RESUMO
Two new HLA-C alleles were identified in a volunteer bone marrow donor by sequence-based typing.
Assuntos
Transplante de Medula Óssea , Antígenos HLA-C/genética , Doadores de Tecidos , Alelos , Teste de Histocompatibilidade/métodos , Experimentação Humana , Humanos , Análise de Sequência de DNARESUMO
We describe the decline in islet function, in relation to HLA sensitization, in an islet transplant recipient and the recovery of this function after treatment with anti-CD20 monoclonal antibody and IV immunoglobulins. A 51-year-old woman with type 1 diabetes received one intraportal islet infusion. Following this transplantation, she became insulin independent. A search for HLA antibodies by using an ELISA technique remained consistently negative for HLA class I and II. It was only 2 years after the islet transplantation that this search became positive against class II antigens, reaching a peak of reactivity concomitantly with the appearance of a deterioration of glucose control requiring low-dose insulin therapy. Luminex screening and single-antigen assays then revealed the presence of both nondonor-specific and donor-specific antibodies against HLA class II molecules. This immunization, already present in the pretransplant serum, had increased during the 6 months preceding the clinical deterioration. Since these data nevertheless pointed to antibody-mediated rejection of the islet allograft, treatment with anti-CD20 monoclonal antibody and IV immunoglobulins was initiated. One month later, the search by ELISA for antibodies against HLA class II antigens became negative, the Luminex tests normalizing more gradually. As the result of an improvement in glucose control, the patient was again insulin-free.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Imunidade Humoral/imunologia , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Transplante das Ilhotas Pancreáticas/imunologia , Anticorpos Monoclonais Murinos , Feminino , Humanos , Pessoa de Meia-Idade , Rituximab , Resultado do TratamentoRESUMO
Two cases of splenic peliosis are reported. Clinical and radiographic signs and laboratory results do not contribute greatly to diagnosis which is based on histology findings. Splenic rupture is the major complication and requires immediate splenectomy.
Assuntos
Peliose Hepática/complicações , Esplenopatias/complicações , Ruptura Esplênica/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Evolução Fatal , Feminino , Humanos , Esplenectomia , Esplenopatias/patologia , Esplenopatias/cirurgia , Ruptura Esplênica/cirurgiaRESUMO
A 56 year-old, multiparous woman suffering from a myeloproliferative syndrome, who had received multiple red blood cell and platelet transfusions, was the recipient of an allograft of peripheral blood stem cells derived from her HLA-A, B, DR, DQ and DP and ABO identical sister, following myeloablative conditioning. The persistence of severe, isolated thrombopenia resistant to platelet transfusions led to the discovery of anti-HLA class I allo-immunisation. As HLA compatible platelet transfusions did not result in satisfactory platelet increments, we then discovered the simultaneous presence of anti-HPA-1a allo-immunisation. Genotyping of the HPA-1 systems of the patient (HPA-1B/B) and her sister (HPA-1A/B) enabled us to elucidate the mechanism underlying the persistent thrombopenia and the inefficacy of transfusion. In fact, only transfusion of HPA-1B/B platelets (HLA compatible or incompatible) proved to be efficacious. To reduce the level of anti-HPA-1a antibodies, we performed plasmapheresis sessions and used an anti-CD20 monoclonal antibody. It was only on achieving total haematopoietic chimerism, through rapid interruption of the immunosuppression, that we obtained spontaneous normalisation of the platelet count. The present case emphasises the necessity, before undertaking any allograft of haematopoietic stem cells - even if the latter come from a strictly HLA identical member of the family - of performing a search for eventual anti-HPA allo-immunisation.
Assuntos
Antígenos de Plaquetas Humanas/imunologia , Transplante de Medula Óssea/efeitos adversos , Trombocitopenia/imunologia , Feminino , Humanos , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
In the present article, we report the identification of the first HLA-B*07 null allele found in a Polish patient awaiting a kidney allograft. A discrepant result obtained between serological typing (HLA-B "blank") and high-resolution molecular typing using PCR-SSP method (HLA-B*070201 allele) suggested the presence of a null allele. Genomic DNA sequencing of the HLA-B*07 allele revealed a single nucleotide substitution at the 3' end of the exon 4 leading to a premature stop codon.
Assuntos
Alelos , Antígenos HLA-B/genética , Adulto , Sequência de Bases , Antígeno HLA-B7 , Humanos , Masculino , Dados de Sequência Molecular , Mutação Puntual , Polimorfismo de Nucleotídeo ÚnicoRESUMO
In the present report we describe the laborious identification of the A*02010102L allele found in three healthy individuals of a French family who have shown a reduced A2 antigen expression using serological tests since the 1980s. PCR-SSP typing showed a classical A*0201 allele. Sequencing of exons 2, 3 and 4 confirmed this assignment. Sequencing of the whole gene (promoter, introns and exons 1-8) revealed one single point mutation (T to C) at position -101 in the enhancer B element region compared to the A*02010101 allele. This single mutation appears to be related to the reduced expression of the A2 antigen. This allele segregates with the haplotype Cw*12, B44, DR7, DQ2, which is different to the one described earlier.
Assuntos
Antígenos HLA-A/classificação , Antígenos HLA-A/genética , Alelos , Sequência de Bases , Antígenos HLA-A/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A second HLA-A*68 null allele, HLA-A*6818 N, was identified in our laboratory after discrepant results were obtained between class I serological and molecular typing in a male patient suffering from narcolepsy. HLA-A*6818 N displays a sequence identical to that of the HLA-A*6802 allele, except in exon 2 where 20 nucleotides inserted at codon position 48 are a repeat of the 20 preceding nucleotides. This duplication creates a shift of the reading frame, which leads to a premature non-sense codon at position 59 of the null allele.
Assuntos
Alelos , Antígenos HLA-A/genética , Sequência de Bases , Códon/genética , Éxons/genética , Saúde da Família , Feminino , Mutação da Fase de Leitura/genética , Duplicação Gênica , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Haplótipos/genética , Teste de Histocompatibilidade , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA-A24 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA-A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA-A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.
Assuntos
Alelos , Genes MHC Classe I , Antígenos HLA-A/genética , Íntrons/genética , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Expressão Gênica , Antígenos HLA-A/biossíntese , Antígeno HLA-A24 , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Splicing de RNA , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Testes SorológicosRESUMO
Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.
Assuntos
Primers do DNA , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Reação em Cadeia da PolimeraseRESUMO
The authors describe an A*68 allele present at the molecular level but not expressed at the cell surface. This non expression results from the deletion of one nucleotide in exon 1, which causes a shift of the reading frame leading to an early non-sense codon in the same exon.
Assuntos
Alelos , Células da Medula Óssea/química , Células da Medula Óssea/imunologia , Antígenos HLA-A/genética , Feminino , Humanos , Dados de Sequência Molecular , Pseudogenes/imunologiaRESUMO
HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified whereas the corresponding antigen was not detected on the cell surface. In the present report, we describe four members of a family in whom an HLA-A1 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was undetectable by isoelectric focusing (IEF). Sequencing of the HLA-A*01 allele from the promoter region to the eighth exonic region revealed insertion of a "C" nucleotide at the beginning of the fourth exon as compared to the common HLA-A*0101 allele. This mutation causes a frame shift, giving rise to an early stop codon in the fourth exon.