HLA polymorphisms and risk of red blood cell alloimmunisation in polytransfused patients with sickle cell anaemia.

BACKGROUND: Red blood cell (RBC) alloimmunisation is an event that may occur due to factors such as numerous blood transfusions, age, gender and genetic factors such as human leukocyte antigen (HLA). AIMS/OBJECTIVES: The aim of the present study was to investigate the possibility of alloimmunisation to red blood cell group antigens associated with the HLA of individuals and to relate alloimmunisation to risk factors. METHODS: A total of 172 polytransfused patients with sickle cell anaemia (SCA) (44 alloimmunised, 128 non-alloimmunised) participated in this study. Blood group genotyping was performed by the DNA microarray method and HLA genotyping by polymerase chain reaction - specific sequence of oligonucleotides. RESULTS: The number of transfusions received directly influenced the incidence of alloimmunisation, and the most common alloantibodies were against Rh (48·8%) and Kell (17%) systems. The HLA-C*06 and HLA-DQB1*03 variants were significantly higher in alloimmunised patients. The HLA-DRB1*04 and HLA-DRB1*11 were more often found in individuals who developed the alloantibodies anti-Fya and anti-K, respectively. CONCLUSION: This study suggests that polytransfused patients with SCA possessing the HLA-DQB1*03 and HLA-C*06 allele variants are more susceptible to alloimmunisation. In addition, HLA-DRB1*04 and HLA-DRB1*11 alleles were seen to be associated with the production of anti-Fya and anti-K antibodies, respectively.

New associations: INFG and TGFB1 genes and the inhibitor development in severe haemophilia A.

INTRODUCTION: The development of factor VIII (FVIII) inhibitor is the main complication of replacement therapy in patients with haemophilia A (HA). A ratio of 5-7% of individuals HA develops antibodies (inhibitors) against the FVIII infused during the treatment, thereby reducing their pro-coagulant activity. The immunomodulatory cytokine genes have been related to the risk of development of alloantibodies in several studies, mainly in HA with severe form. AIM: We investigated the polymorphisms in regulatory regions of cytokine genes (IL1A, IL1B, IL1R, IL1RA, IL4RA, IL12, INFG, TGFB1, TNF, IL2, IL4, IL6, IL10) that could influence the risk of developing inhibitors in patients with severe HA. METHODS: The genotyping of cytokine genes of 117 patients with HA was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the protocol recommended by the manufacturer (Invitrogen kit Cytokines(®) , Canoga Park, USA) RESULTS: From the cohort of 117 patients with severe HA, 35 developed inhibitors. There was a higher frequency of +874 T allele in INFG and of +869 TT and TG/TG in TGFB1 genes on patients with inhibitors. CONCLUSION: This suggests that polymorphisms in INFG and in TGFB1 genes are related to risk of developing inhibitor, and could contribute to a genetic profile of the individual HA for the risk of inhibitors development to FVIII.

Allele and haplotype frequencies of HLA-A, B, C, DRB1 and DQB1 genes in polytransfused patients in ethnically diverse populations from Brazil.

The red blood transfusion is a practice often used in patients with haematological and oncological diseases. However, the investigation of human leucocyte antigen (HLA) system frequency in these individuals is of great importance because multiple transfusions may lead to HLA alloimmunization. Brazil is a country that was colonized by many other ethnicities, leading to a mixed ethnicity and regionalized population. In view of the importance of HLA typing in these patients, the aim of this study was to investigate the allele and haplotype frequencies from polytransfused patients from three different regions from Brazil. HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genotyping of 366 patients was performed by PCR-SSO, based on the Luminex technology (One Lambda(®) ), and the anti-HLA class I and class II antibodies were analysed using LabScreen Single Antigen Antibody Detection (One Lambda, Inc.). Allele and haplotype frequencies of polytransfused patients of three regions from Brazil were obtained using the Arlequin program. The most frequent allele frequencies observed were HLA-A*02, A*03, B*15, B*35, B*51, C*07, C*04, C*03, DRB1*13, DRB1*11, DRB1*07, DRB1*03, DRB1*01, DQB1*03, DQB1*02, DQB1*06 and DQB1*05. There were differences between the groups for allele variants HLA-B*57 (between Group 1 and Group 2) and HLA-C*12 (between Group 1 and Group 3). The most frequent haplotypes found in the sample were HLA-A*01B*08DRB1*03, DRBI*07DQB1*02, DRB1*01DQB1*05, DRB1*13DQB1*06 and A*02B*35. HLA class I and II antibodies were detected in 77.9% and 63.9% patients, respectively, while the both alloantibodies were detected in 62 (50.9%) patients. In conclusion, the HLA typing for polytransfused patients in each region has a great importance, as seen in this study; individuals from different regions from Brazil have HLA distribution not completely homogeneous.

Influence of KIR genes and their HLA ligands in susceptibility to dengue in a population from southern Brazil.

Killer cell immunoglobulin-like receptors (KIR) form a group of regulatory molecules that specifically recognise human leukocyte antigen (HLA) class I molecules, modulating the cytolytic activity of natural killer cells. The purpose of this study was to investigate the influence of KIR genes and their class I HLA ligands in susceptibility to dengue fever in a population from southern Brazil through a case-control study. One hundred four subjects with confirmed diagnoses of dengue participated in this study, along with a control group of 172 individuals from the same geographic area. HLA and KIR genotyping was performed by polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP) and with sequence-specific primer (PCR-SSP) techniques, respectively. Data analysis showed significant differences for the KIR2DS1 (54.8% vs 40.7%, P = 0.03), KIR2DS5 (50.0% vs 36.0%, P = 0.03) and KIR2DL5 (76.0% vs 56.4%, P = 0.001) genes. With regard to KIR-ligand pairs, positive associations with dengue were observed in KIR3DS1-Bw4 (45.2% vs 29.7%, P = 0.01), KIR3DL1-Bw4 (80.7% vs 65.1%, P < 0.001), KIR2DL1-C2 (75.0% vs 62.2%, P = 0.03) and KIR2DS1-C2 (40.4% vs 25.6%, P = 0.01) interactions, and a negative association in KIR2DL3-C1/C1 (18.2% vs 33.1%, P = 0.01). Furthermore, the analysis of KIR haplogroups showed a possible protective factor against dengue fever in individuals with the AA genotype. Taken together, these results suggest the existence of genetic predisposition to dengue fever in the population from southern Brazil.

Protective effect of HLA-DRB1 11 and predisposition of HLA-C 04 in the development of severe liver damage in Brazilian patients with chronic hepatitis C virus infection.

The objective of this study was to investigate human leucocyte antigen (HLA) genes in patients chronically infected with hepatitis C virus (HCV) and to analyse the possible role of these genes in the progression of chronic hepatitis C. One hundred and forty-five (145) Brazilian patients infected only with HCV genotype 1 were evaluated. HLA class I (A, B, C) and class II (DRB1, DQA1, DQB1) typing were carried out by PCR-SSO, through Luminex technology. Associations were found with protection against development of liver damage by both DRB1 11 (5.0% versus 18.2%, P=0.0016, OR=0.23, CI 95% = 0.09-0.58; Pc=0.0208) and DRB1 11-DQA1 05-DQB1 03 haplotype (4.2% versus 15.3%, P=0.0032; OR = 0.24, CI 95% = 0.08-0.64). Liver damage was associated with HLA-C 04 in patients with <20 years of infection (38.4% versus 9.1%, P = 0.002, OR = 6.25, CI 95%=1.97-19.7; Pc=0.0238). It is concluded that HLA alleles can influence the development of liver damage in HCV type-1 chronically infected Brazilian patients.

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

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.

Influence of HLA alleles in response to treatment with pegylated interferon-alpha and ribavirin in patients with chronic hepatitis C.

The objective of this study was to analyse the possible role of HLA polymorphism of chronically infected hepatitis C virus patients in the response outcome to treatment with pegylated interferon-alpha plus ribavirin. To that end, 144 Brazilian patients infected only with genotype 1 of the virus were treated with pegylated interferon-alpha at 1.5 µg kg(-1) in conjunction with ribavirin (1000 mg if patient weight was <75 kg and 1250 mg if >75 kg) for 48 weeks. The patients did not have concomitant HBV or HIV infections or liver disease, did not undergo previous antiviral treatment, and were followed up for 24 weeks after the end of treatment to assure they presented a sustained virological response. Patients were classified according to response to treatment in responsive (SVR), nonresponsive (NRS) and relapsers (REL). HLA class I and class II typing were carried out through PCR-SSO using Luminex technology. A statistically higher frequency of DRB1*11 patients was observed in the SVR group (39.6% vs. 14.3%P = 0.0012; Pc = 0.0156; OR = 3.94; 95% CI = 1.8-8.8). HLA-DQB1*03 patients were also more frequent in the SVR group, but the P value lost significance after Bonferroni correction (62.3% vs. 41.7%P = 0.024; Pc = 0.14, OR = 2.3; 95% CI = 1.14-4.60). HLA class II antigens can positively influence the response to treatment with pegylated interferon-alpha and ribavirin.

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.

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.

JAK2 46/1 haplotype is associated with JAK2 V617F--positive myeloproliferative neoplasms in Brazilian patients.

INTRODUCTION: This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. METHODS: The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. RESULTS: The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially those that were JAK2 V617F positive, than in the control population. We also compared the 46/1 haplotype status in each MPN disease entity, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPNu with controls. The G allele frequency relative to controls was significantly enriched in patients with PV and ET, but not in those with PMF and MPNu. PV and ET patients especially, all of whom had the JAK2 V617F mutation, showed significant excess of the G allele. The frequency of JAK2 V617F mutation was associated with elevated hematological parameters, but when we analyze the occurrence of the mutation and the presence of the G allele, just the high hemoglobin was significantly. CONCLUSION: In agreement with previous reports, JAK2 46/1 haplotype for JAK2 V617F was associated with cMPN positive in Brazilian patients.