Evaluation of Toll-like-receptor gene family variants as prognostic biomarkers in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main feature is persistent joint inflammation. Toll-like receptors (TLRs) play critical roles in the activation of innate and adaptive immune responses, and influence the activity of NF?B, a key player in chronic inflammation. We aimed at investigating the association of TLR allelic variants with susceptibility and severity of RA through a systematic, high-throughput, analysis of TLR genes. All coding exons and flanking regions of nine members of the TLR family (TLR1-9) were analyzed in 66 patients with RA and 30 healthy controls by next generation sequencing. We focussed on three single allelic variants, N248S in TLR1, Q11L in TLR7 and M1V in TLR8 based on the allelic frequencies in both patient and control populations, the predicted impact on protein function and the novelty in RA research. Analysis of these selected variants in a larger cohort of 402 patients with RA and in 208 controls revealed no association with susceptibility. However, the M1V allele was associated with a lower need for disease-modifying antirheumatic drugs (DMARDs) (p =0.008) and biologic treatments (p =0.021). Functional studies showed that the M1V variant leads to a reduced production of inflammatory cytokines, IL-1?, IL-6 and TNF?, in response to TLR8 agonists. Thus, the presence of this variant confers a significant protective effect on disease severity. These results show for the first time the association between the M1V variant of TLR8 and reduced disease severity in RA, which could have prognostic value for these patients.This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation grant PI11/02012, and grant RD12/0036/0022 from Red Temática de Investigación Cooperativa en Cáncer, Sociedad Española de Reumatología grant FER13/13 and Instituto de Investigación Valdecilla (IDIVAL) grant APG-03. I.V. is funded by programa Ramón y Cajal, Ministerio de Economia y Competitividad, Spain

A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis

Background: Toll-like receptor (TLR) family members are key players in inflammation. TLR10 has been poorly studied in chronic inflammatory disorders, and its clinical relevance in rheumatoid arthritis (RA) is as yet unknown. We aimed at identifying TLR10 variants within all coding regions of the gene in patients with RA as well as studying their functional and clinical significance. Methods: TLR10 gene variants were studied by performing sequencing of 66 patients with RA and 30 control subjects. A selected variant, I473T, was then analyzed in 1654 patients and 1702 healthy control subjects. The capacity of this TLR10 variant to modify the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) was determined by using a luciferase reporter assay and analyzing the expression of NFkB target genes by quantitative polymerase chain reaction. Differences between groups were analyzed by using the Mann-Whitney U test and the unpaired two-tailed Student’s t test. Results: We detected ten missense variants in the TLR10 gene and focused on the I473T substitution based on allele frequencies and the predicted functional impact. I473T variant is not associated with susceptibility to RA, but it significantly correlates with erosive disease in patients seropositive for antibodies to citrullinated protein antigens (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to infliximab treatment as measured by the change in Disease Activity Score in 28 joints (p = 0.012) and by the European League Against Rheumatism criteria (p = 0.049). Functional studies showed that TLR10 reduced activation of the NFkB inflammatory pathway in hematopoietic cells, whereas the I473T variant lacked this inhibitory capacity. Consistently, after exposure to infliximab, cells expressing the I437T variant showed higher NFkB activity than cells carrying wild-type TLR10. Conclusions: A TLR10 allelic variant, I473T, has impaired NFkB inhibitory activity and is highly associated with disease severity and low response to infliximab in patients with RA

Identificación de variantes génicas relevantes de la vía NFkB y sus consecuencias funcionales en pacientes con artritis reumatoide

NFkB es uno de los principales moduladores de las respuestas inmune e inflamatoria. Diversos estudios en pacientes con artritis reumatoide (AR) sugieren un papel relevante en dicha enfermedad. Nuestra hipótesis de trabajo es que pueden existir mutaciones en los genes involucrados en la regulación de NFB que pueden tener consecuencias funcionales y por tanto asociarse bien con la susceptibilidad a padecer AR, o bien con el pronóstico de la enfermedad. En esta tesis, describimos que el gen ASCC1 es un inhibidor de la ruta NFB y que la variante S78* da lugar a una proteína truncada que bloquea la función del gen y que se asocia con una mayor gravedad de la enfermedad. Describimos también la variante M1V en el gen TLR8 asociada a un mejor pronóstico en pacientes con AR. Por otro lado, definimos funcionalmente al gen TLR10 como un inhibidor de la actividad transcripcional de NFB en células hematopoyéticas y a la variante I473T capaz de modificar esta actividad inhibidora bloqueando la función del gen y asociándose con una mayor gravedad de la enfermedad. Por último, mostramos como esta variante está significativamente asociada a una menor respuesta tanto clínica como biológica al tratamiento con el fármaco infliximab

Método para predecir la respuesta clínica a un tratamiento con agentes antiinflamatorios

ABSTRACT: The present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10, I473T, as biomarkers for predicting gravity or prognosis in inflammatory diseases, including rheumatoid arthritis, and/or for predicting the response to a treatment using anti-inflammatory agents such as anti-TNFα agents.RESUMEN: La presente invención se refiere al uso de variantes alélicas de TLR10 humano, incluyendo a la variante alélica de TLR10 humano I473T: como biomarcador para predecir la gravedad o el pronóstico en enfermedades inflamatorias, incluyendo la artritis reumatoide y/o para predecir la respuesta a un tratamiento con agentes antiinflamatorios, tales como agentes anti-TNFα.Solicitud: PCT/ES2017/000089 (07.07.2017)Nº de Publicación: WO2018/020060A1 (01.02.2018