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Role of HLA alleles polymorphism in systemic lupus erythematosus: A prospective study from North India


1 Department of Pathology, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh, India
2 Department of Medicine, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh, India

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Date of Submission26-Jul-2021
Date of Acceptance05-Jan-2022
Date of Web Publication13-Jan-2023
 

   Abstract 


Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder and has complex etiopathogenesis. The most appropriate hypothesis states that genetic susceptibility in the presence of environmental risk factors predisposes to SLE. HLA class II alleles are critical to immune response and are highly polymorphic. Various alleles in HLA-DR and -DQ regions were analyzed in SLE patients and healthy controls to see their role in susceptibility or protection to SLE. Materials and Methods: This was a prospective observational study, in which a total of 100 SLE patients and 100 controls were analyzed. HLA typing was done by polymerase chain reaction (PCR)-sequence-specific oligonucleotide (SSO) method (SSO probe). Results: DRβ1*0301 was significantly increased in SLE patients when compared to controls and had the highest odds ratio. Other risk factor alleles found to be increased were DRβ1*0701, DQβ1*0202, and DQβ1*0301, which had a significant positive association with SLE, suggesting their role in susceptibility to SLE. In contrast, DRβ1*0401, DRβ1*1401, DRβ1*1404, DRβ1*1501, DQβ1*0501, and DQα1*0201 showed statistically significant reduction in SLE patients, while these were much more common in controls, suggesting their protective role. Conclusion: This study is only the second study in patients from North India and it determines the role of DRβ1*0301, DRβ1*0701, DQβ1*0202, and DQβ1*0301 alleles as risk factors in SLE patients.

Keywords: Autoimmune Diseases, DQ alleles in autoimmune diseases, DR alleles, HLA, systemic lupus erythematosus


How to cite this URL:
Rana RS, Naik B, Yadav M, Singh U, Singh A, Singh S. Role of HLA alleles polymorphism in systemic lupus erythematosus: A prospective study from North India. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Feb 2]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=367705





   Introduction Top


Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease affecting multiple organ systems and has a complex pathogenesis including genetic, immunologic, and environmental factors. Genetic association of SLE has been known for a long time now. There is an increased risk of SLE in first-degree relatives and twins. Monozygotic twins have even more risk than dizygotic twins. Although the etiology of SLE is multifactorial, still the most appropriate hypothesis is the combination of individual susceptibility determined through genetic risk factor alleles along with the presence of an environmental risk factor. Human Leukocyte Antigen (HLA) class II alleles are critical to immune response in an individual and have been found to be significantly associated with SLE. Majority of these studies have been done in USA, UK, and European countries and very few Indian studies are available corroborating the effect of HLA allele polymorphisms on SLE.[1],[2],[3],[4],[5] Ethnicity plays an important role in determining the pathogenesis. The primary aim of this study is to analyze HLA allele polymorphisms by HLA-DRβ1 (DRB1), -DQα1 (DQA1), and -DQβ1 (DQB1) typing on SLE patients and to compare them with healthy controls. Other objectives include estimating the association of these HLA alleles with specific autoantibody like anti-dsDNA and anti-Smith (Sm) antibody. SLE has a complex etiopathogenesis, and determining the HLA alleles conferring genetic susceptibility will have huge impact in disease management.


   Materials and Methods Top


This was a prospective observational study conducted in the UGC Advanced Immunodiagnostic Training and Research Centre of the Department of Pathology during the period 2012–2019. A total of 100 patients of SLE were included. Inclusion criterion was patients diagnosed as SLE as defined by the 2012 revised criteria proposed by the American College of Rheumatology.[6] Patients who were on immunosuppressive treatment were excluded from this study. Detailed clinical history and family history was retrieved from patients. Autoantibodies like antinuclear antibodies (ANA), anti-dsDNA, and anti-Sm antibody were tested by indirect non-competitive enzyme immunoassay (Euro-Diagnostica, Malmö, Sweden).

HLA Typing

HLA-DR and -DQ allele typing was done by polymerase chain reaction (PCR) using Mr. Spot machine (BAG Healthcare, Lich/Germany). The PCR test has four basic steps: first, DNA isolation; secondly, PCR amplification; then, hybridization and detection; and lastly, data interpretation.

Isolation of genomic DNA from peripheral blood was done by phenol–chloroform method. Isolated DNA was added to the master mix and MgCl2 solution, mixed, and amplified in a thermocycler. Hybridization of the amplicon with sequence-specific oligonucleotide (SSO) probes was done in MR.SPOT processor. Hybridization buffer was added to the amplified D NA and then the mixture was transferred to a test well containing all the required SSO probes, which were immobilized. The hybridizing oligonucleotides were marked with biotin and had a DNA sequence specific to the target HLA allele. If the amplified DNA had the target polymorphism and it was complementary to the oligonucleotide sequence, hybridization took place. The unbound DNA was washed off and the bound oligonucleotide could be detected by colorimetric reaction. Conjugate (streptavidin–alkaline phosphatase) was added to the wells to bind with biotin-labeled and SSO-bound amplicon. After washing the unbound conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate was added and after reacting with alkaline phosphatase, it produced a blue-purple reaction product. The colored end products were photographed on MR.SPOT and analyzed using the HISTO MATCH software. The HISTO MATCH software can determine the positive and negative reactions through the intensity of colored end product and HLA typing of the sample based on the specific hybridization pattern.

All statistical analyses were performed using statistical package for social sciences (SPSS) version 20 (IBM SPSS Statistics). The risk association of HLA alleles with SLE was determined by the odds ratio (OR). P value of less than 0.05 was taken to be significant.


   Results Top


Among all 100 SLE patients, more than 80% were females, while males formed less than 20%. Mean age of SLE patients was 30 ± 10 years (range: 13–60 years). The most common clinical manifestations were arthritis, alopecia, nephritis, and anemia.

Among DRβ1 genes, DRβ1*0301 (P value 0.04) and DRβ1*0701 (P value 0.023) had significant positive association with SLE, suggesting their role in its susceptibility. In contrast, DRβ1*0401 (P value 0.044), DRβ1*1401 (P value 0.03), DRβ1*1404 (P value 0.001), and DRβ1*1501 (P value 0.023) were significantly reduced in SLE patients, while these were much more common in controls, suggesting their protective role. Among these, OR (4.85) was the highest in DRβ1*0301 positivity [Table 1].
Table 1: HLA-DRβ1 in SLE cases

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Among the DQ alleles, DQβ1*0202 (P value 0.003) and DQβ1*0301 (P value 0.045) were significantly higher in SLE cases as compared to controls, while DQβ1*0501 (P < 0.001) was significantly reduced in SLE. DQα1*0201 (P value 0.034) had a significant negative association with SLE. OR of SLE was the highest in DQβ1*0202 (3.37%) and DQβ1*0301 (2.37%) among the statistically significant associations [Table 2].
Table 2: HLA-DQα1 and -DQβ1 alleles in SLE cases and control cases

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The HLA alleles significantly associated with SLE were evaluated for any significant association with autoantibodies. DRβ1*1404-positive patients had significant association with anti–Sjögren's-syndrome-related antigen B (SS-B), (P value 0.045), whereas DRβ1*1501-positive patients had negative association with ANA (P value 0.03). Correlation of DQβ1 and DQα1 alleles with autoantibodies showed that DQβ1*0202-positive patients had negative association with anti-SSB Ab (P value 0.031). DQβ1*0501-positive patients had negative association with anti-U1 Ribonucleoprotein (RNP) (P value 0.017) and anti-SSB Ab (P value 0.048), while others had no significant association with any autoantibodies.


   Discussion Top


HLA is a strong candidate gene for susceptibility development of SLE. Earlier studies have been unable to determine the exact genetic association because of high degree of polymorphism in HLA gene. In the present study, we analyzed newly diagnosed SLE patients without any prior immunosuppresive treatment and we observed several HLA alleles associated as a genetic risk factor of SLE and few others offering possible protection from SLE. This study is one of the very few Indian studies investigating the role of polymorphic HLA alleles in genetic sussceptibility to SLE and to the best of our knowledge, it is just the sixth study from India and the second study from North India.[1],[2],[3],[4],[5] Moreover, the role of HLA-DRβ1 has been studied, but HLA-DQα1 and -DQβ1 have not been extensively studied. The presence or absence of these susceptibility alleles and protective alleles can also predict prognosis, organ involvement, and association with other autoantibodies. Guidance of therapy and answers to questions such as whether any nephrotoxic drug can be initiated or not might be possible based on this susceptibility gene testing. In the present study, among the HLA-DRβ1 alleles analyzed in SLE patients and controls, DRβ1*0301 and DRβ1*0701 had significant positive association, whereas DRβ1*0401, DRβ1*1401, DRβ1*1404, and DRβ1*1501 were significantly reduced in SLE patients. Similarly, DQβ1*0202 and DQβ1*0301 were significantly increased in SLE cases as compared to controls, while DQβ1*0501 was significantly reduced in SLE. DQα1*0201 showed significant negative association with SLE. HLA-DRβ1*0301 is almost always associated as a genetic risk factor in majority of western and Indian studies [Table 3].[1],[3],[7],[8],[9],[10],[11] However, in a study done in Saudi Arabia, HLA-DRB3 was found to be protective against SLE. This study showed a contradictory result, but is unique as Saudi Arabia has a high rate of consanguineous marriages.[12]
Table 3: Association of HLA-DR and -DQ in SLE cases

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The present study and another Indian study have shown positive association of DRβ1*0701 with SLE patients.[2] However, few western studies have shown it to be negatively associated with SLE.[10],[11] DRβ1*0401, DRβ1*1401, and DRβ1*1404 showed negative or reduced expression in SLE patients in our study and similar findings were reported in other studies,[1],[2],[7],[8],[10] while one indian study showed increased expression.[3] However, DRβ1*1501 was negatively associated with SLE in the present study, but majority of western studies have reported it to be positively associated with SLE.[7],[8],[9],[13] None of the other Indian studies have reported it to be significant. A meta-analysis was performed by Niu et al.[7] on 25 studies from different countries (not in Indian population) to analyze the association of HLA-DRB1 gene polymorphism and SLE. HLA-DR4, -DR11, and -DR14 were negatively associated with SLE and can be considered protective. HLA-DR3, -DR9, and -DR15 showed significant positive association with SLE and can be attributed as the genetic risk factors for SLE. For lupus nephritis, HLA-DR4 and -DR11 showed possible protection and HLA-DR3 and -DR15 conferred increased risk.

A study done on Japanese population concluded that increased association or risk of SLE was found with DRB1*5:01 and decreased association or protection with DRB1*13:02 and DRB1*14:03.[14]

Association of HLA-DQ in SLE Cases

In the present analysis of HLA-DQβ1 and -DQα1 in SLE patients in our population, we demonstrated that HLA-DQβ1*0202 and -DQβ1*0301 were positively associated with SLE patients as compared to control, whereas HLA-DQβ1*0501 and -DQα1*0201 were negatively associated with SLE as compared to control. Similarly, in 2004, Cortes et al.[13] found HLA-DQβ1*0402 was positively associated with SLE, whereas HLA-DQβ1*0501 and -DQβ1*0303 were negatively associated. McHugh et al.[11] studied DQ alleles in British population and found that HLA-DQβ1*0201 was positively associated, while HLA-DQβ1*0603 was negatively associated with SLE. Shankarkumar et al.[1] showed a significant increase in the frequency of DQβ1*0302 and DQβ1*0601 among Indian patients with SLE and a decrease in DQβ1*0203. The results are fairly variable and much larger studies will be needed to corroborate the findings of the present study.


   Conclusion Top


The present study focuses on the multiple alleles of HLA as potential genetic risk factors for SLE. Alleles like DRβ1*0301 have been found to be widely associated with SLE patients in studies done on Indian and western population. This study also conclusively determines the positive role of DRβ1*0301 in SLE. Other alleles also show significant associations with SLE and the clinicopathological features of SLE, but variable results were obtained in previous studies. Ethinicity has a very important role in determining the genetic susceptibility, so larger studies done on Indian population are required to support the findings of the present study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Shankarkumar U, Ghosh K, Badakere SS, Mohanty D. HLA-DRB1*03 and DQB1*0302 associations in a subset of patients severely affected with systemic lupus erythematosus from western India. Ann Rheum Dis 2003;62:92-3.  Back to cited text no. 1
    
2.
Katkam SK, Rajasekhar L, Kutala VK. The influence of functional polymorphic positions of HLA-DRβ1 molecules on risk for South Indian systemic lupus erythematosus patients. Lupus 2018;27:991-1000.  Back to cited text no. 2
    
3.
Mehra NK, Pande I, Taneja V, Uppal SS, Saxena SP, Kumar A, et al. Major histocompatibility complex genes and susceptibility to systemic lupus erythematosus in northern India. Lupus 1993;2:313-4.  Back to cited text no. 3
    
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Pradhan VD, Devaraj JP, Shankarkumar U, Badakere SS. Autoimmune studies and HLA associations in SLE patients from Mumbai. Int J Hum Genet 2004;4:141-6.  Back to cited text no. 4
    
5.
Dedhia L, Pradhan V, Ghosh K, Nadkar M, Parekh S. HLA and KIR gene polymorphism in lupus nephritis patients from Western India. J Mol Sci 2018;2:3.  Back to cited text no. 5
    
6.
Petri M, Orbai AM, Alarcón GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677–86.  Back to cited text no. 6
    
7.
Niu Z, Zhang P, Tong Y. Value of HLA-DR genotype in systemic lupus erythematosus and lupus nephritis: A meta-analysis. Int J Rheum Dis 2015;18:17-28.  Back to cited text no. 7
    
8.
Ayed K, Gorgi Y, Ayed-Jendoubi S, Bardi R. The involvement of HLA -DRB1*, DQA1*, DQB1* and complement C4A loci in diagnosing systemic lupus erythematosus among Tunisians. Ann Saudi Med 2004;24:31-5.  Back to cited text no. 8
    
9.
International MHC and Autoimmunity Genetics Network, Rioux JD, Goyette P, Vyse TJ, Hammarström L, Fernando MM, et al. Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases. Proc Natl Acad Sci U S A 2009;106:18680-5.  Back to cited text no. 9
    
10.
Flåm ST, Gunnarsson R, Garen T; Norwegian MCTD Study Group, Lie BA, Molberg Ø. The HLA profiles of mixed connective tissue disease differ distinctly from the profiles of clinically related connective tissue diseases. Rheumatology (Oxford) 2015;54:528-35.  Back to cited text no. 10
    
11.
McHugh NJ, Owen P, Cox B, Dunphy J, Welsh K. MHC class II, tumour necrosis factor alpha, and lymphotoxin alpha gene haplotype associations with serological subsets of systemic lupus erythematosus. Ann Rheum Dis 2006;65:488–94.  Back to cited text no. 11
    
12.
Wadi W, Elhefny NE, Mahgoub EH, Almogren A, Hamam KD, Al-Hamed HA, et al. Relation between HLA typing and clinical presentations in Systemic Lupus Erythematosus patients in Al-Qassim region, Saudi Arabia. Int J Health Sci (Qassim) 2014;8:159-65.  Back to cited text no. 12
    
13.
Cortes LM, Baltazar LM, Lopez-Cardona MG, Olivares N, Ramos C, Salazar M, et al. HLA Class II haplotypes in mexican systemic lupus erythematosus patients. Hum Immunol 2004;65:1469-76.  Back to cited text no. 13
    
14.
Furukawa H, Kawasaki A, Oka S, Ito I, Shimada K, Sugii S, et al. Human leukocyte antigens and systemic lupus erythematosus: A protective role for the HLA-DR6 alleles DRB1*13:02 and *14:03. PLoS One 2014;9:e87792. doi: 10.1371/journal.pone. 0087792.  Back to cited text no. 14
    

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Correspondence Address:
Mahima Yadav,
D63/13A, 6 Annapoorna Nagar, Mahmoorganj, Varanasi, Uttar Pradesh - 221010
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_764_21




 
 
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  [Table 1], [Table 2], [Table 3]



 

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