Análise dos polimorfismos da região 3' não traduzida do gene HLA-G e inserção AluyHG em pacientes de Santa Catarina com histórico de artrite reumatoide

Abstract

A Artrite Reumatoide (AR) é uma doença autoimune caracterizada por inflamação crônica que causa erosões e deformidades nas articulações, podendo ter acometimento de órgãos como pulmão e coração. A molécula HLA-G está relacionada com a inibição de células do sistema imune, como células T e NK, conferindo proteção contra respostas inflamatórias. Entre os polimorfismos ligados à sua expressão, estão os localizados na região 3´UTR do gene HLA-G, que incluem dez sítios polimórficos descritos: um polimorfismo de inserção ou deleção (InDel) de um fragmento de 14pb e nove SNPs (+3001, +3003 T/C, +3010 C/G, +3027 A/C, +3035 C/T, +3142 G/C, +3187 A/G, +3196 G/C e +3227 A/G). A partir disso, o objetivo desse estudo será realizar um estudo caso-controle e verificar a influência desses polimorfismos na manifestação da doença. A família Alu é o elemento móvel mais comum em genomas de primatas, com mais de 1,1 milhões de cópias no Homo sapiens. A inserção AluyHG encontra-se entre os loci HLA-A e HLA-G, podendo ser um marcador da região. A partir disso, o objetivo desse estudo foi genotipar 115 pacientes e 115 controles (classificados por faixa etária) e verificar a influência desses polimorfismos na manifestação da doença. Para isso, foi realizado um levantamento de dados epidemiológicos e clínicos relacionados à manifestação da doença (hábito tabagista, fator reumatoide, proteína C-reativa e velocidade de hemossedimentação) e estes foram testados. O DNA foi extraído de sangue total e a genotipagem feita por PCR e visualização em eletroforese em gel vertical de poliacrilamida em concentração de 7%, corado com nitrato de prata, no caso do polimorfismo de 14pb do HLA-G, e em gel de agarose 1%, corado com GelRedTM, no caso do polimorfismo AluyHG (com 322pb). Os SNPs do HLA-G foram identificados por meio de sequenciamento pelo método de Sanger. Foram realizados cálculos das frequências alélicas e genotípicas, análises de associações e inferência de haplótipos. Após as análises foram encontrados valores significativos de associação dos polimorfismos e a manifestação de AR para os genótipos +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). Para os modelos de herança, foram encontradas associações com: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). Para os dados clínicos, foram encontradas as seguintes associações: FR e +3010*GG (OR=4,685, p=0,032) e +3196*GC (OR=0,388, p=0,049), CRP e +3196*GC (OR=0,301, p=0,011) e +3196*CC (OR=2,713, p=0,035), VHS e +3003*C (OR=0,296, p=0,042) e +3003*T (OR=3,382, p=0,042). Para os haplótipos e combinações haplotípicas, foram encontrados: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); nos modelos de herança UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). Em conclusão, este estudo permitiu investigar a possibilidade de associação do gene HLA-G em relação à Artrite Reumatoide, corroborando com dados encontrados na literatura e trazendo alguns dos resultados de associação com doenças autoimunes, que não haviam sido encontrados em nenhuma literatura até o momento. Mais estudos funcionais devem ser realizados para melhor compreender como os polimorfismos genéticos podem agir na patogênese da AR.Palavras chave: Artrite Reumatoide. HLA-G. AluyHG. Doença autoimune. Estudo de associação. Genética epidemiológica. ABSTRACTRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation causing erosions and deformity in the joints, and may be of involvement of organs such as lung and heart. The HLA-G is associated with the inhibition of immune cells, such as NK and T cells, conferring protection against inflammatory responses. Among the polymorphisms linked to its expression are the ones located in the 3'UTR region of the gene that include ten polymorphic sites described: a polymorphism of insertion or deletion (InDel) with a fragment of 14pb and nine SNPs (+3001C/T, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G, +3196G/C and +3227A/G). From this, the objective of this study will be to conduct a case-control study and the influence of these polymorphisms in disease manifestation. Alu family is the most common mobile element in primate genomes, with more than 1.1 million copies in Homo sapiens. The insertion AluyHG located between the loci HLA-A and HLA-G, may be a marker region. From this, the objective of this study was to genotype 115 patients and 115 controls (classified by age group) and the influence of these polymorphisms in the demonstration and development of the disease. For this, a survey was conducted of epidemiological and clinical data related to the development of the disease (smoking habits, rheumatoid factor, C-reactive protein and erythrocyte sedimentation rate) and these were tested. DNA was extracted from whole blood by PCR and genotyping made and viewed in vertical gel electrophoresis in polyacrylamide concentration of 7% stained with silver nitrate in the case of polymorphism of 14bp of the HLA-G, and 1% agarose gel in AluyHG case polymorphism (with 322pb). The HLA-G SNPs were identified through sequencing by the Sanger method. Calculations were made of the allele and genotype frequencies, analyzes of associations and haplotype inference. The analysis found associations of the polymorphisms and the risk of developing RA for genotypes +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). For heritage models, associations were found with: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). For the clinical data, the following associations were found: RF and +3010*GG (OR=4,685, p=0,032) and +3196*GC (OR=0,388, p=0,049), CRP and +3196*GC (OR=0,301, p=0,011) and +3196*CC (OR=2,713, p=0,035), ESR and +3003*C (OR=0,296, p=0,042) and +3003*T (OR=3,382, p=0,042). For haplotypes and haplotype combinations were found: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); in heritage models: UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). In conclusion, this study allowed us to investigate the possible association of HLA-G gene in relation to rheumatoid arthritis, corroborating with the data in the literature and bringing some of the association results with autoimmune diseases who had not found any literature to date. More functional studies should be conducted to better understand how genetic polymorphisms can act in the pathogenesis of RA.<br>

Abstract : Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation causing erosions and deformity in the joints, and may be of involvement of organs such as lung and heart. The HLA-G is associated with the inhibition of immune cells, such as NK and T cells, conferring protection against inflammatory responses. Among the polymorphisms linked to its expression are the ones located in the 3'UTR region of the gene that include ten polymorphic sites described: a polymorphism of insertion or deletion (InDel) with a fragment of 14pb and nine SNPs (+3001C/T, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G, +3196G/C and +3227A/G). From this, the objective of this study will be to conduct a case-control study and the influence of these polymorphisms in disease manifestation. Alu family is the most common mobile element in primate genomes, with more than 1.1 million copies in Homo sapiens. The insertion AluyHG located between the loci HLA-A and HLA-G, may be a marker region. From this, the objective of this study was to genotype 115 patients and 115 controls (classified by age group) and the influence of these polymorphisms in the demonstration and development of the disease. For this, a survey was conducted of epidemiological and clinical data related to the development of the disease (smoking habits, rheumatoid factor, C-reactive protein and erythrocyte sedimentation rate) and these were tested. DNA was extracted from whole blood by PCR and genotyping made and viewed in vertical gel electrophoresis in polyacrylamide concentration of 7% stained with silver nitrate in the case of polymorphism of 14bp of the HLA-G, and 1% agarose gel in AluyHG case polymorphism (with 322pb). The HLA-G SNPs were identified through sequencing by the Sanger method. Calculations were made of the allele and genotype frequencies, analyzes of associations and haplotype inference. The analysis found associations of the polymorphisms and the risk of developing RA for genotypes +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). For heritage models, associations were found with: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). For the clinical data, the following associations were found: RF and +3010*GG (OR=4,685, p=0,032) and +3196*GC (OR=0,388, p=0,049), CRP and +3196*GC (OR=0,301, p=0,011) and +3196*CC (OR=2,713, p=0,035), ESR and +3003*C (OR=0,296, p=0,042) and +3003*T (OR=3,382, p=0,042). For haplotypes and haplotype combinations were found: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); in heritage models: UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). In conclusion, this study allowed us to investigate the possible association of HLA-G gene in relation to rheumatoid arthritis, corroborating with the data in the literature and bringing some of the association results with autoimmune diseases who had not found any literature to date. More functional studies should be conducted to better understand how genetic polymorphisms can act in the pathogenesis of RA.