Genotipagem do Human Leucocyte Antigen (HLA): Técnicas atuais e aplicabilidade da abordagem de Next Generation Sequencing
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2019-10-29
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O complexo MHC (Major Histocompatibility Complex) está presente em todos os mamíferos e, no homem é designado HLA (Human Leucocyte Antigen). Este cluster de genes está localizado no braço curto do cromossoma 6 (6p21) e possui mais de 220 genes, sendo uma das regiões mais intensivamente estudadas no genoma humano há mais de 20 anos, sendo que ainda nos dias de hoje é alvo de vários estudos. O complexo génico HLA é um dos mais polimórficos do genoma humano e divide-se em três regiões: HLA classe I, II e III. A principal função das moléculas de MHC é a apresentação de fragmentos peptídicos provenientes de agentes patogénicos e exibi-los à superfície das células de forma a serem reconhecidos e eliminados adequadamente pelos linfócitos T. Desde a descoberta do HLA, vários estudos contribuíram para a melhor compreensão da resposta imunitária. As primeiras associações entre a região HLA e determinadas doenças reforçaram a ideia já existente, da complexidade deste cluster de genes e o seu extenso polimorfismo. Com o passar do tempo, foi possível verificar que determinados genótipos HLA estão associados com um grande número de doenças, bem como a reações adversas a certos fármacos e com a compatibilidade HLA na transplantação. O estudo desta região permitiu perceber muitas das suas características únicas, despertando um grande interesse tanto a nível clínico como científico, de tal forma que várias técnicas de genotipagem HLA foram desenvolvidas. Inicialmente a genotipagem era feita através de métodos serológicos, no entanto não eram completamente satisfatórios. Foi necessário desenvolver métodos de tipagem mais robustos e específicos: PCR-SSP (Sequence Specific Primer), PCR-SSO (Sequence Specific Oligonucleotide) e PCR-SBT (Sequencing based typing). No entanto, estes também apresentam resultados com ambiguidades, dependendo do tipo de metodologia e kit usado. Mais recentemente o desenvolvimento da tecnologia NGS (Next Generation Sequencing) permitiu um maior nível de resolução entre outras vantagens discutidas ao longo deste trabalho. O presente trabalho teve como objetivos a aplicação das técnicas clássicas de genotipagem de HLA e a revisão sobre a aplicabilidade de NGS nesta área. Neste estudo, após utilização das técnicas, foram calculadas as frequências alélicas para as amostras de dadores de medula e de sangue do cordão umbilical. Para os genes HLAA, -B, -C e –DRB1, os alelos mais frequentes foram os mesmos em ambos os tipos de amostras, tendo sido o HLA-A*02 com frequências alélicas de 26,83% e 27,55%; HLA-B*44 com 14,34% e 13,96%; HLA-C*07 com 24,66% e 25,66%; e o gene HLA-DRB1*07 com 18,09% e 16,60%, para as amostras de dadores de medula e sangue do cordão, respetivamente. Estas frequências alélicas descritas relativamente às amostras de dadores de medula óssea voluntários e amostras de cordão a nível do Norte de Portugal mostram uma semelhança com as frequências alélicas obtidos por outros autores relativamente à população Portuguesa. Com base na literatura, a introdução da tecnologia NGS nos laboratórios clínicos trará inúmeros benefícios, principalmente ao nível da compatibilidade dador-recetor. Esta tecnologia acarreta menos ambiguidades, menos custos associados à medida que a tecnologia evolui e para além disso, permitirá aumentar o nosso conhecimento relativamente à relevância biológica não só das regiões codificantes, mas principalmente das regiões não codificantes, já que permite a sequenciação mais alargada dos genes. Os estudos apresentados revelam dados bastantes satisfatórios quanto à aplicabilidade a nível laboratorial do NGS, com taxas de precisão acima dos 97% na genotipagem HLA.
The Major Histocompatibility Complex (MHC) is present in all mammals and is called HLA (Human Leucocyte Antigen) in humans. This gene cluster is located in the short arm of chromosome 6 (6p21) and has more than 220 genes, being one of the most intensively studied regions in the human genome for over 20 years, and is still the subject of many studies today. The HLA gene complex is one of the most polymorphic in the human genome and is divided into three regions: HLA class I, II and III. The primary function of MHC molecules is to present peptide fragments from pathogens and display them on the cell surface in order to be recognized and properly deleted by T lymphocytes. Since the discovery of HLA, several studies have contributed to a better understanding of the immune response. The first associations between the HLA region and certain diseases reinforced the existing idea of the complexity of this gene cluster and its extensive polymorphism. Over time, it has been found that certain HLA genotypes are associated with a large number of diseases, as well as adverse reactions to certain drugs and HLA compatibility in transplantation. The study of this region has made it possible to see many of its unique characteristics, arousing great interest both clinically and scientifically, so that various HLA genotyping techniques have been developed. Initially the genotyping was done by serological methods, however they were not completely satisfactory. More robust and specific typing methods were needed: PCR-SSP (Sequence Specific Primer), PCR-SSO (Sequence Specific Oligonucleotide) and PCR-SBT (Sequencing Based Typing). However, these also yield ambiguous results, depending on the type of methodology and kit used. More recently, the development of Next Generation Sequencing (NGS) technology has enabled a higher level of resolution among other advantages discussed throughout this work. The present work aimed to apply the classic HLA genotyping techniques and to review the applicability of NGS in this area. In this study, after using the techniques, allele frequencies were calculated for bone marrow and cord blood donor samples. For the HLA-A, -B, -C and –DRB1 genes, the most frequent alleles were the same in both types of samples, being HLA-A*02 with allele frequencies of 26.83% and 27.55%; HLA-B*44 with 14.34% and 13.96%; HLA-C*07 with 24.66% and 25.66%; and the HLA-DRB1*07 gene with 18.09% and 16.60% for cord and cord blood donor samples, respectively. These allelic frequencies described for samples from volunteer bone marrow donors and cord samples in Northern Portugal show a similarity to the allelic frequencies obtained by other authors from the Portuguese population. Based on the literature, the introduction of NGS technology in clinical laboratories will bring numerous benefits, particularly in terms of donor-recipient compatibility. This technology leads to less ambiguity, less cost as technology evolves, and furthermore, it will increase our knowledge regarding the biological relevance of polymorphisms not only of coding sequences, but especially non-coding regions, as it allows for broader gene sequencing. The studies presented reveal quite satisfactory data regarding the NGS laboratory applicability, with accuracy rates above 97% in HLA genotyping.
The Major Histocompatibility Complex (MHC) is present in all mammals and is called HLA (Human Leucocyte Antigen) in humans. This gene cluster is located in the short arm of chromosome 6 (6p21) and has more than 220 genes, being one of the most intensively studied regions in the human genome for over 20 years, and is still the subject of many studies today. The HLA gene complex is one of the most polymorphic in the human genome and is divided into three regions: HLA class I, II and III. The primary function of MHC molecules is to present peptide fragments from pathogens and display them on the cell surface in order to be recognized and properly deleted by T lymphocytes. Since the discovery of HLA, several studies have contributed to a better understanding of the immune response. The first associations between the HLA region and certain diseases reinforced the existing idea of the complexity of this gene cluster and its extensive polymorphism. Over time, it has been found that certain HLA genotypes are associated with a large number of diseases, as well as adverse reactions to certain drugs and HLA compatibility in transplantation. The study of this region has made it possible to see many of its unique characteristics, arousing great interest both clinically and scientifically, so that various HLA genotyping techniques have been developed. Initially the genotyping was done by serological methods, however they were not completely satisfactory. More robust and specific typing methods were needed: PCR-SSP (Sequence Specific Primer), PCR-SSO (Sequence Specific Oligonucleotide) and PCR-SBT (Sequencing Based Typing). However, these also yield ambiguous results, depending on the type of methodology and kit used. More recently, the development of Next Generation Sequencing (NGS) technology has enabled a higher level of resolution among other advantages discussed throughout this work. The present work aimed to apply the classic HLA genotyping techniques and to review the applicability of NGS in this area. In this study, after using the techniques, allele frequencies were calculated for bone marrow and cord blood donor samples. For the HLA-A, -B, -C and –DRB1 genes, the most frequent alleles were the same in both types of samples, being HLA-A*02 with allele frequencies of 26.83% and 27.55%; HLA-B*44 with 14.34% and 13.96%; HLA-C*07 with 24.66% and 25.66%; and the HLA-DRB1*07 gene with 18.09% and 16.60% for cord and cord blood donor samples, respectively. These allelic frequencies described for samples from volunteer bone marrow donors and cord samples in Northern Portugal show a similarity to the allelic frequencies obtained by other authors from the Portuguese population. Based on the literature, the introduction of NGS technology in clinical laboratories will bring numerous benefits, particularly in terms of donor-recipient compatibility. This technology leads to less ambiguity, less cost as technology evolves, and furthermore, it will increase our knowledge regarding the biological relevance of polymorphisms not only of coding sequences, but especially non-coding regions, as it allows for broader gene sequencing. The studies presented reveal quite satisfactory data regarding the NGS laboratory applicability, with accuracy rates above 97% in HLA genotyping.
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Dissertação de Mestrado em:
Biotecnologia para as Ciências da Saúde
Palavras-chave
HLA , genotipagem