HLA Association in SLE patients from Lahore-Pakistan

Th e fi rst genetic factors to be identifi ed as important in the pathogenesis of Systemic lupus erythematosus (SLE) were those of the major histocompatibility complex (MHC) on chromosome . It is now widely accepted that MHC genes constitute a part of the genetic susceptibility to SLE. Th e study population comprised  SLE patients fulfi lling at least four of the American college of Rheumatology criteria for SLE and  healthy blood donors as controls. SLE female versus male ratio was approximately :. Mean age at diagnosis was . ± . (- years). DNA-based HLA Typing for HLA-A, HLA-B, and HLA-DRB was carried out by Polymerase chain reaction with sequence specifi c primers using genomic DNA obtained from blood samples. A total of  alleles have been studied at locus A,  alleles at locus B and  DRB alleles. Th e allelic frequencies of HLA-A, HLA-B, and HLA-DRB antigens in SLE patients from Pakistan were compared with the controls. A signifi cant increase was observed in the frequency of HLA-A*, A*, A*, A*, A* A*, HLA-B*, B*, B*, B*, B*, B*, B*, B*, HLA-DRBI*, DRBI*, DRBI*, DRBI* among SLE patients indicating a positive association of these alleles with SLE. HLA-A*, A*, A*, A*, A*, A*, HLA-B*, HLA-DRB*, were found to be decreased in the patient group as compared to controls indicating a negative association of these alleles with SLE. Th us from this study we can conclude that SLE is associated with certain MHC alleles in Pakistani population. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


Introduction
Th e fi rst genetic factor to be identifi ed as important in the pathogenesis of SLE was the Major Histocompatibility Complex (MHC).Th is complex which resides on chromosome  comprises at least  genes as well as  pseudogenes with many of these genes playing an important role in immune responses [].The MHC shows a high degree of polymorphism and these polymorphisms are associated with a wide range of diseases.Defects in certain MHC genes lead to autoimmune disorders as it is a failure of self tolerance, recognition of an antigen that is self and reacts to it; one of the examples is that of Systemic Lupus Erythematosus (SLE).It is now widely accepted that MHC genes constitute a part of the genetic susceptibility to SLE.MHC is divided in to Class I, Class II, Class III regions, each containing group of genes with related functions.For example, within the MHC, there are  genes that encode Class I molecules, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G and among these HLA-A, B, C are the most polymorphic and probably the most important.Furthermore, fi ve loci encode Class II molecules, among them HLA-DP, DQ and DR are most important and polymorphic.Association of SLE with DRB alleles has been studied and DRB* is found to be positively associated with SLE in most of the populations [].Number of genes resides in the MHC Class III region such as early components of the complement cascade, heat shock  gene complexes, tumor necrosis factor complexes and many other genes.One of the mechanism by which all these alleles may contribute to pathogenic outcomes in SLE is the alterations in the nature of antigen presentation by HLA molecules to T-Helper cells that lead to abnormal T-cell response [].We conducted this study to analyze the contribution of HLA-A, HLA-B, and of DRB alleles to the susceptibility to SLE among Pakistani SLE patients and controls.

Materials and Methods
The human studies reported in this manuscript were approved by the Ethical Committee of the School of Biological Sciences, University of the Punjab, Lahore, Pakistan.A total of  SLE patients fulfi lling American College of Rheumatology (ACR) criteria and  controls were enrolled in this study.Sample number is low because SLE is a quite rare disease in Pakistan.Rheumatology and Nephrology department of different hospitals of Lahore were chosen because most of the patients were referred to these departments for the diagnosis of lupus.Of the , fi fty-fi ve (.) were females and six (.) were males.Th e female versus male ratio was .: indicating that SLE is quite common in females.Mean age at diagnosis was . ± . years and the range was - years.Mucocutaneous involvement was found in lupus patients such as malar rash  (.), discoid rash  (.), photosensitivity  (.).Various syndromes were overlapping SLE and the most common one was Rheumatoid arthritis ( patients: .) but cases of Sjogren's syndrome ( patients: .), Scleroderma ( patients: .), Secondary Antiphospholipid syndrome ( patients: .), and of Budd-Chiari Syndrome ( patients: .) were also found along with SLE.Renal involvement was found in  patients: .) patients characterized by proteinuria and red cell cast.Blood samples drawn by venepuncture from each patient and control were collected in an ethylene diamine tetra acetic acid (EDTA) vial for DNA-based HLA Typing.An informed written consent was taken from all subjects recruited in the study as well as from the matched controls.Genomic DNA was purifi ed by using Genomic DNA purifi cation kit (Fermentas, Cat  K-USA) [].HLA-A, HLA-B, and HLA-DRB typing was carried out by Polymerase chain reaction with sequence specific primers (One Lambda, Lot  -USA) using genomic DNA from SLE patients.Th e concentration of DNA was estimated by taking A/A ratio [].According to One Lambda HLA Class I Class I HLA typing kit protocol, the Polymerase Chain Reaction-Single Stranded Polymorphism (PCR-SSP) methodology was based on the principle that by using recombinant Taq polymerase, completely matched oligonucleotide primers are more effi ciently used in amplifying target sequence.All kit reagents and DNA samples were brought at room temperature and then vortexed.Primer set tray was placed in a PCR tray microtube storage rack and the tray label was removed. μl of deionized water was added to the negative control reaction tube on the primer set tray.Th en . μl of recombinant Taq polymerase ( units/μl) was added to the Micro SSP TM Dmix tube and vortexed.Th e Micro SSP TM D-mix tube was pulse spinned in a microcentrifuge to bring all liquid down from sides of the tube.Micro SSP TM D-mix ( μl) was added to the negative control reaction tube. μl of the DNA sample was then added to the Micro SSP TM D-mix tube, vortexed and again spinned.Sample-reaction mixture ( μl) from the Micro SSP TM D-mix tube was added in to each reaction tube of the Micro SSP TM primer set tray except the negative control reaction tube.Th e reaction tubes were then sealed with the tray seal.Th e Micro SSP TM primer set tray was placed in a thermocycler and PCR program was started.Primer pairs were designed to have perfect matches only with a single allele or group of alleles.Under strictly controlled PCR conditions (Table ), perfectly matched primer pairs resulted in the amplifi cation of target sequences.Th e primer set tray was removed from the thermocycler and was unsealed gently without splashing the samples. μl of the PCR reaction was transferred to a . agarose gel and the samples were electrophoresed at  volts for  minutes, bands were fi nally visualized by UV illuminator (Figure ).The control primer pair amplifies a conserved region of the Human β-globin gene, which is present in all human DNA samples and is used to verify the integrity of the PCR reaction.The amplified DNA fragments of the specific HLA primer pairs were smaller than the product of the internal control primer pair, but larger than the diff use, unincorporated primer band.The typing results were interpreted by using the worksheet provided with the trays.

Statistical analysis
The allelic frequencies of HLA-A, HLA-B, and HLA-DRB antigens in Pakistani SLE patients were com-   pared with the controls by using online MedCalc software for calculating Odds Ratios [].Fisher's exact test was calculated by using only online available Fisher's exact test calculator described by Agresti in .

Results
Th e typing results were interpreted by using the worksheet provided with the trays (Appendix-VI).Th e allelic frequencies of HLA-A, HLA-B, and HLA-DRB alleles in Pakistani SLE patients were compared with the controls by using online MedCalc software for calculating Odds Ratio [].It is conventional to create confi dence interval (CI) at  level as it provides a range about the observed eff ect size.Nomenclature of HLA alleles included in this chapter was listed in the January  update of the IMGT database (A sequence database for the human major histocompatibility complex) [].

HLA-A Alleles
A total of  alleles were studied at locus A,  alleles at locus B and  DRB alleles.Frequency of HLA-A alleles in control as well as in case groups is mentioned in Figure .
An increase was observed in the frequency of HLA-A*, -A*, -A*, -A*, -A*, -A* alleles among total SLE patients.Odds Ratio for HLA-A*, -A*, -A*, -A*, -A*, -A* alleles was more than  and the lower, upper bound of confidence interval didn't include  that's why these alleles can act as a signifi cant risk to SLE.For example, Odds ratio for HLA-A* allele was ., it means that the odds of HLA-A* allele was four times higher in cases than controls.There was a negative association of HLA-A*, A* alleles with SLE as the OR was less than  and the confidence interval did not include

Association of HLA alleles with SLE
In order to prove the signifi cant association of HLA alleles with SLE, Fischer's exact test was applied and it was found that HLA-A*, HLA-A*, HLA-A*, HLA-A*, HLA-DRB* were signifi cantly associated with SLE (Table ).

Discussion
SLE is a genetically complex disease and the major contribution is from MHC genes.A lot of work has been done to fi nd out the role of genetic factors both in murine lupus models and in human SLE.Genetic analysis of SLE in humans and in murine lupus models, suggest that SLE is a polygenic disorder.The actual genes identified in human and murine SLE may not be identical but immunological pathways can be similar [].Th e precise genetic cause of the association has been diffi cult to defi ne because of the high degree of polymorphism within the genes of HLA.In the present study, those MHC genes were identified which were involved in the development of SLE in a relatively defined population in Lahore, Pakistan.Th e relationships between HLA and disease are of considerable importance as they provide new tools for studying the inheritance, classification, diagnosis and pathogenesis of the disease in question.
It is universally accepted that genetic factors play an important role in the susceptibility to develop SLE and varies among populations.Particular attentions have been focused on the association of the disease with genetic markers located within the MHC region.Several studies have illustrated the fact that SLE is associated with certain MHC alleles in different populations.Vargas-Alcaron et al. [] studied heat shock  gene polymorphism in Mexican patients with autoimmune disease.Sirikong et al. [] investigated the association of DRB and DQB with Th ai patients having SLE and found signifi cant increase in the frequency of DRB* and DQB*.Ramal et al. [] reported that HLA associations between HLA-DRB-DQB and SLE were diff erent for Gypsy people as he found DRB*, DQB* and DR to be signifi cantly associated with SLE.Endreff y et al. [] studied DRB, DQA, and DQB allele polymorphisms along with their clinical features in Hungarian patients having SLE while Azizah et al. [] reported them in Malaysian-Chinese population, the diff erence is that the later study showed strong association of DQA* with anti-Ro/La while DQA* was found to be associated with dsDNA in Malaysian Chinese population.In this study, HLA-DRB, HLA-DRB, HLA-DRB, HLA-DRB alleles were studied but few HLA-DRB alleles were found to be associated with SLE while HLA-DRB, HLA-DRB, HLA-DRB alleles were absent in Pakistani SLE patients as well as in the healthy controls.Pradhan et al. [] studied HLA associations in SLE patients from Mumbai and showed a significant two-fold increase in the odds-ratio for HLA A, A, and B alleles with increase Ro, La, Sm and dsDNA autoantibodies while there is significant two-fold decrease in the odds-ratio for HLA A, A, B B alleles with decreased Ro, La, Sm and dsDNA autoantibodies when compared to normal controls.
In   could not be demonstrated in racially mixed population []. In

FIGURE 1 .
FIGURE 1.A positive reaction for a specifi c HLA allele or allele group was visualized on the gel as in the wells 2, 8, and 15, an amplifi ed DNA fragment can be observed between the internal control product and the unincorporated primer band.NC: Negative control in the well 1, representing the primer band.
this study, HLA-A*, -A*, -A*, -A*, -A*, -A*, HLA-B*, -B*, -B*, -B*, -B*, -B*, -B*, -B*, HLA-DRBI*, -DRBI*, -DRBI*, -DRBI*, -DRBI* were significantly increased in SLE patients as compared to the controls.HLA-A*, -A* HLA-B*, -B*, -B*, -B*, HLA-DRB*, -DRB*, -DRB*, -DRB* pro- the present study, HLA-DRBI*, -DRBI*, -DRBI*, -DRBI*, -DRBI* were signifi cantly increased in SLE patients as compared to controls.HLA-DRB*, -DRB*, -DRB*, -DRB* provided protection from the disease while HLA-DRB* had no aff ect on the disease.Morimoto et al.[]  studied the relation of Class II with Cutaneous manifestations in Japanese population and found a positive malar rash associations with DRB* and skin ulcers with DRB*.In addition most studies showed an association between HLA DRBI*, HLA DRB* and lupus nephritis, although HLA alleles have rarely been associated with SLE renal histopathology.In the work presented here, HLA-DR-BI*, -DRBI*, -DRBI*, -DRBI* were found to be significantly increased in SLE patients.DRB* showed a strongest association (OR: .;  CI: .-.)with SLE patients.Castano-Rodriguez et al.[]  found HLA-DRB gene as a major factor for the development of SLE in Latin Americans and showed the strongest association of HLA-DRB* (OR: .;  CI: .-.)with SLE.Our results, HLA-DRB* (OR: .;  CI: .-.)also showed a positive association with SLE but DRB* disclosed the strongest association (OR: .;  CI: .-.).Wong and Tsao[]  suggested that HLA Class II containing DRB and DQB alleles are strong risk factors for SLE.Furthermore, Farjadian et al.[]  reported that the most frequent Class I and Class II alleles in Baloch population with SLE of Iran and Pakistan were A* (.),B* (.),DQA* (.),DQB* (), and DRB* ().In the work being discussed here, HLA-B* (OR: .;  CI: .-.)and HLA-DRB* (OR: .;  CI: .-.)were also found to be signifi cantly increased in SLE patients but variation lies in HLA-A* allele which was found to provide protection from the disease in the studied SLE patients.Th us diff erences also lie within the Asian population but all of them showed HLA association with SLE.Th us this case-control study highlights the importance of HLA polymorphism in lupus susceptibility.ConclusionThis study further confirmed and extended the association of SLE with certain MHC alleles in Pakistani population like HLA-A*, -A*, -A*, -A*, -DRB* were significantly associated with SLE.Identification of genes involved in the development of SLE will provide new insight into the development of disease in susceptible individuals by HLA typing.As new therapies are developed our studies will open up new opportunities to improve diagnosis and treatment of SLE patients in this population.

TABLE 1 .
One Lambda PCR Program , -A*, -A*, -A*, -A*, -A* alleles with SLE was not proved by this study at  signifi cance level (Table).Frequency of HLA-A alleles in control and case groups FIGURE 3. Frequency of HLA-B alleles in control and case groups . HLA-A* had no aff ect because OR was equal to , means that this was not signifi cant at  level for  confi dence intervals.HLA-A*, -A*, -A*, -A*, -A*, -A* alleles were neither found in cases nor in controls.Association of HLA-A*HLA-B Alleles There was an increase in the frequency of HLA-B*, -B*, -B*, -B*, -B*, -B*, -B*, -B* among total SLE patients indicating a positive association of these alleles with SLE.Frequency of HLA-B alleles in control as well as in case groups is mentioned in Figure .Odds Ratio for HLA-B*, -B*, -B* and -B* was less than  indicating a negative association between these alleles and SLE.Similarly, Odds ratio for HLA-B*, -B*, 2.
The frequency of HLA-DRBI*, -DRBI*, -DRBI*, -DRBI*, -DRBI* was increased among total SLE patients, thus showed positive association between the alleles and SLE disease at the  confi dence level as the Odds of these alleles was greater in cases than in controls.HLA-DRB*, -DRB*, -DRB*, -DRB* alleles were the protective factors in the causation of SLE disease or in other words showing a negative association with SLE at the  signifi cance level because the Odds of these alleles was smaller in cases than in controls.It was observed that HLA-DRB* had no aff ect representing no relationship between these alleles and SLE.HLA-DRBI*, -DRBI*, -DRB*, -DRB*, -DRB*,-DRB* were not found in both cases and in controls.Further more, association of HLA-DRB* allele with SLE was not proved by this study at  signifi cance level (Table).

TABLE 3 .
HLA Alleles at Locus B

TABLE 5 .
Association of HLA alleles with SLE by Fischer Exact test NAGEEN HUSSAIN ET AL.: HLA ASSOCIATION IN SLE PATIENTS FROM LAHOREPAKISTAN vided protection from the disease while HLA-A* HLA-B*, -B*, -B*, -B*, HLA-DRB* had no affect.HLA-DR is expressed on a signifi cantly high percentage of both CD and CD circulating T cells in active cutaneous lupus erythematosus[].HLA DRB* and the allelic variant DRB* have been associated with SLE in Blacks and Asians and HLA-DRBI*,  and  have been associated with lupus in Caucasians but clear associations with HLA and SLE BOSNIAN JOURNAL OF BASIC MEDICALSCIENCES 2011; 11 (1): 25-26