1. INTRODUCTION

Systemic lupus erythematosus (SLE) is a complex, multifaceted autoimmune disease that can present as fever, skin rashes, fatigue, and pain or swelling of the joints; it is associated with the production of autoantibodies, and is known to be associated with both environmental and genetic factors. Some of these environmental factors include infection as well as exposure to cigarette smoke, ultraviolet light, and certain chemicals and medications,1–4 whereas genetic factors include a family history of SLE.5,6

Over the past several decades, numerous loci across the human genome have been identified as being associated with SLE. Genetic loci located on chromosome 1 play important roles in immune surveillance, immune clearance, and involved in the pathogenesis of SLE. Tsao et al7 presented evidence for linkage of a chromosome 1 region to SLE, whereas Moser et al8 reported evidence for linkage of chromosome 1q to SLE in African-American pedigrees. In a meta-analysis, Karassa et al9 showed the polymorphism of Fcγ receptor IIa, which is encoded on chromosome 1, to be associated with SLE risk in multiple ethnic groups. In addition to genes located on chromosome 1, the human leukocyte antigen (HLA) system, a complex of several hundred genes that encodes a variety of cell surface proteins, is located on the short arm of chromosome 6 and also plays a crucial role in the adaptive immune response and the pathogenesis of SLE.

The HLA system was first reported in the 1970s by Grumet et al10 to be associated with autoimmune disorders. Since the mid-1980s, different HLA genotypes have been demonstrated to be related to the pathogenesis of different autoimmune diseases, including SLE.11 Previous studies have shown the possession of different HLA-DR genotypes to be associated with SLE in different populations; for example, possession of HLA-DR3 is associated with SLE in Caucasians,12–14 whereas HLA-DR15 is associated with SLE in southern Chinese15 and Japanese16 populations. Several linkage analysis studies have showed that 6p11-21, the location of HLA, was the risk locus of SLE.5,6,17–19 A study using transmission disequilibrium testing demonstrated the association of HLA-DRB1*1501, and not DRB1*0301, with SLE in Americans20; however, a similar study showed HLA-DRB1*0301 to be a risk factor in Caucasian SLE patients in the United Kingdom.21 Multiple genome-wide association studies also revealed an association between the HLA region on chromosome 6p21 and the development of SLE,22,23 although a study reported by Lu et al24 concluded that, in Taiwanese patients, no HLA-DRB1 allele was significantly associated with SLE. This study aims to investigate whether HLA-DR genotypes are associated with SLE in Taiwanese patients, as well as whether any HLA-DR genotypes that are associated with an increased risk of SLE (hereafter referred to as risk alleles) are also associated with specific clinical manifestations of SLE.

2. METHODS

We performed a review of 234 SLE patients (208 females, 26 males) and 346 healthy controls (301 females, 45 males) who underwent follow-up at the Rheumatology clinic at Kaohsiung Medical University Hospital. The collection of patient data for the purpose of this study was performed in accordance with the WMA Declaration of Helsinki, and was approved by the Institutional Review Board at Kaohsiung Medical University Hospital. All patients meet the 2019 European Alliance of Associations for Rheumatology/American College of Rheumatology (EULAR/ACR) Classification Criteria for SLE.25 The symptoms and signs of SLE for each patient were reviewed, as well as patient age at disease onset and laboratory findings.

HLA-DR genotyping was done with the commercially available HLA FluoGene DRDQ kit (Inno-Train Diagnostik GmbH, Kronberg, Germany), which uses a sequence-specific primer for amplification with polymerase chain reaction.

Anti-dsDNA, anti-Sm, anti-RNP, anti-Ro, anti-La, anti-ENA, anticardiolipin, and anti–β2-glycoprotein 1 antibodies were measured with commercial kits by using fluoro-enzyme-immunoassay (EliA kit; Phadia AB, Uppsala, Sweden). Lupus anticoagulant was detected using a commercial kit (simplified dilute Russell’s viper venom test; Siemens Healthineers, Erlangen, Germany). We further investigated the association of risk alleles (defined as HLA-DR genotypes that significantly increase SLE incidence) with the age of disease onset and the individual clinical manifestations of SLE. We defined renal involvement (lupus nephritis) in accordance with the 2019 EULAR/ACR classification criteria for SLE.25

Statistical analyses were performed with SPSS Statistics software (IBM, Armonk, NY) and G*Power software (Heinrich Heine University Düsseldorf, Düsseldorf, Germany). Chi-square tests were performed to evaluate the differences in HLA-DR genotype frequencies between SLE patients and healthy controls. Alpha level was set at 0.05; p-values were corrected for multiple comparisons, whereas a t test was performed to compare the mean age of disease onset between patients with and without risk alleles, and chi-square tests were performed to compare the strength of association between different disease manifestations and the presence of risk alleles.

3. RESULTS

The demographics and clinical characteristics of the SLE patients are described (Table 1). The average age of disease onset was found to be 28.9 ± 12.2 years.

Table 1 - Demographics and clinical characteristics of SLE Sex F 208 M 26 Onset age (mean ± SD) 28.9 ± 12.2 y Clinical manifestations n = 234 (%)  Fever 92 (39.3)  Mucocutaneous 169 (72.2)   Alopecia 83 (35.5)   Oral ulcer 73 (31.2)   Malar rash 126 (53.8)   Discoid 27 (11.5)   SCLE 8 (3.4)  Photosensitivity 64 (27.4)  Raynaud’s 42 (17.9)  Arthritis 150 (64.1)  Cutaneous vasculitis 51 (21.8)  Lymphadenopathy 46 (19.7)  Deep vein thrombosis 8 (3.4)  Neuropsychiatric 7 (3.0)  Avascular necrosis of bone 36 (15.4)  Serositis 60 (25.6)   Pleurisy 58 (24.8)   Pericarditis 28 (12.0)   Peritonitis 3 (1.3)  Renal involvement 129 (55.1) Laboratory data n = 234 (%)  Hematological involvement 153 (65.4)   Leukopenia 128 (54.7)   Thrombocytopenia 72 (30.8)   Hemolytic anemia 29 (12.4)  SLE-specific antibodies 175 (74.8)   Anti-dsDNA 169 (72.2)   Anti-Sm 42 (17.9)  Anti-RNP 77 (32.9)  Anti-Ro 142 (60.6)  Anti-La 47 (20.1)  Anti-ENA 182 (77.8)  Antiphospholipid antibodies 76 (32.5)   Anticardiolipin 51 (21.8)   β2-glycoprotein 1 12 (5.1)   Lupus anticoagulant 40 (17.1)

SCLE = subacute cutaneous lupus erythematosus; SLE = systemic lupus erythematosus.

Clinically, fever was noted in 39.3% of the patients, whereas mucocutaneous lesions and arthritis were noted in 72.2% and 64.1% of the patients, respectively. Renal involvement (lupus nephritis) was found in 55.1% of the patients. Serologically, SLE-specific antibodies were found in 74.8% of the patients, whereas antiphospholipid antibodies were found in 32.5%.

The genotype frequency of HLA-DR2 was found to be significantly higher (OR = 2.05, 95% CI, 1.44-2.92) in SLE patients than in controls (p < 0.001) (Table 2). We plotted the data (HLA-DR vs uncorrected odds ratio) as a forest plot to better illustrate the higher genotype frequency of HLA-DR2 (Fig. 1). This increased frequency was still significant after p-value correction (pcorr < 0.010). Notably, the HLA-DR6 genotype appeared to trend toward negative correlation with SLE incidence (OR = 0.57, 95% CI, 0.36-0.90, p = 0.015, pcorr = non-significant), whereas the HLA-DR8 genotype appeared to trend toward positive correlation (OR = 1.63, 95% CI, 1.10-2.42, p = 0.014, pcorr = non-significant) (Table 2).

Table 2 - Frequencies of HLA-DR alleles in patients with SLE and healthy controls DR SLE, n = 234 (%) Control, n = 346 (%) OR (95% CI) p p corr 1 1 (0.4) 2 (0.6) 0.74 (0.07-8.19) 0.804 NS 2 98 (41.9) 90 (26.0) 2.05 (1.44-2.92) <0.001 <0.010 3 37 (15.8) 56 (16.2) 0.97 (0.62-1.53) 0.904 NS 4 49 (20.9) 81 (23.4) 0.87 (0.58-1.29) 0.484 NS 5 76 (32.5) 130 (37.6) 0.80 (0.56-1.13) 0.209 NS 6 32 (13.7) 75 (21.7) 0.57 (0.36-0.90) 0.015 NS 7 14 (6.0) 14 (4.0) 1.51 (0.71-3.23) 0.286 NS 8 65 (27.8) 66 (19.1) 1.63 (1.10-2.42) 0.014 NS 9 55 (23.5) 91 (26.3) 0.861 (0.59-1.27) 0.447 NS 10 4 (1.7) 5 (1.4) 1.186 (0.32-4.47) 0.801 NS

HLA = human leukocyte antigen; NS = not significant; OR = odds ratio; pcorr = p-values corrected by Bonferroni method; SLE = systemic lupus erythematosus.

Fig. 1:

Forest plot of uncorrected odds ratios of HLA-DR alleles in SLE patients vs healthy controls. HLA = human leukocyte antigen; SLE = systemic lupus erythematosus.

Further exploratory analysis was performed after identifying HLA-DR2 as a risk allele. In SLE patients, HLA-DR2 was associated with a lower age of disease onset, as well as a higher prevalence of oral ulcer (OR = 2.092, 95% CI, 1.19-3.67), avascular necrosis of bone (OR = 3.74, 95% CI, 1.76-7.93), and renal involvement (lupus nephritis) (OR = 2.05, 95% CI, 1.19-3.51) (Table 3). We then plotted these clinical manifestations against the odds ratios that would be conferred by possession of HLA-DR2 (Fig. 2).

Table 3 - Comparison of clinical manifestations between HLA-DR2(+) and HLA-DR2(−) SLE patients Clinical manifestations DR2(+), n = 93 (%) DR2(−), n = 141 (%) p OR (95% CI) Onset age (mean ± SD) 26.0 ± 9.9 y 30.8 ± 13.2 y 0.002 NA Malar rash 52 (55.9) 74 (52.5) NS Oral ulcer 38 (40.9) 35 (24.8) 0.01 2.092 (1.19-3.67) Photosensitivity 28 (30.1) 36 (25.5) NS Arthritis 63 (67.7) 87 (61.7) NS Cutaneous vasculitis 22 (23.7) 29 (20.6) NS Serositis 23 (24.7) 37 (26.2) NS Avascular necrosis of bone 24 (25.8) 12 (8.5) <0.001 3.74 (1.76-7.93) Hematologic 59 (63.4) 94 (66.7) NS Neuropsychiatric 3 (3.2) 4 (2.8) NS Renal involvement 61 (65.6) 68 (48.2) 0.009 2.05 (1.19-3.51) SLE-specific antibodies 71 (76.3) 104 (73.8) NS Antiphospholipid antibodies 28 (30.1) 43 (30.5) NS

t test used for comparison of onset age; chi-square test used for comparison of other clinical manifestations.

NA = not applicable; NS = not significant; OR = odds ratio; SLE = systemic lupus erythematosus.

Fig. 2:

Odds ratios of clinical manifestations in DR2-positive vs DR2-negative SLE. SLE = systemic lupus erythematosus.

4. DISCUSSION

In this study, HLA-DR2 was found to be positively correlated with susceptibility to SLE in Taiwanese patients. In addition, we found that the presence of HLA-DR2 in SLE patients was associated with a higher prevalence of oral ulcer, avascular necrosis of bone, and renal involvement.

Many genes and immune pathways are involved in the pathogenesis of SLE. As a classical autoimmune disease, endogenous autoantigens bound to HLA class I trigger an immune response in SLE. HLA-DR, an HLA class II receptor that binds with exogenous antigens, is also known to be associated with various autoimmune diseases. A discrepancy seemingly arises between these mechanisms, but can be explained by the involvement of autophagy and cross-presentation in antigen presentation. During antigen presentation in general, endogenous antigens are presented by HLA class I molecules, while exogenous antigens are presented by HLA class II molecules; however, autophagosomes containing damaged organelles and autoantigens also serve as a source of endogenous peptides that also bind to HLA class II molecules.26 SLE is linked to large amounts of apoptotic cells in various tissues,27 and autoantigens are known to be translocated into apoptotic bodies during the process of apoptosis.28 Autoantigens derived from phagocytosed apoptotic cells are efficiently expressed on HLA class II molecules on dendritic cells,29 which phagocytose apoptotic cells more efficiently than macrophages.27,30 It has been proposed that a defect in the clearance of apoptotic cells leads to an overload of apoptotic cells and a breakdown of self-tolerance in SLE.31

Various studies conducted in different populations have shown that susceptibility to SLE is influenced by different HLA-DR genotypes in different populations. Our findings are compatible with previous studies performed in East Asian populations that include Chinese, Japanese, and Korean patients.15,16,32 Furukawa et al33 suggested that, in Japanese SLE patients, increased DR15, decreased DR13, and decreased DR14 were associated with SLE. Lee32 suggested in 2003 that, in Koreans, HLA-DRB1*15 polymorphism is an independent risk factor for SLE.

In our study, we found that HLA-DR2 (ie, HLA-DR15 and HLA-DR16 taken together) is positively correlated with susceptibility to SLE. Conversely, we also noted that possession of HLA-DR6 appeared to trend toward negative correlation with SLE, and although this result was not statistically significant, it was consistent with findings from previous studies, such as the protective effects conferred by HLA-DR6 alleles including DRB1*13 and *14 as reported by Furukawa et al,33 and HLA-DR6 allele HLA-DRB1*13 as reported by Bettencourt et al.34

SLE affects the kidneys in about 50% of patients, with lupus nephritis being the most common cause of SLE-related kidney injury, of which 10% of patients will progress to end-stage renal disease.35 HLA-DR2, specifically the variant HLA-DR15, has been associated with lupus nephritis in many discrete populations36–42; additionally, the subvariant HLA-DRB1*1503 has been found by Bastian et al43 to be associated with new or worsening proteinuria. Chung et al44 previously reported association of HLA-DR2 and HLA-DR3 with lupus nephritis in a meta-analysis of three genome-wide association studies of 2000 unrelated European women with SLE, whereas Niu et al45 noted in a review that HLA-DR4 and HLA-DR11 (a component of HLA-DR5) conferred a protective effect against lupus nephritis, and that HLA-DR3 and HLA-DR15 (a component of HLA-DR2) conferred an increased risk. Our study revealed that possession of HLA-DR2 was associated with development of lupus nephritis in Taiwanese SLE patients, which is compatible with previous studies.

Our study revealed an association between possession of HLA-DR2 and avascular necrosis. We performed a literature review for avascular necrosis and its association with different HLA genotypes. Morimoto et al46 reported that HLA-DQA1*0601 is negatively associated with aseptic bone necrosis, but little other literature is available. Our findings may be novel or might be confounded by the fact that patients possessing HLA-DR2 generally have a more severe clinical course, and the incidence of avascular necrosis in these patients could be related to more aggressive treatment with high doses of glucocorticoids.47 Prasad et al had investigated whether any clinical, laboratory, or therapeutic differences other than use of glucocorticoids existed between SLE patients who did and did not develop osteonecrosis, but could not identify any other factors which conferred risk or protection.48,49 Several previous studies attempted to examine whether the presence of antibodies related to antiphospholipid syndrome (APS antibodies, ie, lupus anticoagulant, anticardiolipin antibodies, and anti–β2-glycoprotein 1 antibodies) was related to the incidence of osteonecrosis, but results were controversial. Jones et al50 investigated whether abnormal levels of coagulation factors were associated with osteonecrosis, and reported that osteonecrosis patients were more likely to have high levels of anticardiolipin antibody IgG. Conversely, Mok et al48 investigated whether the presence of APS antibodies was associated with avascular necrosis in SLE patients, but no link could be established. We examined whether the presence of APS antibodies was associated with the incidence of avascular necrosis in our study group, but found no correlation (APS antibody positive vs negative, OR = 0.731, 95% CI, 0.325-1.647, p-value not significant).

We found possession of HLA-DR2 to be positively associated with the formation of oral ulcers. Smikle et al51 previously reported a positive association between DRB1*15/16 and oral ulcers in Jamaican SLE patients, and Selvaraja et al52 reported that HLA-DRB1*15 was significantly associated with oral ulcer in Malay SLE patients. Similarly, Bang et al53 reported that HLA-DRB1*15:01 is associated with oral ulcers and renal disorders. Our findings are consistent with the results from these studies.

HLA genotypes have long been known to be correlated with age at disease onset of SLE, as well as its presentation.54,55 In addition, several studies have observed a correlation between a lower age at disease onset and more serious manifestations of disease.56,57 We found that in SLE patients, possession of HLA-DR2 was associated with an earlier onset of disease and a more severe disease course.

Recent studies have also outlined the role of type I interferon as a possible genetic signature for SLE,58 and therapeutics targeting the type I interferon pathway have been approved.59 Stickler et al60 previously reported that a proliferative response of CD4+ T cells to interferon-beta (IFN-β)-1b was associated with the presence of HLA-DR2, specifically the subtype HLA-DR15. The presence of HLA-DR2 is thus associated with increased CD4+ T cell activity, leading to increased secretion of pro-inflammatory cytokines, including IL-17 and interferon-gamma (IFN-γ). These cytokines activate the adaptive immune response and play a major role in immune clearance. This might also explain in part the propensity for increased SLE risk in HLA-DR2 carriers.61

The results of our study are in contrast with that by Lu et al,24 which showed no association of HLA-DRB1 with SLE susceptibility in Taiwan. The strength of our study is its relatively large sample size: a post-hoc analysis of test power using a two-tailed z test comparing HLA-DR2-positive and HLA-DR2-negative patients using G*Power revealed that a power of over 0.97 was achieved; however, a post-hoc analysis of test power using the same parameters as our study revealed a test power of less than 0.80 in Lu et al’s24 study group. In a similar study, Rudwaleit et al62 examined HLA-DRB1 and HLA-DQB1 alleles in 26 Chinese SLE patients (with 77 controls) attending the National University Hospital in Singapore. The study revealed a lack of DR2 association with SLE in these patients, and the authors proposed two possible explanations: genetic admixture in the Singapore Chinese population, or ethnic heterogeneity among the southern Chinese.62 It is also possible that this finding was due to sampling bias; however, due to the low number of patients enrolled (power = 0.27).

In conclusion, of the HLA-DR genotypes tested, HLA-DR2 was found to be positively correlated with susceptibility to SLE in Taiwanese, as well as an earlier and more severe clinical course. These findings contrast with that of Lu et al.24 In addition, our study revealed that carriage of HLA-DR6 appeared to trend toward negative correlation with SLE, while carriage of HLA-DR8 appeared to trend toward positive correlation with SLE. SLE is a systemic disease with heterogeneous clinical manifestations, and the effects of different HLA-DR genotypes on disease susceptibility appear rather subtle; as a result, the discrepancy between our study and that by Lu et al24 could be due to sampling bias. In any case, a relatively low number of cases might not be enough to reveal the true nature of this disease.

ACKNOWLEDGMENTS

This work was supported by grants from the National Science and Technology Council, R.O.C. (MOST-111-2314-B-037-084) and Kaohsiung Medical University Hospital (KMUH111-1R18).

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