Investigação de polimorfismos nos genes C4A e C4B (Sistema complemento) da região de classe III do MHC nos transplantes de células-tronco hematopoiéticas com doador não aparentado

Orientador : Prof. Dr. Ricardo PasquiniCoorientador : Profª. Drª. Noemni Farah PereiraDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Medicina Interna. Defesa : Curitiba, 16/12/2016Inclui referências : f. 68-75Resumo: Introdução: A compatibilidade HLA entre receptor e doador é insuficiente para evitar complicações como a doença do enxerto-contra-hospedeiro (DECH) após o transplante de células-tronco hematopoiéticas (TCTH), e isto sugere que a variabilidade não HLA pode influenciar desfechos deste procedimento terapêutico. A região de classe III do MHC abriga genes relacionados à resposta imune e aos processos inflamatórios cujas variantes alélicas têm sido associadas a doenças autoimunes e a algumas complicações pós-TCTH como a DECH. Objetivo: Este estudo retrospectivo investigou se incompatibilidades em 23 polimorfismos de único nucleotídeo (SNPs) nos genes C4A e C4B do MHC classe III estariam associados à maior incidência da DECH aguda (DECHa), crônica (DECHc) e mortalidade pós-TCTH não-aparentado. Casuística: 238 receptores(R), com idade entre 4 meses e 74 anos, 59% do sexo masculino, 52% com doenças malignas, transplantados entre 1996 e 2015, com células-tronco de medula óssea (83%) ou de sangue periférico (17%) obtidas de doadores(D) HLA-A, -B, -C, -DRB1, -DQB1 compatíveis, 61% com condicionamento mieloablativo, 90% receberam imunoprofilaxia com ciclosporina e metotrexato. Métodos: A tipagem dos 23 SNPs dos genes C4A e C4B foi realizada por amplificação alelo-específica (PCR-SSP) em 129 pares R-D e por sequenciamento de segunda geração em 109 pares R-D. Também avaliou-se o polimorfismo de HLA-DPB1 por sequenciamento de Sanger, uma vez que poderia haver associação de incompatibilidades não permissíveis deste gene com a DECH e ou mortalidade. Resultados: Dentre os pares R-D, 42,9% apresentaram 1-7 SNPs incompatíveis, cuja presença não foi associada com DECHa (RRS 0,99; p=0,948), DECHc (RRS 1,36; p=0,250) e mortalidade (RR 0,92; p=0,714). Somente 12 SNPs foram incompatíveis em 10 ou mais pares e puderam ter seu efeito isolado investigado, mas nenhum deles mostrou-se associado com DECH ou mortalidade. Na amostra total, 85,3% dos pares R-D apresentaram incompatibilidades DPB1, 41,6% permissíveis e 43,7% não permissíveis, mas sem associação com DECHa, DECHc ou mortalidade. Mesmo assim, estratificou-se os pares R-D conforme a compatibilidade DPB1: o grupo I incluiu pares R-D compatíveis e aqueles com incompatibilidades permissíveis, e o grupo II foi constituído somente de pares com incompatibilidades não permissíveis. Não houve associações entre incompatibilidades C4 e DECHa (RRS 1,03; p=0,922), DECHc (RRS 1,59; p=0,168) ou mortalidade (RR 1,21; p=0,544) no grupo I, e tampouco com respeito à DECHa (RRS 0,94; p=0,860), DECHc (RRS 1,08; p=0,855) ou mortalidade (RR 0,68; p=0,260) no grupo II . Conclusão: Apesar da identificação de incompatibilidades nos SNPs C4A e C4B entre R-D, esta característica não se mostrou fator de risco das formas aguda ou crônica da DECH e nem do aumento da mortalidade nos pacientes desta casuística. A ausência de associação pode ser devido ao pequeno tamanho amostral. Por outro lado, a diversidade nos genes C4A e C4B pode não influir na resposta imunológica da DECH. Palavras-chave: Polimorfismos de único nucleotídeo (SNPs). Genes C4 e C4B. MHC Classe III. Doador não aparentado. Transplante de Células-Tronco Hematopoiéticas.Abstract: Introduction: Patient and donor HLA matching is not enough to avoid post-hematopoietic stem cell transplant (HSCT) complications such as graft-versus-host disease (GVHD), and this suggests that non-HLA diversity can also impacts transplant outcomes. MHC class III region hosts genes related to immune/inflammatory responses whose allelic variants have been associated to autoimmune diseases and some post-HSCT complications such as GVHD. Objective: This retrospective study investigated whether incompatibilities in one or more of 23 single-nucleotide-polymorphisms (SNPs) in the C4A and C4B genes (MHC class III) would be associated with higher incidence of GVHD and mortality post-unrelated HSCT. Casuistic: 238 recipients(R), age ranging from 4m to 74y, 59% male, 52% with malignant diseases, transplanted between 1996 and 2015 with hematopoietic stem cells from bone marrow (83%) or peripheral blood (17%) collected from HLA-A, -B, -C, -DRB1, -DQB1 matched donors (D), 61% underwent myeloablative conditioning and 90% received cyclosporine plus methotrexate as immunoprophylaxis. Methods: Typing of the 23 SNPs was done by allele-specific amplification (PCR-SSP) in 129 R-D pairs, and by next-generation sequencing in the remaining 109 R-D pairs. HLA-DPB1 polymorphism was also investigated by Sanger sequencing, once there could be associations between non-permissive mismatches on this gene and GVHD or mortality. Results: 42.9% R-D pairs had 1 to 7 C4 SNPs mismatched, but the presence of these mismatches was not associated with aGVHD (SHR 0.99; p = 0.948), cGVHD (SHR 1.36; P = 0.250) or mortality (HR 0.92; p = 0.714). Only 12 SNPs were mismatched in 10 or more R-D pairs and could have their isolated impact investigated, but none of them showed an association with GVHD or mortality. In the total sample, 85.3% R-D pairs showed DPB1 mismatches, 41.6% were permissibile and 43.7% non-permissible, but no association with aGVDH, cGVHD and mortality was found. Despite of the lack of association, R-D pairs were stratified according to DPB1 compatibility status: group I included matched pairs as well as those with permissive mismatches; and group II hosted only non-permissible mismatches. No association between C4A and C4B SNPs mismatches and aGVHD (SHR 1.03; p=0.922), cGVHD (SHR 1.59; p=0.168) or mortality (HR 1.21; p=0.544) was found in group I, and neither with aGVHD (SHR 0.94; p=0.860), cGVHD (SHR 1.08; p=0.855) or mortality (HR 0.68; p=0.260) in group II. Conclusion: Despite of the identification of C4 mismatches between R/D, this characteristic was not shown to be a risk factor for acute or chronic GVHD nor for the increase in mortality in this series of patients. The absence of associations may be due to the small sample size. On the other hand, the C4A and C4B genes diversity may not influence the immune response of the GVHD. Key words: Single nucleotide polymorphisms (SNPs). C4A and C4B genes. MHC Class III. Unrelated donor. Hematopoietic stem cell transplantation

Tissue distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing mouse

Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E-13.5, E-16.5 fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes