Undifferentiated arthritis (UA) is a common inflammatory type of arthritis which is known by joint swelling, pain, and stiffness and is not classified as a specific rheumatologic disease. It is estimated that 32% of UA patients will develop rheumatoid arthritis (RA) that is a chronic, inflammatory, autoimmune disease which could cause joint damage and unchangeable disability. RA has heterogeneous presentations and affects 0.5 to 1.0% in white populations. Several risk factors have been reported for RA development. These include genetic, sex, and environmental factors such as slilica exposure, infectious agents, smoking, deficiency of vitamin D, obesity, and microbiota changes. Genetic factors play a significant role in rheumatoid arthritis development, and probably account for 50–60% of disease susceptibility which are classified as Human Leukocyte Antigen (HLA) and non-HLA genes. Methotrexate (MTX) is a first-line treatment in rheumatoid arthritis management. It is the most important drug in combination therapies, and is considered as a gold standard among RA therapies. Epigenetics as a promising and growing research field in rheumatoid arthritis not only contributes to the RA development but also could be involved in disease management and therapy. Objectives: The current study aimed in a) evaluating the association of well-known single nucleotide polymorphisms (SNPs) belonging to human leukocyte antigen (HLA) and non-HLA genes with early rheumatoid arthritis (ERA) development among UA diagnosed patients; b) assessing the response to therapy of ERA patients according to genetic background based on SNPs involved in MTX/folate pathway, and c) studying long interspersed transposable element 1 (LINE1) methylation status among healthy control, ERA and RA cases and also its methylation changes in response to MTX therapy among ERA patients. Subjects, Material and Methods: A total of 471 patients with UA have been recruited. Genomic DNA from white blood cells (WBC) was extracted and genotyping for 7 polymorphisms belonging to HLA genes including rs1233334, rs1063320, 14 bp Insertion/deletion, rs660895, rs6910071, rs9275595 and rs10807113 and another 7 SNPs belonging to non-HLA genes including PTPN22 rs2476601, PADI4 rs2240340, STAT4 rs7574865, CTLA4 rs231775, TRAF1 rs3761847, IL-10 rs1800871 and IL-6 rs1800795 have been performed. Regarding response to the therapy, 6 SNPs involving in MTX/Folate pathway including ATIC rs2372536, MTRR rs1801394, SHMT1 rs1979277, SLC19A1 rs1051266, MTHFR rs1801133 and MTHFR rs1801131 had been genotyped. In respect of epigenetics, LINE1 methylation status of synovial cells and WBC was evaluated. Besides, the methylation status of 50 healthy controls, 50 ERA and 30 RA patients were compared. Finally, LINE1 methylation of ERA patients according to the response to therapy have been investigated. Statistical analyses for genetic association, gene-gene interaction and methylation level has been carried out. Results: Regarding the association of HLA variants with ERA development, no significant association was found. With respect to non-HLA SNPs there was not significant association with ERA development, too. After stratification according to sex, there was a significant association of TRAF1 rs3761847 GA heterozygous in males (p<0.05). The association was under dominant model. Multifactorial Dimensionality Reduction (MDR) analysis also revealed that there was an association between smoking and anti-Citrullinated Protein Antibody (ACPA) with ERA development. Regarding association of MTX/folate-related SNPs, there was a significant association of CC homozygous genotype of SLC19A1 rs1051266 in ACPA-positive patients with response to the therapy (p<0.02).

L’artrite reumatoide (AR) è una patologia autoimmune infiammatoria cronica che colpisce prevalentemente le articolazioni ed è potenzialmente invalidante. L’AR presenta manifestazioni eterogenee e colpisce lo 0.5-1.0% della popolazione caucasica. Nello sviluppo di tale patologia concorrono diversi fattori di rischio, come quelli genetici, legati al sesso e fattori ambientali come l’esposizione al silice, agenti infettivi, fumo di sigaretta, carenza di vitamina D, obesità e alterazioni del microbiota umano. I fattori genetici giocano un ruolo chiave nello sviluppo e mantenimento dell’artrite reumatoide nel circa 50-60% dei casi. I geni coinvolti sono classificati come i geni per l’Antigene Leucocitario Umano (HLA) e geni non-HLA. Il Methotrexate (MTX) è uno dei farmaci più utilizzati per la terapia dell’AR e viene considerato il gold standard per il trattamento di tale patologia. Può essere utilizzato in combinazione con altre terapie. L’epigenetica sta rappresentando un ambito promettente per l’AR, poiché può contribuire non solo nell’arrestare il decorso di tale patologia, ma può aiutare nel migliorare le terapie e quindi la sua gestione. Scopo: Lo studio presentato in questa tesi ha lo scopo di: a) valutare l’associazione di polimorfismi a singolo nucleotide (SNP) noti, dei geni HLA e geni non-HLA, con lo sviluppo dell’artrite precoce (ERA) tra pazienti a cui è stata diagnosticata artrite indifferenziata; b) valutare la risposta alla terapia nei pazienti ERA in base al risultato genetico basato sugli SNP coinvolti nella via MTX/ciclo dei folati, c) studiare lo stato di metilazione dei Long Interspersed Transposable Element 1 (LINE-1) nei controlli sani e nei casi di ERA e RA. Inoltre si vuole studiare una eventuale modificazione nello stato di metilazione tra i pazienti ERA sottoposti al trattamento con MTX . Materiali e Metodi: I pazienti totali con artrite indifferenziata reclutati e analizzati in questo studio sono in totale 471. Il DNA genomico è stato estratto delle cellule bianche del sangue (WBC). La genotipizzazione è stata effettuata su 7 polimorfismi tra i gene HLA, come: rs1233334, rs1063320, 14 bp Insertion/deletion, rs660895, rs6910071, rs9275595 e rs10807113, mentre per i geni non-HLA sono: PTPN22 rs2476601, PADI4 rs2240340, STAT4 rs7574865, CTLA4 rs231775, TRAF1 rs3761847, IL-10 rs1800871 e IL-6 rs1800795. Per quanto riguarda lo studio della risposta alla terapia MTX/ciclo dei folati sono stati genotipizzati i seguenti geni: ATIC rs2372536, MTRR rs1801394, SHMT1 rs1979277, SLC19A1 rs1051266, MTHFR rs1801133 e MTHFR rs1801131. Le analisi epigenetiche hanno visto lo studio dello stato di metilazione di LINE-1 nelle cellule sinoviali e WBC. Lo stato di metilazione è stato valutato e comparato in 50 pazienti sani, 50 pazienti con ERA e 30 con AR. Inoltre è stato valutato lo stato di metilazione di LINE-1 nei pazienti ERA in base alla risposta alla terapia a cui sono stati sottoposti. Sono state condotte analisi statistiche per lo studio di associazione genetica, interazione gene-gene e lo studio di metilazione. Risultati: L’associazione delle varianti HLA coinvolte nello sviluppo di ERA non ha mostrato alcun risultato significativo. Anche per quanto riguarda gli SNP non-HLA, non si è riscontrata alcuna associazione significativa con lo sviluppo di ERA. Dopo la stratificazione in base al sesso, si è osservata un’associazione significativa con il gene TRAF1 rs3761847 per il genotipo eterozigote G/A nei maschi (p<0.05), secondo il modello dominante. Le analisi Multifactorial Dimensionality

Genetic, pharmacogenetic and epigenetic of early rheumatoid arthritis

RAVAEI, AMIN
2020

Abstract

Undifferentiated arthritis (UA) is a common inflammatory type of arthritis which is known by joint swelling, pain, and stiffness and is not classified as a specific rheumatologic disease. It is estimated that 32% of UA patients will develop rheumatoid arthritis (RA) that is a chronic, inflammatory, autoimmune disease which could cause joint damage and unchangeable disability. RA has heterogeneous presentations and affects 0.5 to 1.0% in white populations. Several risk factors have been reported for RA development. These include genetic, sex, and environmental factors such as slilica exposure, infectious agents, smoking, deficiency of vitamin D, obesity, and microbiota changes. Genetic factors play a significant role in rheumatoid arthritis development, and probably account for 50–60% of disease susceptibility which are classified as Human Leukocyte Antigen (HLA) and non-HLA genes. Methotrexate (MTX) is a first-line treatment in rheumatoid arthritis management. It is the most important drug in combination therapies, and is considered as a gold standard among RA therapies. Epigenetics as a promising and growing research field in rheumatoid arthritis not only contributes to the RA development but also could be involved in disease management and therapy. Objectives: The current study aimed in a) evaluating the association of well-known single nucleotide polymorphisms (SNPs) belonging to human leukocyte antigen (HLA) and non-HLA genes with early rheumatoid arthritis (ERA) development among UA diagnosed patients; b) assessing the response to therapy of ERA patients according to genetic background based on SNPs involved in MTX/folate pathway, and c) studying long interspersed transposable element 1 (LINE1) methylation status among healthy control, ERA and RA cases and also its methylation changes in response to MTX therapy among ERA patients. Subjects, Material and Methods: A total of 471 patients with UA have been recruited. Genomic DNA from white blood cells (WBC) was extracted and genotyping for 7 polymorphisms belonging to HLA genes including rs1233334, rs1063320, 14 bp Insertion/deletion, rs660895, rs6910071, rs9275595 and rs10807113 and another 7 SNPs belonging to non-HLA genes including PTPN22 rs2476601, PADI4 rs2240340, STAT4 rs7574865, CTLA4 rs231775, TRAF1 rs3761847, IL-10 rs1800871 and IL-6 rs1800795 have been performed. Regarding response to the therapy, 6 SNPs involving in MTX/Folate pathway including ATIC rs2372536, MTRR rs1801394, SHMT1 rs1979277, SLC19A1 rs1051266, MTHFR rs1801133 and MTHFR rs1801131 had been genotyped. In respect of epigenetics, LINE1 methylation status of synovial cells and WBC was evaluated. Besides, the methylation status of 50 healthy controls, 50 ERA and 30 RA patients were compared. Finally, LINE1 methylation of ERA patients according to the response to therapy have been investigated. Statistical analyses for genetic association, gene-gene interaction and methylation level has been carried out. Results: Regarding the association of HLA variants with ERA development, no significant association was found. With respect to non-HLA SNPs there was not significant association with ERA development, too. After stratification according to sex, there was a significant association of TRAF1 rs3761847 GA heterozygous in males (p<0.05). The association was under dominant model. Multifactorial Dimensionality Reduction (MDR) analysis also revealed that there was an association between smoking and anti-Citrullinated Protein Antibody (ACPA) with ERA development. Regarding association of MTX/folate-related SNPs, there was a significant association of CC homozygous genotype of SLC19A1 rs1051266 in ACPA-positive patients with response to the therapy (p<0.02).
RUBINI, Michele
DI VIRGILIO, Francesco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2478828
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