Class-I human leukocyte alleles in leprosy patients from Southern Brazil

Introduction: The present study was designed to investigate a possible role of HLA (histocompatibility leucocyte antigen) class-I alleles (HLA-A, -B, and -C) in leprosy patients from Southern Brazil. Methods: Two hundred and twenty-five patients with leprosy and 450 individuals for the control group were involved in this research. HLA genotyping was performed through PCR-SSO protocols (One Lambda, USA); the frequency of these alleles was calculated in each group by direct counting, and the frequencies were then compared. Results: There was an association between HLA-A*11 (6.9% vs 4.1%, p=0.0345, OR=1.72, 95% CI=1.05-2.81), HLA-B*38 (2.7% vs. 1.1%, p=0.0402, OR=2.44, 95% CI=1.05-5.69), HLA-C*12 (9.4% vs. 5.4%, p=0.01, OR=1.82, 95% CI=1.17-2.82), and HLA-C*16 (3.1% vs. 6.5%, p=0.0124, OR=0.47, 95% CI=0.26-0.85) and leprosy per se. In addition, HLA-B*35, HLA-C*04, and HLA-C*07 frequencies were different between lepromatous (LL) and tuberculoid (TT) patients. However, after adjusting for the number of alleles compared, Pc values became nonsignificant. Conclusions: Although our results do not support the previous findings that HLA class-I alleles play a role in leprosy pathogenesis, we suggest new studies because of the importance of the association between the HLA and KIR in the innate immune response to leprosy.


Article/Artigo
Leprosy is a chronic mycobacterial infection caused by the intracellular macrophage pathogen Mycobacterium leprae 1 , with about 260,000 new cases per year 2 .A major proportion of the leprosy cases remaining in the world are in India and a few other countries such as Brazil.
The more benign paucibacillary (PB) forms, borderline tuberculoid (BT) and tuberculoid tuberculoid (TT), are characterized by the predominance of a Th1-type immune response.In contrast, in the multibacillary (MB) forms, borderline borderline (BB), borderline lepromatous (BL) and lepromatous leprosy (LL), Th2-type immune response is responsible for the high antibody levels 3 .
The variability of the host response to infection seems to be genetically and environmentally influenced.The major histocompatibility complex (MHC), located on the short arm of human chromosome 6p21, is a candidate region for controlling disease susceptibility.The influence of the MHC on antigen presentation to T cells may be directly responsible for the genetic susceptibility to disease 4 .
In humans, histocompatibility leucocyte antigen (HLA) molecules are cell surface glycoproteins divided into two classical groups: HLA class-I (A, B, and Cw) and class-II (DR, DQ, and DP) antigens.The class-I loci encode molecules that bind antigenic epitopes usually derived from intracellular pathogens and present them to CD8+ T cells, thereby initiating a cytotoxic T cell response.The class-II loci specify molecules that primarily bind peptides of extracellular origin and present them to CD4+T cells, resulting in cytokine production and T cell help in the antibody production.
Therefore, this study aims at evaluating the influence of HLA class-I alleles on the susceptibility or resistance to leprosy and their clinical forms in Southern Brazilians.

Patients and controls
Two hundred and twenty-five patients with leprosy assisted at 15 th Regional de Saúde do Estado do Paraná, Maringá, Brazil were involved in this study.All the patients were from Paraná, Brazil.The individuals were classified in four distinct groups according to clinical and laboratorial observations offered by the dermatologist responsible for leprosy diagnosis: TT (46; 20.4%), LL (88; 39.1%), B (66; 29.3%), and indeterminate (23; 10.2%); two patients had no defined observation.Patient's age varied between 22 and 82 years (53.7±13.0);97 were female, and 128 were male.Volunteers who were identified through a questionnaire as positive for other infectious or auto-immune diseases were excluded from the study.
The control group was constituted of 450 healthy subjects matched according to age, sex, ethnic group, and occupation, with no history of HLA-associated diseases and other demographic parameters.They were typified by Laboratório de Imunogenética at Universidade Estadual de Maringá.Control's age varied between 13 and 89 years (37.1±12.3);240 were female, and 210 were male.
Paraná's population is predominantly of European origin (80.6%), with a small but significant contribution of African (12.5%) and Amerindian (7%) genes 22 .In this study, both patients (60.4% of European and 39.6% African origin) and controls (63.6% of European and 36.4% African origin) were classified as mixed ethnic groups, according to phenotypic characteristics, as according to Parra et al. 23 , in Brazil, at an individual level, skin color determined by physical evaluation is a poor predictor of genomic African ancestry.
The risk of population stratification bias due to differences in the ethnic background between patients and controls and variations of allele frequencies according to ethnic background was minimized by including patients and controls matched for the same ethnic background, residing in the same geographical area of leprosy prevalence.

DNA extraction
Venous blood samples (10 mL) were collected from the subjects, centrifuged for obtaining the buffy-coat and conserved at -20ºC until the use.The genomic DNA was extracted by EZ-DNA (Biological Industries  , Kibbutz Beit Haemek, Israel) or Neoscience (One Lambda  , CA, USA) kits from 150 µL of frozen blood, and then stored in a freezer at -20ºC.

Determination of alleles of the HLA system
After the evaluation of DNA purity and adjusting the concentration by optical density, HLA class-I alleles were genotyped by polymerase chain reaction-sequence specific of oligonucleotides (PCR-SSO) protocols with the Luminex 100xMAP flow cytometry dual-laser system to quantify fluorescently labelled oligonucleotides attached to color-coded microbeads (One Lambda®, CA, USA), according to the manufacturer's instructions.

Statistical analysis
The frequency of alleles was obtained through direct counting of the alleles and expressed as percentage frequency.The data were tested for their fit to Hardy-Weinberg equilibrium by calculating the expected frequencies of the genotypes and comparing them with the observed values using the Arlequin software, version 3.1, available at http://cmpg.unibe.ch/software/arlequin3.
The analysis of the association between the HLA variables and the occurrence of leprosy types was performed using Fisher's exact test, with the Graph Pad Software program (San Diego, CA, USA), available at http://www.graphpad.com/quickcalcs/contingency1.cfm.
The odds ratio (OR) with 95% confidence intervals (95% CI) was calculated using SISA statistics online, available at http://www.quantitativeskills.com/sisa/index.htm, to evaluate the risk of the individual to develop the disease having an HLA type.Data were considered statistically significant when p<0.05.
As each individual is tested for several HLA alleles and the same data were used for comparing the frequencies, it is possible that one of the alleles will by chance deviate significantly.To overcome this error, the p value is corrected by the use of Bonferroni inequality method, that is, by multiplying it with the number of alleles compared in each locus.
Ethical considerations All the participants signed written informed consent forms, authorizing the use of their samples in this study, which was approved by the Ethics Committee for Human Research of the Universidade Estadual de Maringá.
Table 1 summarizes the allele frequencies of class-I HLA alleles in leprosy per se and control populations and the results of the association analyses.Conformation to Hardy-Weinberg proportions was examined at all loci in the control group, and the examination did not show any deviation (p>0.05).
Percent allele frequencies of class-I HLA alleles in opposite poles of leprosy are represented in Table 2.The frequencies of HLA-B*35 and HLA-C*04 were lower in LL as compared with TT patients, while the frequency of HLA-C*07 was higher.Therefore, our association analysis of stratified patient groups indicates that HLA-B*35 and HLA-C*04 could be protective alleles against LL leprosy, while HLA-C*07 could predispose to this form of disease.Several studies have shown association of specific HLA class-II antigens/alleles with decreased or increased risk of leprosy in populations around the world (reviewed recently by Francheschi et al. 17 ).However, consistent association of HLA class-I antigens/alleles with leprosy or its progression toward clinical forms has not been well documented.This control-case study investigated the genetic variation present in HLA-A, -B, and -C genes and its relation with leprosy and subtypes.
Although after the correction of probabilities by multiplying p values by the numbers of variants tested in each locus, the results have been clearly statistically insignificant, we can note that the same association with HLA-A*11 and leprosy was also observed in other studies in Korea 24 and South India [20][21] .HLA-A11 was also significantly increased in erythema nodosum leprosum (ENL) leprosy patients from North India 25 .
In the Turkish population, different results were observed.HLA class-I antigens A9, A10, A32, B5, B21, Bw4, Bw6, Cw1, and Cw2 were found to be significantly more frequent in patients with leprosy, while HLA class-I antigens A3, B44, B49 were significantly more frequent in controls 19 .
When the groups were stratified, HLA-B*35 and HLA-C*04 showed to be protective against LL leprosy, perhaps due to the best presentation of bacillus to T cell, while HLA-C*07 showed to be a

TABLE 1 -Human leukocyte antigens class-I allelic frequencies in patients with leprosy per se compared with those in control subjects.
DISCUSSION

TABLE 2 -Comparison of human leukocyte antigens class-I allelic frequencies between patients with lepromatous and those with tuberculoid clinical forms of leprosy.
HLA: human leukocyte antigens, N: number of patients, n: allelic number, LL: lepromatous leprosy, TT: tuberculoid leprosy,