Abstract
Abstract
Background
Previous studies have suggested an influence of HLA molecules on the regulation of the anti Mycobacterium leprae immune response.
Methods
DNA typing of HLA-DRB1 alleles in 71 leprosy patients and 81 healthy controls was performed. Genomic DNA was extracted from peripheral blood and used as a template to amplify the polymorphic second exon of the HLA-DRBl by the ploymerase chain reaction (PCR). PCR products were hybridized separately with sequence-specific oligonucleotides.
Results
DRB1*1401 and DRB1*1406 alleles were significantly more prevalent in leprosy patients, whereas a decreased frequency of DRB1*0808 and DRB1*1103 alleles was found, by comparison with the group control.
Conclusions
The HLA-DRB1 alleles could act alone or in combination with other genes to confer differential susceptibility and also protection to leprosy disease in endemic areas of the American continent.
Introduction
Leprosy is the clinical syndrome that occurs in genetically predisposed individuals as a result of infection with Mycobacterium leprae. It is a chronic infectious disease accompanied by irreversible peripheral nerve damage. 1 Leprosy is characterized by a spectrum of clinical manifestations, resulting from interactions between the host immune response and the invading M. leprae. 2
Although the immunopathology of leprosy has received much attention over the years, the proper reasons underlying individual differences in resistance and response to the bacilli are less characterized. Family and population studies have indicated that in humans host genetic factors are major determinants of susceptibility to leprosy. 3
Previous studies 4,5 have suggested an influence of HLA molecules on the regulation of the anti M. leprae immune response. Thus, the aim of the present study was to investigate the contribution of the HLA-DRB1 alleles in the development of leprosy disease in subjects that were born in Rosario, Santa Fe Province, Argentina. Since the majority of its inhabitants are descended from immigrants, mainly from European countries, the present study offered the opportunity to analyse the genetic association in a sample population mostly composed of European descendants.
Materials and methods
The study included 71 unrelated individuals with a diagnosis of leprosy (56 multibacillary and 15 paucibacillary forms), based on clinical assessment and detection of acid-fast bacilli in skin slit smears and skin lesion histopathology. They were made up of men and women of various age groups ranging from 49.17 ± 11.97 years. Cases were from Rosario city, which is the second largest urban area in the country. Additionally, 81 non-related healthy individuals with neither symptoms nor previous diagnosis of leprosy disease were included as a control group. Non-infected individuals were ethnically matched patients, from the same geographic area and had an average age of 48.62 ± 13.71 years.
The majority of Rosario inhabitants are descended from immigrants, mainly from Italy and Spain and other European countries and the Middle East. The city is situated on the Paraná river and serves as the commercial centre and port for a large agricultural and cattle-raising area, the so-called ‘wet-pampa’, a region that has most of the Argentine inhabitants.
All subjects gave informed consent to participate in the study, and the protocol was approved by the Ethic Committee of the School of Medical Sciences of Rosario, Argentina.
Genomic DNA was extracted from peripheral blood using the standard salting out method and used as a template to amplify by the polymerase chain reaction (PCR), the polymorphic second exon of the HLA-DRB1. Adequacy of PCR amplification was verified on 2% agarose gel electrophoresis with 2% ethidium bromide incorporated in the gel to facilitate visualization of amplified DNA bands. PCR products were hybridized separately with sequence-specific oligonucleotides (SSOs). The protocols of the 12th International Histocompatibility Workshop were used for the analysis of DRB1 associated alleles. The analysis was performed using primers specially designed for these genes. PCR products were dot-blotted and probed with SSOs to assign the DRB1 alleles. 6 Allelic level typing requires a two-stage process in which a primary (allele group specific) panel of probes/primers is employed followed by a secondary panel, selected in reference to results obtained with the primary panel, which affords discrimination of individual alleles within the group.
Differences in the distribution of HLA-DRB1 alleles between leprosy disease patients and controls were analysed by or Pearson's χ2-test with Yates correction or Fisher's exact test, if applicable. Odds ratios and relative risks were also calculated. P < 0.05 values were corrected according to the number of comparisons (α/n).
Results
The distributions of the HLA-DRB1 alleles in patients and controls are summarized in Table 1. Individuals with leprosy and healthy controls revealed 26 and 20 HLA-DRB1 different alleles, respectively.
Occurrence of HLA-DRB1 alleles in leprosy patients and controls
OR, odds ratio; RR, relative risk; CI, confidence interval; AF, allelic frequencies, NS, not significant
Data represent the number of each allele detected (n) and the AF with respect to the total number of studied alleles. P values <0.05 were considered significant and further corrected according to the number of comparisons
Among leprosy patients alleles occurring at higher frequencies (%) were DRB1*1501 (11.99%), DRB1*0801 (9.87%), DRB1*1401 (9.15%), DRB1*1406 (9.15%) and DRB1*1001 (8.45%). With reference to controls the most prevalent alleles were as follows: DRB1*0701 (14.20), DRB1*1103 (12.96), DRB1*0808 (12.35), DRB1*1303 (9.26) and DRB1*1101 (8.02).
In comparison with the control group, leprosy patients revealed a greater occurrence of the DRB1*1401 and DRB1*1406 alleles (P < 0.002), for which they may be implied in leprosy susceptibility. By contrast, DRB1*0808 and DRB1*1103 alleles were less frequent among patients (P < 0.002) and hence more involved in disease resistance.
In no case, did comparisons in HLA-DRB1 allelic frequencies between multibacillary and paucibacillary forms attain statistical significance.
Conclusions
The fact that not all individuals living in endemic areas are resistant to the disease supports the important participation of genetic factors in the development of leprosy disease. 3 The present study reports the analysis of HLA-DRB1 genes in a group of leprosy individuals. Molecular typing allowed the identification of increased frequencies of DRB1*1401 and DRB1*1406, in infected individuals with respect to the seronegative controls, suggesting that these antigens could be related with the infection by M. leprae. In this, patients also observed that the DRB1*0808 and DRB1*1103 alleles were statistically less frequent than in the group control.
The phenotype of susceptibility to infection by M. leprae is complex and is influenced by a variety of both host and parasitic factors and by the environmental conditions. The possible role of host genetic factors has been considered for many years. Many investigators have tried to find an association between leprosy and its subtypes with genetic markers, with the purpose of identifying susceptibility genes. More attention has been given to the HLA system, because alleles of its highly polymorphic loci encode HLA molecules, crucial to cellular interactions. In leprosy, both protective immunity and immunopathology are dependent on antigen-specific of major histocompatibility complex (MHC) class II restricted T-cell responses.
Previous studies 4 have demonstrated that leprosy is associated strongly with HLA-DR2, especially the DRB1*1501 and DRB5*0101 alleles. In addition other reports 5 have been published on the relationship between arginine at position 13 or 70–71 of DRB1 alleles with susceptibility to tuberculoid leprosy in patients from India. In our study, the alleles found with increased frequency in leprosy patients (DRB1*1401 and DRB1*1406) also show arginine at position 71 but this aminoacid was present in DRB1*0808 which was found with a decreased frequency in these patients. These findings indicate that further studies should be performed to evaluate the association of arginine in pocket 4 of DRB1 alleles with leprosy.
To determine a possible association between HLA specificities and leprosy and/or its clinical forms in an Argentinean population, frequencies of HLA specificities were compared between a group of patients and a group of controls and between the same controls and each subgroup of patients.
The MHC genes have an important role in T-cell repertoire selection, participate in antigen presentation and regulate immune response to infections, which in the case of M. leprae may be detrimental for the infected host 2 . As such these genes may participate not only in the clearance on infecting organisms but also on the mechanisms underlying tissue damage. 3–5 The HLA-DRB1 alleles could act alone or in combination with other genes to confer differential susceptibility and also protection to leprosy disease in endemic areas of the American continent.