Região promotora e codificadora no gene HLA-E: estrutura, variabilidade e haplótipos
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Data
2017-02-20
Autores
Ramalho, Jaqueline [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os genes do Complexo Principal de Histocompatibilidade (MHC) codificam moléculas envolvidas com a regulação do sistema imune, participando do reconhecimento do que é próprio ou estranho ao indivíduo. O gene HLA-E, parte do MHC, é caracterizado por apresentar baixa, porém, ampla expressão pelos tecidos do corpo. O HLA-E é extremamente conservado e um dos menos polimórficos entre os genes HLA de classe I. A principal função da molécula HLA-E está relacionada ao mecanismo de imunovigilância, através da interação com receptores de células Natural Killer e linfócitos T. Pontos de variação nas regiões regulatórias e codificadora de HLA-E podem alterar sua função através da modificação da expressão gênica ou estrutura da molécula codificada, influenciando a interação com peptídeos e receptores. O presente trabalho propôs a avaliação da variabilidade do segmento estendido do gene HLA-E, incluindo promotor e íntrons, e estrutura de haplótipos por meio de sequenciamento de nova geração ou sequenciamento massivo paralelo. A metodologia foi aplicada para avaliação da variabilidade de 420 amostras do Estado de São Paulo. Considerando um segmento de mais de 7kb, o gene HLA-E mostrou-se conservado, apresentando poucas sequências diferentes e frequentes. Ao todo, 63 pontos de variação foram encontrados e caracterizados em 75 haplótipos estendidos. Foram encontrados 37 haplótipos de região promotora, porém apenas 10 apresentam frequência superior a 1%. Para a região codificadora, foram encontrados 27 haplótipos, sendo que 15 caracterizam novos alelos HLA-E. No entanto, duas das moléculas codificadas, representadas pelos alelos do tipos E*01:01 e E*01:03, correspondem a mais de 90% de todas as sequências. Essa característica reforçou a conservação da região codificadora, na qual a maioria dos pontos de variação ocorreram em segmentos regulatórios e íntrons, ou caracterizavam mutações sinônimas em éxons. Na região 3’ não traduzida, 12 haplótipos foram encontrados, porém cerca de 90% das sequências são representadas por apenas dois haplótipos, 3UTR-1 e 3UTR-2. A diversidade nucleotídica foi maior no segmento promotor do que na região codificadora e 3’ não traduzida. Também foi observado fraco desequilíbrio de ligação ao longo de HLA-E, provavelmente devido a presença de diversos elementos Alu que pode influencia a taxa de recombinação entre os segmentos e, por causa disso, um mesmo haplótipo regulatório pode ocorrer associado a diferentes sequências de codificadora. Análises futuras relacionados a ligação de fatores de transcrição e RNAs não codificadores são necessárias para averiguar se essas variações influenciariam o perfil de expressão do gene.
The Major Histocompatibility Complex (MHC) genes encode molecules involved mainly in recognition of self and non self antigens. The HLA-E gene is characterized by low but wide expression among tissues. Thus far, HLA-E is considered a conserved gene and one of the least polymorphic class I HLA genes. The main HLA-E function is related to immune surveillance by the interaction with natural killer cell receptors and T lymphocytes. Variable sites within the HLA-E regulatory and coding segments may influence gene function by modifying its expression pattern or encoded molecule, thus, influencing its interaction with receptors and the peptide. Here we propose an approach to evaluate de complete HLA-E variability, including promoter and introns segments, and the evaluation of the HLA-E haplotype structure by using massively parallel sequencing, or next-generation sequencing. The methodology was applied to survey the variability of a very admixed population such as Brazilians counting 420 samples of São Paulo State. Considering a segment of about 7kb, the HLA-E gene is indeed conserved with few different and frequent sequences. In general, 63 variable sites were detected, arranged 75 extended haplotypes. We found 37 promoter haplotypes, but only 10 presented a frequency greater that 1%. For the coding region, 27 haplotypes were detected, with 15 representing new HLA-E alleles. Nevertheless, the HLA-E conding alleles did encode mainly two different full-length molecules, known as alleles E*01:01 and E*01:03, which corresponds to about 90% of all. This feature reinforced the conservation at the coding region, where most of the variable sites detected are intronic mutations, synomymous mutations in exons, or mutations at regulatory elements. In the 3’ untranslated region, 12 haplotypes were found, but only two different sequences (3UTR-1 and 3UTR-2) presented a summed frequency of 90%. Nucleotide diversity was higher in the distal promoter segment than in the coding and 3’ untranslated segments. HLA-E present a weak linkage disequilibrium along the entire segment, mainly because of the presence of many Alu elements that may influence recombination within the gene. Because of that, the same regulatory haplotype may occur associated with different coding sequences. Further analysis involving the binding of transcription factors and non-coding RNAs are necessary to evaluate whether these variable sites at regulatory segments (or even in the coding sequence) may influence the gene expression profile.
The Major Histocompatibility Complex (MHC) genes encode molecules involved mainly in recognition of self and non self antigens. The HLA-E gene is characterized by low but wide expression among tissues. Thus far, HLA-E is considered a conserved gene and one of the least polymorphic class I HLA genes. The main HLA-E function is related to immune surveillance by the interaction with natural killer cell receptors and T lymphocytes. Variable sites within the HLA-E regulatory and coding segments may influence gene function by modifying its expression pattern or encoded molecule, thus, influencing its interaction with receptors and the peptide. Here we propose an approach to evaluate de complete HLA-E variability, including promoter and introns segments, and the evaluation of the HLA-E haplotype structure by using massively parallel sequencing, or next-generation sequencing. The methodology was applied to survey the variability of a very admixed population such as Brazilians counting 420 samples of São Paulo State. Considering a segment of about 7kb, the HLA-E gene is indeed conserved with few different and frequent sequences. In general, 63 variable sites were detected, arranged 75 extended haplotypes. We found 37 promoter haplotypes, but only 10 presented a frequency greater that 1%. For the coding region, 27 haplotypes were detected, with 15 representing new HLA-E alleles. Nevertheless, the HLA-E conding alleles did encode mainly two different full-length molecules, known as alleles E*01:01 and E*01:03, which corresponds to about 90% of all. This feature reinforced the conservation at the coding region, where most of the variable sites detected are intronic mutations, synomymous mutations in exons, or mutations at regulatory elements. In the 3’ untranslated region, 12 haplotypes were found, but only two different sequences (3UTR-1 and 3UTR-2) presented a summed frequency of 90%. Nucleotide diversity was higher in the distal promoter segment than in the coding and 3’ untranslated segments. HLA-E present a weak linkage disequilibrium along the entire segment, mainly because of the presence of many Alu elements that may influence recombination within the gene. Because of that, the same regulatory haplotype may occur associated with different coding sequences. Further analysis involving the binding of transcription factors and non-coding RNAs are necessary to evaluate whether these variable sites at regulatory segments (or even in the coding sequence) may influence the gene expression profile.
Descrição
Palavras-chave
HLA-E, Sequenciamento de nova geração, Variabilidade, Região promotora, Região codificadora, Haplótipos