 + and DR4-patients in RA patients have been questioned [10]. Meanwhile, research also suggests that the SE's principal function is linked to disease severity than susceptibility [11].
Diverse populations have various HLA-DRB1 risk alleles. Some meta-analyses have been undertaken in speci c ethnic groupings of the population. In Asian populations, a meta-analysis of the association between the HLA-DRB1 allele and rheumatoid arthritis susceptibility was conducted [12]. It was also done in Latin American [13] and in Caucasian [14]. However, no meta-analysis has been reported in multi-ethnic groups with the most up-to-date information.
In this study all reports relating to the risk of HLA-DRB1 alleles with RA were considered. This metaanalysis aggregated all data from different ethnic groups to produce a more general and comprehensive results. To our knowledge, this is the only study that takes into account multi-ethnic groups data to infer new insights between the HLA-DRB1 allele and the chance of developing RA.

Search strategy and Study selection
We used the search terms "HLA-DRB1" or "HLA-DRB1 allele" AND "Associated polymorphism" AND "Rheumatoid arthritis" to explore for studies published before June 2021 in the Google Scholar, PubMed, and Springer databases. The following criteria were used to determine which should be included: 1) casecontrol (RA patients vs healthy) studies; 2) if su cient data for each allele was given; and 3) NOS featured studies that were of high quality; 4) conventional molecular techniques were applied. Metaanalysis studies and non-original papers were not included in the study.

Quality assessment and data extraction
The rst author's last name, publication year, study area, sample size, gender frequency, average age, and HLA-typing method were all obtained from each study. The Newcastle-Ottawa Scale (NOS) [15] was used to assess the quality of these investigations, with articles scoring more than ve out of a possible eight being considered high-quality and included in this meta-analysis (Supplementary)

Risk of bias assessment
The funnel plots were used to reveal the distribution of odds ratios (ORs) from separate studies in relation to their respective standard error (SE). The regression test proposed by Egger et al.[16] was used to analyze publication bias statistically. In addition, for a sensitivity test, we compared trim and ll [17] estimates from xed-effect (FE) and random-effect (RE) model.

Analysis
All statistical analysis was performed with Review Manager 5.4.1. The Mantel-Haenszel technique was used to analyze the effect of HLA-DRB1 alleles in RA. We used odds ratio (OR) and 95 percent con dence interval (CI) to measure the effect of HLA-DRB1 alleles in RA. The forest plot was also shown, along with an explanation of the total pooled P-value. The diamond at the bottom of the forest plot represents the outcome of combining and averaging all of the individual studies. The horizontal points of the diamond are the 95 % CI and they can be interpreted the same way as any of the other individual studies on the plot. I 2 was used to assess statistical heterogeneity between the included studies. The I 2 value varies from 0 to 100% (I 2 = 0-25%, no/low heterogeneity; I2 = 25-50%, moderate heterogeneity; I 2 = 50-75%, substantial/high heterogeneity; I 2 = 75-100%, extreme heterogeneity) [18].For I 2 ≤ 50%, a xed-effect (FE) model was utilized otherwise a random-effect model was used since I 2 > 50% appeared to be that did not happen by chance. GraphPad Prism 9 (https://www.graphpad.com/) was also used to do simple statistical tests and combine scienti c graphing, which served as a cross-check.

Characteristics of included studies
This meta-analysis contained nine reports published between 2011 and 2019. The initial database search yielded 232 articles based on PRISMA 2020 checklist (http://www.prisma-statement.org/), as shown in  Table 1 shows the characteristics of the included studies. There were 3004 cases and 2384 controls in total, with a case-to-control ratio of 1.26. In all groups, the average age was 46 years old, with females accounting for 3823 and males accounting for 960, with the exception of one study [5] that did not provide gender frequency.

Association between HLA-DRB1 alleles and RA
In this meta-analysis, a forest plot showing the correlation between HLA-DRB1 allele and the chance of RA is presented in Fig. 2. Table 2 shows the relationship between the HLA-DRB1 allele and the risk of RA; Fig. 3 popped up the general frequency of the cases and control.
The total effect of HLA-DRB1*04 reveals that those who carry this allele are 2.38 times more likely to develop RA (OR = 2.38, 95%CI = 1.73-3.29, p < 0.00001) and the random-effect model was utilized in the included studies because of high-heterogeneity (I 2 = 85%).
Because of the moderate-heterogeneity (I 2 = 54%) in the HLA-DRB1*10 allele, the random-effect model was employed for meta-analysis. According to the overall data, the allele is associated with a signi cantly increased risk of RA. (OR = 1.88, 95%CI = 1.25-2.83, p = 0.002).
We discovered no relationship between a few HLA-DRB1 alleles and the chance of developing RA in our Because two studies from South Africa [19] and Japan [20] had no HLA-DRB1*16 allele frequency, only seven studies were included in the analysis and there is no link between the alleles and the risk of RA on the total outcome.

Subgroup analysis
We further assessed at each phenotypic group separately to see if there was a speci c link between HLA-DRB1 and the risk of getting RA at an individual level (Table 3) The relationship between HLADRB1 and RA appeared to be somewhat symmetric in a funnel plot.
Furthermore, the ndings of Egger's test indicate that there was no bias (p > .05).

Discussion
Meta-analyses can assist in determining statistical signi cance across studies that appear to have contradictory results. The statistical signi cance established when multiple researches are considered at once is signi cantly higher than when one study is considered alone [21]. Some of the bene ts of metaanalysis include a pooled estimate of impact, an objective assessment of evidence, and the ability to explain heterogeneity between research results [22].
Despite the fact that there is no signi cant link (P = 0.006), the forest plot HLA-DRB1*01 has showed that is more likely to develop RA than controls. This may be because of the longer con dence interval on individual studies. Closer to our ndings, two studies from Syria (OR = 2.29, 95%CI = 1.11-4.75, P = 0.022) [14] and Turkey (OR = 1.99, 95%CI = 1.19-3.33, P = 0.04) [6] both found a robust link of RA in the allele group. HLA-DRB1*01 has also been linked to the risk of RA in areas of northeastern Spain [23] and India [24].
Our ndings on the DRB1*0401 genotype group demonstrate a predisposition to RA, which is consistent with research in European populations [16] that reveal DRB1*0401 is one of the most important genetic risk factors for RA. Also the prevalence of DRB1*0401 is signi cantly higher in RA patients, according to a report in the Chinese population [25]. In contrast to research DRB104:05 is negatively related with RA in Japanese people [20], however our pooled ndings reveal no connection at all with this subgroup across included studies.
Interestingly, unlike many other research, HLA-DRB1*14 was found to be protective in our pooled effects.
There are research that do not complement our ndings of alleles include: The HLA-DRB1*14 (OR = 2.74) allele and RA are linked in a study of a mixed Peruvian population (n = 65) [26]. Similarly, according to another research the HLA-DRB1*14 allele has demonstrated a tendency to be associated with RA was observed in Yakima Indians, a Native American tribe [27].
The HLA-DRB1*09 alleles, which are found to be a predisposing factor on RA patients of Malaysia [28], are not observed in our study. No correlation was found in our study (OR = 1.42, 95%CI = 0.68-2.96, p = 0.34). This difference could be due to the fact that these studies had a larger sample size, resulting in statistical signi cance in this association.
HLA-DRB1*04 was found to be predisposing against RA in our study, as well as in studies of RA patients from Portuguese [29], Spain [30], and United Kingdom [31]. There are, of course, some multiple theories about this allele. For instance, some study revealed a signi cant rise in the frequency of HLA-DRB1*04 alleles in RA patients [32], whereas, others found no such associations [25]. Curiously, like many other research, HLA-DRB1*04 was found to be positive association with RA in our pooled effects. But HLA-DRB1*01 and HLA-DRB1*04 alleles appear to be linked to a decreased chance of developing RA [33,34], which is not consistent with our ndings.
Differences in ethnic groupings, geographic region, and HLA genotype might explain the contradictory results. Differences in ethnic groupings, geographic location, HLA genotyping methods, and sample size may be to blame for the mixed results. Because of the high shared epitope (SE) in the area, it would be best to examine at all genotype level in future study.
The lack of a link between HLADRB1*04 and RA in Greek patients was con rmed in one study. In general, our ndings provide a tremendous insight into HLA-DRB1 and the risk of having RA, which are both consistent with and contradictory to the ndings of many research around the world. This could be because our analysis did not include a European group by default, or because the majority of these studies focus on early disease start.
However, this meta-analysis has certain limitations. We do not take European HLA-DRB1 into account by chance, which is something that other researchers may do in the future. Since ethnicity and geographical differences are important variables in gene-association research.

Conclusion
In conclusion, the pooled results of the meta-analysis reveal a robust association between HLA-DRB1 genetic variants and the chance of developing RA in both allele and phenotype groups. In this metaanalysis study, we identi ed a correlation between HLADRB1 alleles and the chances of having RA. This may contribute in disease identi cation and prevention through early detection and screening of the risk alleles. High allele expression of HLA-DRB1*04 and HLA-DRB1*10 is signi cantly associated to RA in this nding, suggesting that it could be a risk factor for the disease. Low HLA-DRB1*03, HLA-DRB1*07, HLA-DRB1*11, HLA-DRB1*13, and HLA-DRB1*14 allele expression may be linked to RA and may contribute as a protective factor against disease onset. DRB1*0401 and DRB1*0404 have indeed been found to be predisposing to RA, while DRB1*0403 and DRB1*0407 were found to be protective. Tables   Tables 1 Characteristics of the nine included  Phenotype group: studies in which phenotype counts or frequencies were applied as a statistical approach Figure 1 Flow diagram of the study selection process Overall forest plot association of HLA-DRB1 and RA  Supplementary.docx