Marco A. Braga

Association between killer‐cell immunoglobulin‐like receptor genotypes and leprosy in Brazil

The aim of this study was to investigate the role of killer cell immunoglobulin-like receptor (KIR) genes in leprosy immunopathogenesis. Genotyping of KIR and human leukocyte antigen (HLA) genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes in 165 leprosy patients. Both activating KIR2DS2 and KIR2DS3 frequencies were higher in tuberculoid leprosy (TT) patients than in lepromatous leprosy (LL) patients, and the inhibitory KIR with its ligand, KIR2DL1-C2/C2, was elevated in TT patients in comparison to all other leprosy subgroups and controls. However, a negative association between KIR2DL3-C1 and KIR2DL3-C1/C1 and the TT group was identified. Borderline patients exhibited a higher frequency of KIR3DL2-A3/11 than the controls and LL patients, and a lower frequency of KIR2DL1-C2 than the controls and TT subgroup. Some KIR-HLA genotypes could be associated to the development of clinical forms of leprosy and should be investigated further.

DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors

The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.

Influence of class I and II HLA alleles on inhibitor development in severe haemophilia A patients from the south of Brazil.

Summary.  Congenital haemophilia A is a chromosome‐linked recessive disorder caused by the deficiency or reduction of factor VIII (FVIII) pro‐coagulant activity. During treatment, some patients develop alloantibodies (FVIII inhibitors) that neutralize the action of exogenously administered FVIII. Currently, the presence of these inhibitors is the most serious adverse event found in replacement therapy. Some studies have suggested that genetic factors influence the development of the FVIII coagulation inhibitors. To identify the class I and II alleles that may be influencing the formation of inhibitors in severe haemophilic patients. Genotyping of the class I (HLA‐A, ‐B and ‐C) and class II (HLA‐DRB1, ‐DQA1 and ‐DQB1) alleles of 122 patients with severe haemophilia A, including 36 who had developed antibodies to factor VIII, was performed. After the comparison of the group without inhibitors and the group with inhibitors, HLA‐C*16 [Odds ratio (OR) = 7.73; P = 0.0092] and HLA‐DRB1*14 (OR = 4.52; P = 0.0174) were found to be positively associated with the formation of the inhibitors. These results confirm that HLA alleles are involved in inhibitor production and could be used as a tool for recognition of groups at high risk of possible inhibitor development in Southern Brazilian haemophilic patients.

Frequencies of MICA alleles in patients from southern Brazil with multibacillary and paucibacillary leprosy

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which mainly affects the skin and nervous system. The disease has several clinical forms. This study investigated the MICA and HLA‐B genes in 223 samples from leprosy patients and 201 samples from healthy individuals matched for age, gender and ethnical background. Of the patients, 153 had multibacillary, 45 paucibacillary and 25 indeterminate leprosy. The aim of this case–control study was to assess whether the MICA alleles influence susceptibility for leprosy or affect the subtype of the disease in a population of southern Brazil. There were significant differences in frequencies of the MICA*027 allele (4.7% vs 1.8%, P‐value = 0.01, OR = 0.37; 95% CI = 0.16–0.85) between leprosy patients and controls, and of the MICA*010 (4.5% vs 1.6%, P‐value = 0.05, OR = 0.35, 95% CI = 0.13–0.97) and MICA*027 alleles (4.7% vs 1.3%, P‐value = 0.01; OR = 0.27; 95% CI = 0.09–0.79) between multibacillary leprosy patients and the control group. There were no significant differences in the frequency of MICA alleles between paucibacillary leprosy patients and controls. Thus, the MICA*027 allele is associated with a protective effect for leprosy per se, while the MICA*010 and MICA*027 alleles are associated with protection against multibacillary leprosy, the most severe clinical subtype.

Investigation of deletion of 22pb in KIR2DS4 gene in a population of southern Brazil.

The aim of this study was to investigate the distribution of full‐length and deleted variants of KIR2DS4 in a population of southern Brazil and compare the results with other populations, as well as comparing two techniques, PCR‐SSP and PCR‐SSO, for typing of variants.

KIR and HLA ligands demonstrate genetic inheritance diversity in Japanese descendants from Paraná, Brazil

In order to investigate killer immunoglobulin-like receptors (KIR) and their ligands, human leukocyte antigen (HLA), diversity in the Brazilian population influenced by migrations, unrelated Brazilian Japanese descendants were selected and genotyped for the KIR genes and HLA class I allele groups. Genetic heterogeneity in Brazil Paraná Japanese was observed for KIR genes, whose frequency distributions demonstrated similarity with mixed Brazilian populations and with the Japanese population, suggesting gene flow. The data contributed to the identification of the genetic constitution of the Brazilian population influenced by immigrations and two new genotypes were defined.