RESUMO
OBJECTIVE: We investigated the association of single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes and transporters (DMETs) with the response to azathioprine (AZA) in patients affected by myasthenia gravis (MG) to determine possible genotype-phenotype correlations. PATIENTS AND METHODS: Genomic DNA from 180 AZA-treated MG patients was screened through the Affymetrix DMET platform, which characterizes 1931 SNPs in 225 genes. The significant SNPs, identified to be involved in AZA response, were subsequently validated by allelic discrimination and direct sequencing. SNP analysis was carried out using the SNPassoc R package and the haploblocks were determined using haploview software. RESULTS: We studied 127 patients in the discovery phase and 53 patients in the validation phase. We showed that two SNPs (rs8058694 and rs8058696) found in ATP-binding cassette subfamily C member 6, a subfamily member of ATP-binding cassette genes, constituted a new haplotype associated with AZA response in MG patients in the discovery cohort (P=0.011; odds ratio: 0.40; 95% confidence interval: 0.20-0.83) and in the combined cohort (P=0.04; odds ratio: 1.58). CONCLUSION: These findings highlight the role that the ATP-binding cassette subfamily C member 6 haplotype may play in AZA drug response. In view of the significant effects and AZA intolerance, these novel SNPs should be taken into consideration in pharmacogenetic profiling for AZA.
Assuntos
Azatioprina/administração & dosagem , Estudos de Associação Genética/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Miastenia Gravis/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Azatioprina/farmacocinética , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/genética , Variantes Farmacogenômicos , Análise de Sequência de DNARESUMO
Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the 'infectious disease' functional category and 46% with 'inflammatory disease' and 'inflammatory response-associated' categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the 'inflammatory' categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis.
Assuntos
Infecções/complicações , Inflamação/complicações , Leucócitos Mononucleares/metabolismo , Miastenia Gravis/etiologia , Adulto , Fatores Etários , Idade de Início , Biomarcadores , Estudos de Casos e Controles , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Infecções/etiologia , Inflamação/etiologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , RNA não Traduzido/genética , Receptores Colinérgicos/metabolismo , TranscriptomaRESUMO
Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-ß. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG.
Assuntos
Antígenos Virais/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Miastenia Gravis/imunologia , Timo/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Adolescente , Adulto , Antígenos Virais/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Centro Germinativo/virologia , Herpesvirus Humano 4 , Humanos , Interferon beta/genética , Interferon beta/imunologia , Antígeno Ki-67/genética , Antígeno Ki-67/imunologia , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/patologia , Macrófagos/virologia , Masculino , Miastenia Gravis/complicações , Miastenia Gravis/patologia , Miastenia Gravis/virologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Timo/patologia , Timo/virologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genéticaRESUMO
INTRODUCTION: We validated the scale for myasthenia gravis (MG) developed at the Neurological Institute Foundation of Milan (INCB-MG scale). METHODS: A total of 174 patients were evaluated with the INCB-MG and compared with the MG Composite (MGC) as the gold standard. Dimensionality, reliability, and validity of the INCB-MG scale were studied by principal component factor analysis, Cronbach alpha, and Pearson correlation coefficients; interobserver reliability was calculated by the weighted Cohen K coefficient. RESULTS: Generalized and bulbar INCB-MG subscales were unidimensional with excellent consistency; the INCB-MG and MGC scales were strongly correlated. Fatigability assessment was correlated with the INCB-MG generalized, bulbar, and respiratory subscales. CONCLUSIONS: The INCB-MG scale is a reliable tool to assess MG and is strongly correlated with the MGC. The INCB-MG scale is a valid tool for every day practice and should be further investigated for its application in clinical trials.