==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 2379342310.5144/0256-4947.2013.229asm-3-229Original ArticleAssociation of HLA-DRB1*15 and HLA-DQB1* 06 with SLE in Saudis Al-Motwee Saleh aJawdat Dunia bJehani Ghassan S. aAnazi Hanan cShubaili Abdullah cSutton Paul cUyar Aytul F. dHajeer Ali H. c a Medicine, King Abdulaziz Medical City, Riyadh, Saudi Arabia b Immunology, King Abdulla International Medical Research Center, Riyadh, Saudi Arabia c Pathology, King Abdulaziz Medical City, Riyadh, Saudi Arabia d Department of Physiology, Istanbul University, Istanbul, TurkeyCorrespondence: Dr. Ali H. Hajeer, Pathology, King Abdulaziz Medical City, PO Box 22490 Riyadh, 11426 Saudi Arabia, T: +966 11 801 11 11, F: +966 11 252 0130, hajeera@ngha.med.saMay-Jun 2013 33 3 229 234 Copyright © 2013, Annals of Saudi Medicine2013This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND AND OBJECTIVES Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by humoral autoimmunity. The etiology of SLE is thought to be multifactorial including environmental, hormonal, and genetic factors. The human leukocyte antigen (HLA) has extensively been associated with the susceptibility to SLE; however, the association is heterogeneous among different ethnic groups. The aim of this study was to determine the association of HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 with SLE susceptibility in the Saudi population. DESIGN AND SETTINGS A total of 86 consecutive SLE patients attending the rheumatology clinic at King Abdulaziz Medical City, Riyadh, were recruited for this study. METHODS HLA types were determined by the polymerase chain reaction sequence-specific oligonucleotide (PCR-SSP) method in 86 patients and 356 control subjects. RESULTS The following HLA alleles were found to be positively associated with SLE: HLA-A*29 (OR=2.70; 95% CI=1.03–7.08; P=.0035), HLA-B*51 (OR=1.81; 95% CI=1.17–2.79; P=.0066), HLA-DRB1*15 (OR=1.45; 95% CI=0.98–2.29; P=.063), and HLA-DQB1*06 (OR=1.67; 95% CI=1.19–2.36; P=.0032), whereas HLA-DRB1*16 was negatively associated with the disease (OR=0.18; 95% CI=0.02–1.3; P=.055). HLA-DRB1*15 haplotypes were significantly associated with SLE (OR=2.01, 95% CI=1.20–3.68, P=.008); this was mainly due to the HLA-DRB1* 15-DQB1*06 association. CONCLUSIONS Our data suggest an association between MHC class I and class II (HLA-A*29, HLA-B*51, HLA-DRB1*15, and HLA-DQB1*06) and susceptibility to SLE in the Saudi population. HLA-DRB1*15-DQB1*06 haplotype showed the highest risk factor for the disease that is similar to what was seen in the African American patients, suggesting shared susceptibility genetic factors among these ethnic groups. ==== Body Systemic lupus erythmatosus (SLE) is a chronic autoimmune disease characterized by inflammation of various tissues and organs of the body due to the production of autoantibodies.1 SLE mostly affects the heart, lung, skin, joints, kidney, liver, blood vessels, and nervous system. Diagnosing SLE can be difficult as the symptoms come and go unpredictably. The disease is treatable mostly with corticosteroids and immunosuppressants, but thus far it is incurable and can be fatal. The prevalence of SLE varies among ethnicity and gender. It has been found to occur more frequently among African Americans and those of non-European descent. Also it is more common in women than in men.2 The etiology of SLE is unknown; however, several factors that have been considered to trigger as well as exacerbate the disease are hormones,3 pathogens,4 medications, and UV light.5 Moreover, genetic factors have been strongly suggested to contribute to the disease. These were mainly shown by studies on identical twins6 in addition to observing a higher disease frequency in the relatives of patients with SLE7 and children of mothers with SLE.8 Extensive studies have linked SLE susceptibility to genes of the human leukocyte antigen (HLA) region. However, the extent of this association varies among different population. The aim of this study is to investigate the association between HLA alleles and SLE in our Saudi population. METHODS Patients and controls A total of 86 consecutive SLE patients attending the rheumatology clinic at King Abdulaziz Medical City, Riyadh, were recruited for this study. All patients met at least 4 of the 11 American College of Rheumatology criteria.9 Patients were consented and file review was conducted to collect all clinical and laboratory data. HLA results were compared with 356 ethnically matched controls.10 HLA typing A total of 5 mL peripheral blood was collected in EDTA. DNA was prepared from blood leukocytes using the salting out procedure. White cells were separated using Ficoll Hypaque followed by lysis of erythrocytes in red blood cell lysis buffer and protein digestion in proteinase K solution. Finally, DNA was extracted by precipitating proteins in a saturated salt solution using the QIAamp DNA Blood Mini Kit from Qiagen (Valencia, California). All individuals were DNA typed for HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 using polymerase chain reaction-sequence specific primer (PCR-SSP) (Deutsche Dynal AG, Hamburg, Germany) using low-resolution typing method. Statistical analysis The maximum likelihood estimates of allele frequencies and haplotype frequencies were computed using an expectation maximization algorithm by the Arlequin software.11 To compare the differences between the allele frequencies in the controls and SLE groups, a 2×2 contingency table analysis was performed using the Pearson chi-square tests with Fisher exact test, when the expected value for an HLA marker was <5. The strength of association between HLA alleles and SLE was estimated by odds ratios (OR) and 95% confidence intervals (95% CI). P<.05 was considered to be statistically significant. For the 2-locus haplotypes, the the standardized disequilibrium coefficient (D′) and the chi-square values were also calculated. RESULTS We investigated HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genes in 86 Saudi SLE patients and compared their results with 356 healthy controls. The female gender dominated showing a ratio of female to male as 10.7:1, with the mean age of 26.1 years at onset and the mean disease duration of 8.8 years (Table 1). Arthritis was the most common clinical presentation, followed by renal involvement, malar rash, leucopenia, and alopecia (Table 2). Most patients presented with ANA (98.9%), anti-DNA (98.9%), and low complement C3 and C4 (93.0%), (Table 3). Tables 4 to 7 show the HLA class I and class II allele frequencies in both SLE cases and controls. The following HLA types were significantly increased in cases versus controls: HLA-A*29 (OR 2.70, 95% CI 1.03–7.08, P=.035) and HLA-DQB1*06 (OR 1.67, 95% CI 1.19–2.36, P=.032). However, HLA-B*51 (OR 1.81, 95% CI 1.17–2.79, P=.0066) and HLA-DRB1* 15 (OR 1.49, 95% CI 0.98–2.29, P=.063) were marginally significant. HLA-DRB1*01 (OR 0.16, 95% CI 0.02–1.2, P=.041) was protective, whereas HLA-A*02 (OR 0.69, 95% CI 0.47–1.02, P=.06) and HLA-DRB1*16 (OR 0.18, 95% CI 0.02–1.3, P=.055) were marginally protective. Table 8 describes the association between HLA-DRB1* 15 haplotypes and SLE. Apparently, HLA-DRB1* 15-DQB1*06 haplotype carried a significant risk for SLE (OR 2.01, 95% CI 1.20–3.68, P=.008) in our Saudi population. DISCUSSION We investigated the association of HLA genes in a Saudi cohort of SLE patients. This is the first description of immunogenetics of SLE in Saudi Arabia. The age at onset and the preponderance of females over males in this cohort were similar to other populations.12 Two major HLA haplotypes have been shown repeatedly to be associated with SLE worldwide: HLA-DR3 and HLA-DR2 (DR15 and DR16) bearing haplotypes.13–16 Different HLA-DR alleles were reported in different ethnics groups: HLA-DRB1* 0301 with Caucasians, HLA-DRB1*1503 with African Americans, and HLA-DRB1*08 alleles with Hispanics. In our population, HLA-DRB1*15 haplotypes were found to be associated with SLE in Saudis, while HLA-DRB1*16 was protective. In Mexicans, DR15 haplotypes were found to be associated with risk for SLE,17 while, 1 study showed that HLA-DRB1*16 was associated with chronic discoid lupus in Mexicans.18 HLA-DRB1*04 was protective in this Saudi population; the same result was observed in patients from Northwest Spain.19 Several studies analyzed the MHC region for genetic risk of SLE. Graham et al20 narrowed the disease-associated haplotypes HLA-DRB1*1501-HLA-DQB1*0602 and HLA-DRB1*0801-HLA-DQB1* 0402 to a region of 500 kb. Fernando et al21 using British SLE families and TdT analysis, narrowed the susceptibility region in MHC to 180 kb that involved the HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1* 0201. Our own results suggested that HLA-DRB1* 15-HLA-DQB1*06 haplotype is a risk factor for SLE in Saudis; however, looking at the allele frequencies we find that the frequency of HLA-DRB1*15 is nearly 20% whereas that of HLA-DQB1*06 is 40%, suggesting that HLA-DQB1*06 is associated with SLE independent of HLA-DRB1*15. Thus narrowing the risk area of SLE to the DQB1 region, it still remains elusive whether HLA-DQB1*06 is the culprit or another gene polymorphism is in linkage disequilibrium with it. One third of our patients have renal involvement; whereas, in other Asian populations, renal involvement ranged from 18% to 100%, majority reporting >50% of their patients.22 In Italians, lupus nephritis was found to be associated with the HLA-DR15-bearing haplotypes; 23 this was also reported in other studies.24–26 In our patients, there was no association between HLA-DR15-bearing haplotypes and lupus nephritis (data not shown). Alarcón et al27 analyzed factors influencing the development of lupus nephritis. Their results suggested that younger, hypertensive, and of African American or Hispanic ethnicity were predictors of lupus nephritis risk. Moreover, end-stage renal disease was also predicted by the presence of homozygosity for the valine allele of FcγRIIIa (FCGR3A*GG).27 This finding suggested that HLA-DR15 is not the only predictor of lupus nephritis risk and thus further analysis is required to determine the risk factors for the development of lupus nephritis in the Saudi patients. In conclusion, this is the first study to show HLA-DRB1 and HLA-DQB1 associations with SLE in the Saudi population. Acknowledgments We acknowledge the support extended by King Abdullah International Medical Research Center. Table 1 Demographics of the SLE patients. Characteristics Total patient number 86 F:M 10.7:1 Age range of patients 9–60 y Mean age at onset (SD) 26.1 10.2 y Disease duration mean (SD) 8.8 (5) y SLE: Systemic lupus erythematosus, F:M: female to male ratio, SD: standard deviation. Table 2 Clinical presentation of SLE patients. Clinical presentation No. (%) Arthritis 71 (82.6) Renal involvement 30 (34.9) Alopecia 19 (22.1) Raynaud phenomenon 7 (8.1) Photosensitivity 11 (12.8) Hemolytic anemia 13 (15.1) CNS involvement 4 (4.7) Malar rash 25 (29.1) Oral ulcers 13 (15.1) Leukopenia 25 (29.1) Thrombocytopenia 9 (10.5) Serositis 4 (4.7) Pleuritis 4 (4.7) SLE: Systemic lupus erythematosus, CNS: central nervous system. Table 3 Number and percentage of autoantibody positive SLE patients. Autoantibody No. (%) ANA 85 (98.9) Anti-DNA 85 (98.9) Low C3 or C4 80 (93.0) RNP antibodies 12 (14.0) Anti-Sm 5 (5.8) Anti-cardiolipin 39 (45.3) Anti-Ro 23 (26.7) Anti-La 10 (11.6) SLE: Systemic lupus erythematosus. Table 4 HLA-A associations with SLE in Saudi patients. SLE Controls OR 95% CI P N Frequency N Frequency HLA-A*01 17 0.099 51 0.072 HLA-A*02 40 0.233 217 0.305 0.69 0.47–1.02 .06 HLA-A*03 7 0.041 46 0.065 HLA-A*11 8 0.047 27 0.038 HLA-A*23 10 0.058 38 0.053 HLA-A*24 16 0.093 53 0.074 HLA-A*25 0 0.000 1 0.001 HLA-A*26 11 0.058 33 0.046 HLA-A*29 7 0.041 11 0.015 2.70 1.03–7.08 .035 HLA-A*30 7 0.041 39 0.055 HLA-A*31 16 0.093 50 0.070 HLA-A*32 4 0.023 37 0.052 HLA-A*33 7 0.041 43 0.060 HLA-A34 2 0.012 0 0.003 HLA-A66 1 0.006 1 0.001 HLA-A68 15 0.081 55 0.077 HLA-A69 2 0.012 0 0.000 HLA-A74 2 0.012 8 0.011 SLE: Systemic lupus erythematosus, OR: odds ratio, HLA: human leukocyte antigen. Table 5 HLA-B associations with SLE in Saudi patients. SLE Controls OR 95% CI P N Frequency N Frequency HLA-B*07 20 0.116 69 0.097 HLA-B*08 13 0.076 57 0.08 HLA-B*13 1 0.006 10 0.014 HLA-B*14 1 0.006 7 0.01 HLA-B*15 7 0.041 32 0.045 HLA-B*18 1 0.006 22 0.031 HLA-B*27 0 0 6 0.008 HLA-B*35 14 0.081 58 0.081 HLA-B*37 2 0.012 8 0.011 HLA-B*38 4 0.023 9 0.013 HLA-B*39 5 0.029 6 0.008 HLA-B*40 6 0.035 11 0.015 HLA-B*41 9 0.052 28 0.039 HLA-B*42 3 0.017 8 0.011 HLA-B*44 4 0.023 26 0.037 HLA-B*45 0 0 2 0.003 HLA-B*46 0 0 2 0.003 HLA-B*47 0 0 1 0.001 HLA-B*49 1 0.006 27 0.038 HLA-B*50 23 0.134 137 0.192 HLA-B*51 35 0.203 88 0.124 1.81 1.17–2.79 .0066 HLA-B*52 3 0.017 11 0.015 HLA-B*53 7 0.041 31 0.044 HLA-B*54 0 0 1 0.001 HLA-B*55 2 0.012 5 0.007 HLA-B*56 3 0.017 0 0 HLA-B*57 2 0.012 15 0.021 HLA-B*58 6 0.035 27 0.038 HLA-B*67 0 0 2 0.003 HLA-B*73 0 0 5 0.007 HLA-B*78 0 0 1 0.001 SLE: Systemic lupus erythematosus, OR: odds ratio, HLA: human leukocyte antigen. Table 6 HLA-DRB1 associations with SLE in Saudi patients. SLE Controls OR 95% CI P N Frequency N Frequency HLA-DRB1*01 1 0.006 25 0.035 0.16 0.02–1.20 .041 HLA-DRB1*15 35 0.203 104 0.146 1.49 0.98–2.29 .063 HLA-DRB1*16 1 0.006 23 0.032 0.18 0.02–1.30 .055 HLA-DRB1*03 30 0.174 103 0.145 HLA-DRB1*04 17 0.099 114 0.16 HLA-DRB1*11 12 0.07 49 0.069 HLA-DRB1*12 0 0 5 0.007 HLA-DRB1*13 29 0.169 94 0.132 HLA-DRB1*14 1 0.006 7 0.01 HLA-DRB1*07 33 0.192 144 0.202 HLA-DRB1*08 5 0.029 7 0.01 HLA-DRB1*09 1 0.006 0 0 HLA-DRB1*10 7 0.041 37 0.052 SLE: Systemic lupus erythematosus, OR: odds ratio, HLA: human leukocyte antigen. Table 7 HLA-DQB1 associations with SLE in Saudi patients. SLE Controls OR 95% CI P N Frequency N Frequency HLA-DQB1*02 60 0.349 245 0.344 HLA-DQB1*03 34 0.198 164 0.23 HLA-DQB1*04 3 0.017 19 0.027 HLA-DQB1*05 5 0.029 77 0.108 HLA-DQB1*06 70 0.407 207 0.291 1.67 1.19–2.36 .0032 SLE: Systemic lupus erythematosus; OR: odds ratio, HLA: human leukocyte antigen. Table 8 Haplotypes in association with SLE. SLE Controls DRB1*15 Haplotype N Frequency N Frequency HLA-A*02 HLA-B*07 HLA-DRB1*15 HLA-DQB1*06 8 0.047 27 .038 HLA-A*01 HLA-B*51 HLA-DRB1*15 HLA-DQB1*06 4 0.023 0 0 HLA-A*02 HLA-B*51 HLA-DRB1*15 HLA-DQB1*06 3 0.017 8 .011 HLA-A*31 HLA-B*35 HLA-DRB1*15 HLA-DQB1*06 3 0.017 6 .008 HLA-A*02 HLA-B*50 HLA-DRB1*15 HLA-DQB1*06 2 0.012 1 .001 SLE: Systemic lupus erythematosus, human leukocyte antigen. 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