Investigation of MHC gamma block C4A and C4B polymorphisms in unrelated hematopoietic stem cell transplantation

ABSTRACT Background: Immunological life-threatening complications frequently occur in post-hematopoietic stem cell transplantation (HSCT), despite matching recipient and donor (R/D) pairs for classical human leukocyte antigens (HLA). Studies have shown that R/D non-HLA disparities within the major histocompatibility complex (MHC) are associated with adverse effects post-HSCT. Methods: We investigated the impact of mismatches of single-nucleotide polymorphisms (SNPs) in C4A/C4B genes, for showing the highest diversity in the MHC gamma block, on 238 patients who underwent HLA 10/10 unrelated donor (URD) HSCT. The endpoints were acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and mortality. One hundred and twenty-nine R/D pairs had 23 C4-SNPs typed by PCR-SSP (Gamma-Type™v.1.0), and 109 R/D pairs had these 23 SNPs identified by next-generation sequencing (NGS) using the Illumina platform. Results: The percentage of patients who received HSC from HLA 10/10 donors with 1-7 mismatches was 42.9%. The R/D pairs were considered C4 mismatched when bearing at least one disparity. These mismatches were not found to be risk factors for aGVHD, cGVHD or mortality after unrelated HSCT when SNPs were analyzed together (matched or mm ≥ 1), independently or according to the percentage of incompatibilities (full match for 23 SNPs; 1-3 mm and >3 mm). An exception was the association between 1-3 mismatches at the composite of SNPs C13193/T14952/T19588 with the development of aGVHD (P = 0.012) and with grades III-IV of this disease (P = 0.004). Conclusion: Our data are not consistent with the hypothesis that disparities in C4A/C4B SNPs increase the risks of post-HSCT adverse effects for the endpoints investigated in this study.

HLA-Matched Unrelated Donors for Patients with Sickle Cell Disease: Results of International Donor Searches.

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy. Hematopoietic stem cell transplantation (HCT) is the sole curative therapy for SCD, but few patients will have a matched sibling donor. Patients with SCD are mostly of African origin and thus are less likely to find a matched unrelated donor in international registries. Using HaploStats, we estimated HLA haplotypes for 185 patients with SCD (116 from a Brazilian center and 69 from European Society for Blood and Marrow Transplantation [EBMT] centers) and classified the ethnic origin of haplotypes. Then we assessed the probability of finding an HLA-matched unrelated adult donor (MUD), considering loci A, B, and DRB1 (6/6), in international registries. Most haplotypes were African, but Brazilians showed a greater ethnic admixture than EBMT patients. Nevertheless, the chance of finding at least one 6/6 potential allelic donor was 47% for both groups. Most potential allelic donors were from the US National Marrow Donor Program registry and from the Brazilian REDOME donor registry. Although the probability of finding a donor is higher than previously reported, strategies are needed to improve ethnic diversity in registries. Moreover, predicting the likelihood of having an MUD might influence SCD management.

Investigation of MHC gamma block C4A and C4B polymorphisms in unrelated hematopoietic stem cell transplantation.

BACKGROUND: Immunological life-threatening complications frequently occur in post-hematopoietic stem cell transplantation (HSCT), despite matching recipient and donor (R/D) pairs for classical human leukocyte antigens (HLA). Studies have shown that R/D non-HLA disparities within the major histocompatibility complex (MHC) are associated with adverse effects post-HSCT. METHODS: We investigated the impact of mismatches of single-nucleotide polymorphisms (SNPs) in C4A/C4B genes, for showing the highest diversity in the MHC gamma block, on 238 patients who underwent HLA 10/10 unrelated donor (URD) HSCT. The endpoints were acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and mortality. One hundred and twenty-nine R/D pairs had 23 C4-SNPs typed by PCR-SSP (Gamma-Type™v.1.0), and 109 R/D pairs had these 23 SNPs identified by next-generation sequencing (NGS) using the Illumina platform. RESULTS: The percentage of patients who received HSC from HLA 10/10 donors with 1-7 mismatches was 42.9%. The R/D pairs were considered C4mismatched when bearing at least one disparity. These mismatches were not found to be risk factors for aGVHD, cGVHD or mortality after unrelated HSCT when SNPs were analyzed together (matched or mm≥1), independently or according to the percentage of incompatibilities (full match for 23 SNPs; 1-3mm and >3mm). An exception was the association between 1-3 mismatches at the composite of SNPs C13193/T14952/T19588 with the development of aGVHD (P=0.012) and with grades III-IV of this disease (P=0.004). CONCLUSION: Our data are not consistent with the hypothesis that disparities in C4A/C4B SNPs increase the risks of post-HSCT adverse effects for the endpoints investigated in this study.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

HLA-typing analysis following allogeneic bone grafting for sinus lifting.

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6 months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.

Four-year clinical remission of type 1 diabetes mellitus in two patients treated with sitagliptin and vitamin D3.

Type 1 diabetes mellitus (T1DM) is a chronic disease characterized by autoimmune destruction of pancreatic beta cells and inadequate insulin production. Remission criteria in T1DM take into account serum levels of C-peptide and glycosylated hemoglobin, as well as the dose of insulin administered to the patient. However, remission of T1DM lasting longer than 1 year is rare. We describe here the cases of two young women who presented with positive glutamic acid decarboxylase (GAD) antibody and classic clinical manifestations of T1DM. Both patients had a prior history of Hashimoto's thyroiditis. They were initially treated with a basal-bolus regimen of insulin (glargine and lispro/glulisine). Once their blood glucose levels were controlled, they were started on oral sitagliptin 100 mg and vitamin D3 5000 IU daily. After this therapy, both patients achieved clinical diabetes remission for 4 years, along with a decrease in anti-GAD antibody levels. These benefits were probably associated with immunological effects of these medications. Inhibition of dipeptidyl peptidase 4 (DPP-4) in animal models deregulates Th1 immune response, increases secretion of Th2 cytokines, activates CD4+CD25+FoxP3+ regulatory T-cells and prevents IL-17 production. Vitamin D3 also activates CD4+CD25+FoxP3+ regulatory T-cells, and these medications combined can improve the immune response in patients with new-onset T1DM and probably promote sustained clinical remission. LEARNING POINTS: The use of sitagliptin and vitamin D3 in patients with new-onset type 1 diabetes mellitus (T1DM) may help decrease the daily insulin requirement by delaying beta cell loss and improving endogenous insulin production.The use of sitagliptin and vitamin D3 in new-onset T1DM could help regulate the imbalance between Th17 and Treg cells.Age 14 years or above, absence of ketoacidosis and positive C-peptide levels in patients with T1DM are good criteria to predict prolonged T1DM remission.The determination of anti-GAD antibodies and C-peptide levels could be helpful in the follow-up of patients in use of sitagliptin and vitamin D3, which could be associated with prolonged T1DM clinical remission.

Population variation of HLA genes in rural communities in Brazil, the Quilombos from the Vale do Ribeira, São Paulo - Brazil.

In the present study, we characterized the allelic and haplotypic profile of the genes HLA-A, -B, -C and -DRB1 (PCR-SBT) in a population sample of 144 highly admixed individuals, coming from rural communities in Brazil (Quilombos from Vale do Ribeira, in the State of São Paulo). Furthermore, we identified three individuals with a new null allele in the HLA-C gene (HLA-C(∗)02:105N), associated with the haplotype HLA-A(∗)80: 01∼B(∗)18: 01:01G∼DRB1(∗) 07:01.

Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil.

BACKGROUND: Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. METHODS: A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. RESULTS: Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group), partial compatible (one cross-reactive group) or less compatible (two cross-reactive group) donors, respectively. CONCLUSION: The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services.

Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil

ABSTRACT Background: Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. Methods: A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. Results: Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group), partial compatible (one cross-reactive group) or less compatible (two cross-reactive group) donors, respectively. Conclusion: The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services.

HLA imputation in an admixed population: An assessment of the 1000 Genomes data as a training set.

Methods to impute HLA alleles based on dense single nucleotide polymorphism (SNP) data provide a valuable resource to association studies and evolutionary investigation of the MHC region. The availability of appropriate training sets is critical to the accuracy of HLA imputation, and the inclusion of samples with various ancestries is an important pre-requisite in studies of admixed populations. We assess the accuracy of HLA imputation using 1000 Genomes Project data as a training set, applying it to a highly admixed Brazilian population, the Quilombos from the state of São Paulo. To assess accuracy, we compared imputed and experimentally determined genotypes for 146 samples at 4 HLA classical loci. We found imputation accuracies of 82.9%, 81.8%, 94.8% and 86.6% for HLA-A, -B, -C and -DRB1 respectively (two-field resolution). Accuracies were improved when we included a subset of Quilombo individuals in the training set. We conclude that the 1000 Genomes data is a valuable resource for construction of training sets due to the diversity of ancestries and the potential for a large overlap of SNPs with the target population. We also show that tailoring training sets to features of the target population substantially enhances imputation accuracy.

Nomenclatura dos fatores do sistema HLA / Nomenclature for factors of the HLA system

The Nomenclature Committee for Factors of the HLA System of the World Health Organization standardizes the nomenclature of the HLA system and meets regularly during the International Histocompatibility Workshops. During the 15th International Histocompatibility Workshop in Buzios (RJ), Brazil, in September 2008, there was a meeting of the nomenclature committee when new rules were established, whichwere implemented in April 2010.

O Comitê de Nomenclatura dos Fatores do Sistema HLA da Organização Mundial da Saúde normatiza a nomenclatura do sistema HLA e se reúne regularmente durante os Workshops Internacionais de Histocompatibilidade. Durante o 15o Workshop Internacional de Histocompatibilidade, em Búzios (RJ), em Setembro de 2008, houve a reunião do comitê de nomenclatura, quando novas regras foram estabelecidas, sendo implantadas em Abril de 2010.

Nomenclature for factors of the HLA system.

The Nomenclature Committee for Factors of the HLA System of the World Health Organization standardizes the nomenclature of the HLA system and meets regularly during the International Histocompatibility Workshops. During the 15th International Histocompatibility Workshop in Buzios (RJ), Brazil, in September 2008, there was a meeting of the nomenclature committee when new rules were established, which were implemented in April 2010.

Transplante de sangue de cordão umbilical - SCU / Umbilical cord blood transplantation

A frequente utilização de sangue de cordão umbilical - SCU como fonte de células- tronco hematopoéticas - CTH, tanto em crianças, como em adultos, que não dispõem de doador na família, tem levado ao estabelecimento da padronização de critérios em sua seleção, objetivando a obtenção de melhores resultados. A escolha da unidade de SCU deve basear-se no número total de células nucleadas e no número de diferenças de antígenos leucocitários humanos (HLA). Diante de uma unidade com celularidade mínima, deve-se considerar a possibilidade da utilização de duplo cordão. Frente a mais de uma unidade com características semelhantes, a realização da contagem de células CD34 e da compatibilidade ABO, assim como a qualidade e a rapidez para obtenção da unidade, podem definir a escolha.

The frequent use of umbilical cord blood as the source of hematopoietic stem cells, both in children and adults who do not have related donors, has led to the establishment of a better standardization of selection criteria aiming at improving the results. The choice of the umbilical cord blood unit should be based on the total number of nucleated cells and the number of differences in the human leukocyte antigen (HLA) system. When a unit has minimal cellularity, the use of a double cord blood transplant should be considered. When two or more units have similar characteristics, the choice may be determined by the CD34 count, ABO compatibility and the quality and speed to obtain the unit.

Seleção de doador de medula óssea ou sangue periférico / Bone marrow or peripheral blood donor selection

A compatibilidade HLA é o fator mais valorizado na escolha do doador de medula óssea voluntário, preconizando-se a realização de HLA de alta resolução nos locos HLA-A,B,C, DRB1 e DQB1. Tem sido dado preferência para o doador com consanguinidade alélica 8x8 (A,B,C, DRB1). Na presença de incompatibilidade na classe-I sugere-se a busca de doador com compatibilidade DQB1 (9x10). Já as incompatibilidades dos locos DPB1 não constituem critério de exclusão de doador, exceto quando existir presença de anticorpo contra o loco HLA-DP do doador.

The HLA system is considered the most important factor in choosing a volunteer bone marrow donor with the recommendation of performing high resolution HLA tests for the HLA-A, B, C, DRB1 and DQB1 loci. A preference has been given for donor 8x8 (A, B, C, DRB1) allele matching. In the presence of class-I incompatibility a search for DQB1 (9x10) donor compatibility is suggested. The incompatibility of the DPB1 locus does not constitute exclusion of the donor, except when there is the presence of antibodies against the HLA-DP locus of the donor.

Seleção de doador não aparentado / HLA antibodies and donor selection

Apesar da presença de anticorpos anti-HLA em transplantes de órgãos sólidos estar associada à rejeição, essa correlação não havia sido pesquisada em transplante alogênico de células progenitoras hematopoéticas (TCPH). Estudos mais recentes na literatura têm demonstrado que a falência da enxertia no TCPH pode ser mediada por aloanticorpos anti-HLA doador especifico (DSA). A especificidade desses anticorpos pode ser evidenciada pelas técnicas de fase sólida, onde os antígenos HLA únicos são aderidos a pérolas de poliestireno, que permite a realização da prova cruzada virtual. Na presença de DSA, é recomendável selecionar outro doador ou realizar as estratégias de remoção dos anticorpos.

In spite of Anti-HLA antibodies being associated to rejection in solid organs transplantation, this correlation has not been well established yet in allogenic bone marrow transplantation.Recent studies in the literature have demonstrated that engraftment failure in hematopoietic cell transplantation (HCT) can be mediated by donor specific anti-HLA antibodies (DSA). These antibodies specificity can be detected by solid-phase techniques, where single HLA antigens are adhered to microbeads, which allows the interpretation of host reactivity by "virtual crossmatch". In the presence of DSA, it is advisable to either search for another donor or remove the antibodies prior to transplantation.

Antibody-mediated rejection (AMR) after pancreas and pancreas-kidney transplantation.

Antibody-mediated rejection (AMR) requires specific diagnostic tools and treatment and is associated with lower graft survival. We prospectively screened C4d in pancreas (n = 35, in 27 patients) and kidney (n = 33, in 21 patients) for cause biopsies. Serum amylase and lipase, amylasuria, fasting blood glucose (FBG) and 2-h capillary glucose (CG) were also analysed. We found that 27.3% of kidney biopsies and 43% of pancreatic biopsies showed C4d staining (66.7% and 53.3% diffuse in peritubular and interacinar capillaries respectively). Isolated exocrine dysfunction was the main indication for pancreas biopsy (54.3%) and was followed by both exocrine and endocrine dysfunctions (37.1%) and isolated endocrine dysfunction (8.6%). Laboratorial parameters were comparable between T-cell mediated rejection and AMR: amylase 151.5 vs. 149 U/l (P = 0.075), lipase 1120 vs. 1288.5 U/l (P = 0.83), amylasuria variation 46.5 vs. 61% (P = 0.97), FBG 69 vs. 97 mg/dl (P = 0.20) and 2-h CG maximum 149.5 vs. 197.5 mg/dl (P = 0.49) respectively. Amylasuria values after treatment correlated with pancreas allograft loss (P = 0.015). These data suggest that C4d staining should be routinely investigated when pancreas allograft dysfunction is present because of its high detection rate in cases of rejection.