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    • 专利摘要:
    Method to predict the clinical response to a treatment with anti-inflammatory agents. The present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10 I473T, as a biomarker for predicting the severity or prognosis in inflammatory diseases, including rheumatoid arthritis and/or to predict the response to a treatment with anti-inflammatory agents, such as anti-TNFα agents; (Machine-translation by Google Translate, not legally binding)
    公开号:ES2656587A1
    申请号:ES201600636
    申请日:2016-07-26
    公开日:2018-02-27
    发明作者:José Luis FERNANDEZ LUNA；Victor Manuel MARTINEZ TABOADA；Marcos LOPEZ HOYOS；Silvia TORICES DEL VAL；Pedro MUÑOZ CACHO；Ignacio VARELA EGOCHEAGA；Alejandro BALSA CRIADO；Sara Marsal Barril；Antonio Julia Cano
    申请人:Servicio Cantabro De Salud；Fibhulp；Vhir；Universidad de Cantabria；Fundacion Instituto de Investigacion Marques de Valdecilla；
    IPC主号:
    专利说明:

    METHOD FOR PREDICTING CLINICAL RESPONSE TO TREATMENT WITHANTI-INFLAMMATORY AGENTS FIELD OF THE INVENTION
    The present invention can be included in the field of personalized medicine. More specifically, the present invention relates to the use of allelic variants of human TLR10, including the allelic variant of human TLR10 1473T, as a biomarker to predict severity or prognosis in inflammatory diseases, including rheumatoid arthritis and / or to predict disease. response to treatment with anti-inflammatory agents, such as anti-TNFa agents. BACKGROUND OF THE INVENTION
    Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily characterized by chronic inflammation of the synovial lining and Th1 activation, leading to progressive joint destruction. It has been suggested that viruses and bacteria may contribute to initiating or aggravating RA by binding to TolI-like receptors (TLRs) [Iwahashi M, et al., Arthrítís Rheum 2004, 50 (5): 1457-1467 .; Pierer M, et al., J Immunol 2004, 172 (2): 1256-1265).
    TLRs belong to a family of transmembrane proteins that constitute one of the primary defense mechanisms in infectious diseases and some non-infectious in mammals [Gdor 1, et al., Chem Phys 2011, 13 (9): 3782-3787]. In particular, TLRs are type I transmembrane glycoproteins with extracellular leucine-rich repeats (LRRs) and an intracellular TolI / IL-1 receptor (TIR) homology domain [Ulevitch RJ, Nat Rev Immunol 2004, 4 (7 ): 512-520). Inappropriate activation of TLR-mediated pathways has been implicated in loss of self-tolerance leading to autoimmunity and chronic inflammation [Wagner H, Adv Immunol 2006, 91: 159-173; Deighton K, et al., Appetite 2014.81: 52-59). Furthermore, TLRs have been suggested to be involved in responses to pathogens in RA [Cromartie WJ, et al., J Exp Med 1977, 146 (6): 1585-1602).
    Signaling by TLRs involves interaction with TIR domain-containing adapters, including MyD88, TRIF, TRAM, TIRAP, and SARM1 [O'Neill LA, Bowie AG; Nat Rev Immunol 2007, 7 (5): 353-364) which promotes the activation of activated B cell kappa light chain enhancer nuclear factor (NFKB) among other functions. The TLR-dependent activation of NFKB leads to the production of pro-inflammatory chemokines, cytokines, and cell adhesion molecules [O'Neill LA, Bowie AG; Nat Rev Immunol 2007, 7 (5): 353-364 .; Drexler SK, et al., Int J Biochem Cell Biol 2010, 42 (4): 506-518]. There is increasing evidence that NFKB is an important, if not the primary, transcription factor controlling inflammation [Abu-Soud HM, et al., Biochemistry 1998, 37 (11): 37773786] and deciphering the mechanisms that regulate NFKB activity is considered of great importance in understanding the response to inflammatory stimuli.
    TLR10 remains the only orphan member among human TLRs because its ligands remain unknown and there are discordant data about its function [Oosting M, et al., Proc Natl Acad Sci U S A 2014, 111 (42): E4478-4484 .; Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950]. The expression of TLR10 has been described mainly in B cells, dendritic cells, eosinophils, neutrophils and non-immune cells such as trophoblasts [Hasan U et al., J Immunol 2005, 174 (5): 2942-2950 .; Hornung V et al., J Immuno / 2002, 168 (9): 4531-4537 .; Nagase H, et al., J Immunol 2003, 171 (8): 3977-3982 .; Mulla MJ, et al., Am J Reprod Immunol 2013, 69 (5): 449-453]. In mammals, TLR10, TLR1, and TLR6 share a common locus on chromosome 4p14 and are structurally similar to each other [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950]. Despite interacting with MyD88 [Hasan U, et al., J Immuno / 2005, 174 (5): 2942-2950], TLR10 differs from other TLRs due to the lack of a classical posterior signaling pathway [Guan Y, et al. , J Immunol 2010, 184 (9): 5094-5103]. The phylogeny supports the idea that TLR10 arose before the gene duplication that generated TLR1 and TLR6 [Abhishek A, et al., J Clin Rheumato / 2010, 16 (1): 15-18 .; Zhou H, et al., J Mol Evol 2007, 65 (2): 119-123]. TLR1 and TLR6 can form a protein complex with TLR2 and TLR10 [Hasan U, et al., J Immuno / 2005, 174 (5): 2942-2950 .; Mulla MJ et al., Am J Reprod Immuno / 2013, 69 (5): 449-453], although the individual contribution of each protein to the function of the complex is largely unknown.
    Today, TLR10 is the only member of the TLR family without a well-defined biological function. Various studies describe that TLR10 acts as a pro-inflammatory receptor activating NFKB signaling [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950; Regan T, et al., J Immunol 2013, 191 (12): 6084-6092]. Another study showed that TLR10 does not activate typical TLR-induced signaling, including transcriptional activation mediated by NFKB or interferon-beta (IFNP) [Guan Y, et al., J Immunol 2010, 184 (9): 5094-5103]. Recently, TLR10 has been shown to be a modulator with mainly inhibitory effects [Stappers MH, et al., J Infect Dis 2015]. In line with this, block TLR1 OR with
    Antagonistic antibodies potentiate proinflammatory cytokine production [Oosting M, et al., Proc Natl Acad Sci U S A 2014, 111 (42): E4478-4484]. Furthermore, evidence has been provided that TLR1 O induces apoptosis through caspase-3 activation [Kuuliala K, et al., Ann Rheum Dis 2006,65 (9): 1241-1243].
    Genetic variants in members of the TLR family have been mainly associated with disease propensity in RA patients with variable significance level and even discordant results [Kuuliala K, et al., Ann Rheum Ois 2006, 65 (9): 1241- 1243 .; Radstake TR, et al. , Arthritis Rheum 2004,50 (3): 999-1001 .; Etem EO, et al., Rheumatollnt 2011,31 (10): 1369-1374 .; Coenen MJ, et al., PLoS Jan 2010, 5 (12): e14326; Zheng B, et al., Rheumatollnt 2010,30 (9): 1249-1252].
    A previous study investigated the association between TLR 10 variants and RA propensity but no statistical significance was found [Etem EO, et al., Rheumatollnt 2011,31 (10): 13691374]. Although single nucleotide variants of the TLR10 gene have been associated with other autoimmune diseases [Requena T, et al., Immunogenetics 2013, 65 (5): 345-355], tumors [Kutikhin AG, Hum Immuno / 2011, 72 ( 11): 1095-1116], infectious and inflammatory diseases such as extrapulmonary tuberculosis and asthma [Ma X, et al., PLoS One 2007, 2 (12): e1318; Lazarus R, et al., Am J Respir Crit Care Med 2004, 170 (6): 594-600], the functional activity of this protein and the clinical significance of the gene variants are still controversial [Hasan U, et al., J Immunol 2005, 174 (5): 2942-2950 .; Stappers MH, et al., J Infect Ois 2015].
    The allelic variant 1473T has only been described in a previous work associated with a decreased risk of meningioma [Rajaraman P, et al., Cancer Epidemiol Biomarkers Prev 2010, 19 (5): 1356-1361].
    Despite significant advances in recent years in the discovery of prognostic and / or predictive markers related to RA and inflammatory diseases, there is a continuing need for improved methods to assess the severity and prognosis of the disease, to predict the evolution of the disease and the risk of recurrence or relapse, to allow the classification of the patient population according to the prognosis and select the most appropriate treatment accordingly. It is also desired to identify polymorphic regions within a gene, such as human TLR10, that are associated with the response to one or more drugs used in the treatment of RA and others.
    inflammatory diseases, such as disease-modifying antirheumatic drugs (DMARDs) or biological therapies, including TNFo inhibitors. SUMMARY OF THE INVENTION
    NFK8 is a transcription factor implicated in many chronic inflammatory disorders, including RA. For the first time, the inventors have described that TLR10 can inhibit NFK8 signaling in hematopoietic cells. In vitro functional studies showed that TLR10 reduced the activation of the NFK8 inflammatory pathway in hematopoietic cells, while the 1473T variant did not have this inhibitory capacity. The inventors observed that after stimulation of the NFK8 pathway with TNFo. Upon exposure to infliximab (an inhibitor of TNFo.) cells expressing the 1473T variant showed higher NFK8 activity compared to cells bearing wild-type TLR10.
    It is known that TNFo inhibitors. help to control the evolution of RA. However, not all patients respond adequately to anti-TNFo treatment. initial [Alten R, et al., Int J Rheum Dis 2014, 17 (1): 5-18]. The inventors analyzed the
    association of the missense variant of human TLR10, 1473T, which is located in the LRR18 domain, with AR, observing that the 1473T variant is not associated with propensity to AR. However, this variant was found to correlate significantly with erosive disease in seropositive patients for antibodies to citrullinated proteins (ACPA) (p = 0.017 in the total cohort and p = 0.049 in women) and with a lower response to treatment. with infliximab measured by the DAS28 index (p = 0.012) and by the EULAR criteria (p = 0.049), as shown in Figure 28.
    The data show that a genetic variant of TLR10 selects a group of patients with a more severe and resistant disease. Likewise, said TLR10 genetic variant may be a good candidate marker of response to infliximab or other anti-TNFo treatments. in patients with inflammatory disease and in particular with RA.
    In a first aspect, the present invention refers to an in vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFo agent, wherein said method comprises:
    a) determine, in a biological sample isolated from said subject, the presence of one
    or more TLR10 polymorphisms that result in a TLR10 variant that has lost the ability to inhibit NFKB.
    In a preferred embodiment, the present invention refers to an in vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFa agent, wherein said method comprises:
    a) determine, in a biological sample isolated from said subject, the genotype of the single nucleotide polymorphism (SNP) rs11466657.
    In another aspect, the invention relates to an in vitro method for identifying a subject who has or is suspected of having an inflammatory disease with an increased risk of not responding to treatment with an anti-TNFa agent, wherein said method comprises the stage a) according to the first aspect.
    In a related aspect, the invention relates to an in vitro method for selecting a treatment for a subject who has or is suspected of having an inflammatory disease, wherein said method comprises step a) defined above; and also includes:
    b) selecting an anti-TNFa agent when allele (A) is present in at least
    one of the alleles at the SNP locus rs11466657, preferably when the
    allele (A) is present in both alleles.
    In a further aspect, the invention refers to an in vitro method for determining the severity and / or prognosis of the disease in a subject having an inflammatory disease, said method comprising step a) defined above.
    Likewise, it refers to an in vitro method to obtain useful data to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFa agent, the severity and / or prognosis of said disease. where said method comprises step a) defined above.
    Also, the invention provides a method for treating a subject having an inflammatory disease, wherein said method comprises step a) defined above; and also includes:
    b) administering to said subject an anti-TNFa agent when allele (A) is
    present in at least one of the alleles at the SNP locus rs11466657,
    preferably when allele (A) is present in both alleles.
    Furthermore, the present invention relates to an anti-TNFa agent for use in a subject who has or is suspected of having an inflammatory disease, wherein said subject has the (A) allele in at least one of the alleles in the locus. SNP rs11466657, preferably wherein said subject is homozygous for allele (A).
    In a further aspect, the invention provides a kit to determine in a biological sample isolated from a subject the genotype of one or more polymorphisms of TLR10 that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB, where said kit understands:
    - a reagent for determining the genotype of said polymorphisms;
    - optionally, instructions for the use of said reagent in the determination of the
    genotype of said polymorphisms in an isolated biological sample.
    The invention also relates to the use of a kit, as described in the previous aspect, to predict the clinical response of a subject who has an inflammatory disease, to identify a subject who has an inflammatory disease at risk of not responding to a treatment with an anti-TNFa agent, to select a treatment for a subject having an inflammatory disease, or to determine the severity and / or prognosis of an inflammatory disease in a subject, according to the methods of the invention. BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Variants of the TLR10 gene. (A) Schematic representation of the TLR10 protein illustrating the position of the missense variants identified by next generation sequencing (NGS). LRR, leucine-rich repeats; TM, transmembrane domain; IRR, interleukin To "-receptor domain. (B) Genotype distribution of the 1473T variant in the independent cohort of 1493 healthy controls (HC) and 1201 patients with rheumatoid arthritis (RA). (C) TLR10 genotype frequencies in HapMap populations (HapMap is a map of haplotypes in the human genome; International HapMap consortium et al. (2010), Integrating common and rare genetic variation in diverse human populations, Nature, 467, 52-8).
    Figure 2. Association of the 1473T variant with the severity of the disease in two cohorts of (A) 453 and (B) 1201 patients with RA. The beta value (regression coefficient), used to evaluate the effect of the 1473T variant (genetic risk), was estimated for various clinical findings. A positive regression coefficient means that the variant increases the risk. The dashed lines indicate the cut-off values. The clinical parameters were categorized as binary variables. Eccp / years, association with erosions in patients positive for ACPA including the years of evolution of the disease; Eccp / years in women, association with erosions in ACPA-positive female patients, including the years of evolution of the disease; IFX-EULAR, association with response to infliximab (EULAR response criteria: moderate / good versus none); I FX-DAS28, association with change in DAS28 in patients treated with infliximab.
    Figure 3. Regulation of NFKB transcriptional activity by the 1473T variant. A) 3D structure of TLR10 using the Jmol viewer showing the location of residue 1473. NAG, 2- (acetylamino) -2-deoxy-beta-D-glucopyranose. (B) and (C) K562 and U937 cells (respectively) were cotransfected with wild type TLR10 (wild type or wt) or its mutated variant (mut) and a reporter vector containing sequences that respond to NFKB. Cells were stimulated with 10 ng / ml TNFa for 24 h and then cell extracts were prepared and analyzed for luciferase activity. Likewise, (D) K562 AND (E) U937 cells transfected with the indicated TLR10 variants were treated with TNFa in the presence or absence of infliximab and the luciferase activity was analyzed. Histograms show the mean ± SD of three independent experiments. P <0.05.
    Figure 4. Regulation of NFKB target gene levels by the 1473T variant. U937 (A, B, C) and K562 (D, E, F) cells were transfected with TLR10 (wild type or mutated variant) and then treated or not with 20 ng / ml TNFa in the presence or absence of infliximab ( IFX) for 24 h. The target gene expression of NFKB, CCL2 (A, D, F) TRAIL (B, E) and TNFa (C) was determined by quantitative RT-PCR. P-actin was used for normalization. C, cells transfected with empty vector as an additional control. Histograms show the mean ± SD of three independent experiments. * p <0.05. Detailed description of the invention
    Definitions The term "subject" as used herein refers to a mammalian subject. Preferably, it is selected from a human, companion animal, non-domestic livestock, or zoo animal. For example, the subject can be selected from a human, dog, cat, cow, pig, sheep, horse, bear, etc. In a preferred embodiment, said mammalian subject is a human subject.
    The term "subject having a disease" as used herein refers to those subjects who have been diagnosed with such a disease. For example, a "confirmed diagnosis of rheumatoid arthritis" or "definitive rheumatoid arthritis" may be based on the confirmed presence of synovitis in at least one joint, the absence of an alternative diagnosis that better explains synovitis, and the achievement of a total score of 6 or higher (out of a possible maximum of 10) from individual scores in four domains including: number and location of affected joints (range 0-5), serologic abnormalities (range 0-3), elevated acute phase response (range 0-1) AND duration of symptoms (two levels, range 0-1). The meaning of these parameters is as defined in the EULAR classification criteria (Ann Rheum Dis 2010; 69: 15801588).
    The term "subject suspected of having a disease" as used herein refers to a subject exhibiting one or more signs or symptoms indicative of said disease. A subject suspected of having a disease may also have one or more risk factors (ie, a subject suspected of developing or at risk of developing a disease). It also includes an individual who has received a preliminary diagnosis but for whom a confirmatory test has not been performed. In addition, it includes those individuals in remission.
    The term "test" as used in this document refers to identifying, determining or distinguishing those subjects or individuals who present the characteristics or the
    defined phenotype. A "screening" test can be used when the presence of such characteristics or phenotype is suspected.
    The term "treatment" encompasses both prophylactic and therapeutic treatment. The term "therapeutic treatment" or "therapy" as used herein refers to bringing a body from a disease or disease state back to its normal, healthy state. The term "prophylactic treatment" as used herein refers to preventing a disease state.
    The term "response to a treatment" as used herein refers to the degree to which a treatment achieves desired or intended results, for example the ability of a therapy or drug to achieve the desired clinical effect.
    The term "inflammatory disease" as used herein refers to any disease in which there is an excessive or altered inflammatory response leading to inflammatory symptoms. Such inflammatory diseases include, but are not limited to, Addison's disease, acne vulgaris, alopecia areata, amyloidosis, ulcerations, aphthous stomatitis, arteriosclerosis, arthritis, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, spondyloarthropathy, spondylitis, ankylosing asthma. bronchial, Behcet's disease, Boeck's disease, inflammatory bowel disease, Crohn's disease, choroiditis, ulcerative colitis, celiac disease, cryoglobulinemia, macular degeneration, dermatitis, dermatitis herpetiformis, dermatomyositis, insulin-dependent diabetes, juvenile diabetes, inflammatory demyelinating disease , Dupuytren's contracture, encephalomyelitis, allergic encephalomyelitis, endophthalmitis, allergic enteritis, autoimmune enteropathy syndrome, leprous erythema nodosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, cystic fibrosis, gingivitis, glomerulonephritis, Goodpastu syndrome re, Graves syndrome, Hashimoto's disease, chronic hepatitis, histiocytosis, regional ileitis, iritis, systemic lupus erythematosus, cutaneous lupus erythematosus, disseminated lupus erythematosus, lymphogranuloma, infectious mononucleosis, myasthenia gravis, transverse myelitis, myxedema idiopathic, primary obesity , sympathetic ophthalmia, granulomatous orchitis, pancreatitis, panniculitis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradiculitis, psoriasis, chronic obstructive pulmonary disease, purpura, pyoderma gangrenosum, diabetic syndrome, retinopathy rosacea, sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, scleroderma, multiple sclerosis, disseminated sclerosis, uveitis, vitiligo, Whipple's disease, AIDS-associated diseases, severe combined immunodeficiency and Epstein Barr virus: such as the syndrome Sjogren's, osteoarticular tuberculosis r and parasitic diseases: such as leishmaniasis.
    The term "inflammatory rheumatic disease" as used herein refers to a disease of the musculoskeletal system and / or connective tissues in which inflammatory mechanisms play an important role. A rheumatic disease can affect joints, bones, cartilage, tendons, ligaments, and muscles.
    A "sample" in the context of the present invention is a biological sample that contains any type of body cell and may include, as illustrative and non-limiting examples, body fluids and / or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are routinely obtained from a tissue biopsy. A biological sample useful in the present invention includes blood, urine, saliva, synovial fluid, cerebrospinal fluid, bronchial lavage, ascitic fluid, bone marrow aspirate, pleural effusion, as well as any tissue, fluid, or any other body constituent that may contain cells. . In a preferred embodiment, the sample to be tested is saliva or blood. In a more preferred embodiment, the sample is a blood sample.
    The term "allele" has the meaning that is commonly known in the art, that is, an alternative form of a gene (a member of a pair) that is located at a specific position on a specific chromosome which, when translated gives as result in functional or dysfunctional gene products (including nonexistent).
    The term "polymorphism" or "allelic variant" refers to a common sequence variation of a gene. Allelic variants can be found in the exons, introns, untranslated regulatory regions of the gene, or in the sequences that control the expression of the gene. Complete gene sequencing often makes it possible to identify numerous allelic variants for a particular gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype is performed in a sufficiently large population.
    The term "single nucleotide polymorphism" or "SNP" refers to a type of DNA polymorphism that involves the variation of a single base pair. There are millions of SNPs in the human genome. Commonly, these variations are found in coding sequences of genes, non-coding regions of genes, or in intergenic regions between genes. The
    The presence of a SNP within a gene or in a regulatory region near a gene may play a more direct role in disease by affecting the function of the gene.
    The term "probe" as used herein refers to synthetic or biologically produced nucleic acids, between 10 and 285 base pairs in length that contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions for selection. nucleic acid sequences are targeted, and optionally contain a moiety to detect or enhance assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and form stable hybridization products, and a maximum of 285 nucleotides generally represents an upper limit to the length at which the reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization. The probes may optionally contain certain constituents that contribute to their proper or optimal performance under certain test conditions. For example, probes can be modified to improve their resistance to nuclease degradation (for example, by end-capping), to carry detection ligands (for example, fluoroscein), or to facilitate their capture on a solid support (for example, poly-deoxyadenosine "tails").
    The term "primers" as used herein refers to oligonucleotides that can be used for example in an amplification method, such as a polymerase chain reaction ("PCR"), to amplify a nucleotide sequence. Primers are designated based on the polynucleotide sequence of a particular target sequence. The design and validation of primers and probes are well known in the art. For quantitative real-time PCR methods, see for example Rodriguez A et al. (Methods Mol Biol., 2015,1275: 31-56).
    The term "specific" as used herein in relation to a nucleotide sequence means that a nucleotide sequence will hybridize to / amplify a predetermined target sequence and will not substantially hybridize to / amplify a non-target sequence under the conditions In testing, stringent conditions are generally used.
    The term "hybridization" as used herein refers to a process whereby, under predetermined reaction conditions, two partially or completely complementary nucleic acid strands are allowed to join together in an antiparallel manner to form a Stable, specific hydrogen-bonded double-stranded nucleic acid, following explicit rules for which nucleic acid bases can be paired with each other.
    The term "substantial hybridization" means that the amount of hybridization observed will be such that someone observing the results could consider the result positive with respect to the hybridization data in positive and negative controls. Data that are considered "background noise" are not substantial hybridization.
    The term "stringent hybridization conditions" means about 35 ° C to 65 ° C in about 0.9 molar NaCl salt solution. Stringency can also be governed by reaction parameters such as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the hybridization temperature. Generally as hybridization conditions become stricter, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which hybridization is performed will dictate certain characteristics of the preferred probes to be employed.
    The term "identity" as used herein refers to an exact correspondence of nucleotide to nucleotide or amino acid to amino acid of two polypeptide or polynucleotide sequences or, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". The "percent identity" of two sequences, whether they are nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shortest sequence and multiplied by 100. Suitable programs to calculate identity in percentage or similarity between sequences are well known in the art, such as NCBI's BLAST program, used for example with the default parameters (http://www.ncbLnlm.gov/cgi-bin/BLAST).
    The term "partially identical / complementary" as used herein refers to a sequence that is identical / complementary to at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to a reference sequence. In some embodiments, the partially identical / complementary sequence can be substantially identical / complementary to a reference sequence, which is at least about 95%, 96%, 97%, 98%, or 99% identical / complementary. to said reference sequence. In one embodiment, the partially identical / complementary sequence is 100% identical / complementary to a reference sequence.
    The term "kit" indicates a set of reagents and adjuvants required for an analysis. Although a kit consists in most cases of multiple units, the various test items presented in a single unit may also be available and should be considered as kits. Description
    A method to predict the clinical response to an anti-TNFa agent
    In a first aspect, the present invention refers to an in vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFa agent, wherein said method comprises:
    a) determine, in a biological sample isolated from said subject, the presence of one
    or more TLR10 polymorphisms that result in a TLR10 variant that has lost the ability to inhibit NFKB.
    NF-kB (activated B cell kappa light chain enhancer nuclear factor) plays a key role in regulating the immune response due to infection. Defective regulation of NF-kB is also related to cancer, inflammatory and autoimmune diseases, septic shock, viral infections, or inadequate immune development. It is also involved in memory and synaptic plasticity processes.
    In a particular embodiment, said TLR10 variant exhibits at least one polymorphism that gives rise to a misplaced amino acid substitution. Said polymorphisms include but are not limited to those indicated in Table 2. Preferably, said polymorphism is selected from rs11466657 and rs11466650. More preferably, said polymorphism is rs11466657.
    Thus, in a preferred embodiment, the present invention refers to an in vitro method for predicting the clinical response of a subject who has or is suspected of having an inflammatory disease to an anti-TNFu agent, wherein said method comprises:
    a) determine, in a biological sample isolated from said subject, the genotype of the single nucleotide polymorphism (SNP) rs 11466657.
    Likewise, it refers to an in vitro method to identify a subject who has or is suspected of having an inflammatory disease with an increased risk of not responding to treatment with an anti-TNFu agent, in which said method comprises step a) according to the first aspect.
    The invention also refers to an in vitro method for obtaining data useful to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFu agent, where said method comprises step a) described previously.
    The single nucleotide polymorphism (SNP) rs11466657 has been previously described and its sequence is available in public databases (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=11466657 ). As shown in Figure 1A, rs11466657 is located in the region that codes for the LRR18 domain of human TLR10.
    The rs 11466657 is found on chromosome 4, at position 38774173 Y corresponds for example to position 2056 of the human TRLR10 transcript NM_030956.3 (SEO ID NO: 1). For this position (Iocus) the ancestral allele is T and an allelic change from AIT to AQT has been described that translates into a change in the protein sequence (NP _112218.2; SEO ID NO: 2) of isoleucine (! Le) to threonine (Thr) at position 473 (1473T). When said polymorphism is determined in the complementary chain, the allelic change is from A to G (see example 2). In the present invention the allelic variant of SNP rs11466657, which gives rise to the amino acid change 1473T is referred to as allele (G).
    According to the data presented in Example 2, patients with the 1473T variant show a significantly lower response to anti-TNFu agents, according to the DAS28 index (Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA , van de Putte LB, van Riel PL (1995). Modified disease activity scores that include twenty-eight-joint counts.
    Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum., 38, 44-48) and the EULAR response criteria (Karonitsch T, Aletaha D, Boers M, Bombardieri S, Combe B, Dougados M, Emery P, Felson D, GomezReino J, Keystone E, Kvien TK, Martin-Mola E, Matucci-Cerinic M, Richards P, van Riel P, Siegel J, Smolen JS, Sokka T, van der Heijde D, van Vollenhoven R, Ward M, Wells G, Zink A. Landewe R. (2008) . Methods of deriving EULARlACR recommendations on reporting disease activity in clinical trials of patients with rheumatoid arthritis. Ann Rheum Dis, 67 (10), 1365-73), which also indicates that this variant selects a group of patients with one more disease severe or severe.
    Thus, the determination of the presence of the (G) allele in at least one of the alleles at the SNP locus rs11466657, preferably the determination of the (G) allele in both alleles (ie, GG genotype) is indicative of a higher risk. of not responding to the anti-inflammatory agent. In other words, it is predictive of absence of clinical response or resistance to treatment.
    The determination of the presence or absence of said SNP can be determined by DNA sequencing, PCR analysis or any other genotyping method known in the state of the art. Examples of such methods include, but are not limited to, chemical assays, such as allele-specific hybridization, primer extension ("first extension"), allele-specific oligonucleotide ligation, sequencing, enzymatic cleavage, 5 'discrimination (flap ) endonuclease; and detection methods, such as fluorescence, chemiluminescence, and mass spectrometry. For example, the presence or absence of such polymorphisms can be detected in a DNA sample, preferably after amplification. For example, isolated DNA can be subjected to polymerase chain reaction (PCR) amplification, using oligonucleotide primers specific for the polymorphism or that allow amplification of a region containing the polymorphism. For example, the conditions for primer hybridization can be selected to ensure specific amplification; whereby the appearance of an amplification product is indicative of the presence of the polymorphism according to the invention. Alternatively, the DNA can be amplified, so that the SNP can be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
    Numerous strategies for genotype analysis have been described (Cooper et al., 1991; Grompe, 1993). For example, the presence or absence of a restriction site in a nucleic acid can be determined. When a polymorphism creates or eliminates the recognition site of a restriction enzyme, this allows a genotyping of the polymorphism in a simple way by direct PCR. Other strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphisms (RFLPs); allele-specific oligonucleotide (ASO) hybridization, allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; Denaturing gradient gel electrophoresis (OGGE), temperature gradient gel electrophoresis (TGGE), single-chain conformational polymorphism (SSCP), and denaturing high-performance liquid chromatography (Kuklin et al, 1997).
    Direct sequencing can be performed by any method known in the art, including, but not limited to, chemical sequencing, using the Maxam-Gilbert method; enzymatic sequencing, using the Sanger method; pyrosequencing, mass spectrometry sequencing; sequencing using arrays; o Quantitative real-time PCR. Typically, the DNA to be sequenced is first subjected to PCR amplification using specific amplification primers. However, several other methods are available, allowing DNA to be studied independently of the PCR reaction, for example, the rolling circle amplification (RCA), the Invader® assay, or the oligonucleotide ligation assay (OLA).
    Another preferred quantification procedure is quantitative PCR (qPCR). QPCR or real-time PCR is well known to one of ordinary skill in the art. Various instruments are marketed to carry out this reaction, such as ABI Prism 7700 SOS, GeneAmp 5700 SOS, ABI Prism 7900 HT SOS from Applied Biosystems; iCycler iQ from BioRad; Cepheid Smart Cycler; Rotor-Gene from Corbett Research; LightCycler from Roche Molecular Biochemicals and Mx4000 Multiplex from Stratagene. The qPCR procedure enables accurate quantification of the PCR product in real time by measuring the accumulation of the PCR product very early in the exponential phase of the reaction, thus reducing the quantification bias linked to the efficiency of the PCR amplification involved. produced in endpoint PCR. Real-time PCR is well known in the art and therefore is not described in detail herein. An overview of the technology and protocols for qPCR is available, for example, from the vendors mentioned above, for example, http://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr. htmlo
    http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/qpcrtechnical-guide.html. A review of the use of qPCR in mRNA quantification is found for example in Wong ML and Medrano JF, Biotechniques 2005, 39 (1): 75-85.
    Different detection chemistries are available for qPCR. They can all be used with the qPCR instruments mentioned above. The term "detection biochemistry" refers to a procedure for reporting the amplification of the specific PCR product in real-time PCR. These screening chemistries are classified into two main groups. The first group comprises double-stranded DNA intercalating molecules: such as SYBR Green I and EvaGreen; and the second group includes oligonucleotides typically labeled with a fluorophore. The latter, in turn, has been divided into three subgroups: (i) primers-probes (Scorpions, Amplifluor®, LUX TM, Cyclicons, Angler®); (ii) hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon TM, MGB-Pleiades, MGB-Eclipse, ResonSense®, Yin-Yang or displacing) probes; and (iii) nucleic acid analogs (PNA, LNA®, ZNA TM, non-natural bases: Plexor ™ primer, Tiny-Molecular Beacon), see E. Navarro eta 1., Clinica Chimica Acta, Volume 439, 15 January 2015, Pages 231-250.
    For example, such probes are oligonucleotides, they can be double-labeled, such as hydrolysis probes or molecular beacons. The 5 'end of the oligonucleotide is typically labeled with a fluorescent reporter molecule such as FAM, TET or JOE while the 3' end is labeled with a quencher molecule, such as T AM or BHQ 1. The sequence of the probe is specific for a region of interest in the amplified target molecule. In a preferred embodiment, said probe is a hydrolysis probe that is designed so that the length of the sequence brings the 5 'fluorophore and 3' quencher molecule in close enough proximity to suppress fluorescence. Various reporter and quencher molecules for use in qPCR probes are well known in the art. These are available, for example, from https://www.eurofinsgenomics.eu/en/dna-rna-oligonucleotides/optimised-applicationoligos/qpcr-probes.aspx.
    Likewise, the present invention relates to oligonucleotide sequences (primers and / or probes) that specifically hybridize with one of the alleles of the polymorphisms. The term "a primer and / or probe" specifically includes "primers and / or probes", encompassing for example a primer, a probe, a primer and a probe, a primer pair, and a primer and probe pair.
    Preferably, a probe and / or primer is a polynucleotide sequence of between 10 and 30 nucleotides, more preferably between 15 and 26 nucleotides, even more preferably between 18 and 22 nucleotides, and even more preferably of around 20 nucleotides. In a particular embodiment, said primers and / or probes have been modified for detection or to enhance the performance of the assay.
    In a related aspect, the invention relates to an in vitro method for selecting a treatment for a subject who has or is suspected of having an inflammatory disease, wherein said method comprises step a) defined above; and also includes:
    b) selecting an anti-TNFa agent when allele (A) is present in at least
    one of the alleles at the SNP locus rs11466657, preferably when the
    allele (A) is present in both alleles.
    In a preferred embodiment, the subject is ACPA positive. In a more preferred embodiment, said subject is an ACPA positive female.
    An anti-tumor necrosis factor (TNF) agent decreases, blocks, inhibits, abrogates or interferes with TNF activity in vitro and / or in vivo. The terms antiTNFa and TNFa inhibitor are used interchangeably herein. In a preferred embodiment, said anti-TNFa agent is an antibody, preferably a specific anti-TNFa antibody. Methods for obtaining antibodies against a known antigen are part of the state of the art and a person skilled in the art will know how to obtain anti-TNFa antibodies.
    The term "antibody" is used in the broadest sense and includes fully assembled antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), antigen-binding antibody fragments (eg , Fab, Fab ', F (ab'h, Fv, scFv, diabodies), single domain antibodies (sdAb) and recombinant peptides comprising the above, as long as they show the desired biological activity. The term "antibody" is also used refers to a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin.
    Anti-TNFα agents are well known in the state of the art. By way of illustration, anti-TNFa agents include but are not limited to anti-TNFa agents selected from the group consisting of etanercept, adalimimab, infliximab, golimumab, certolizumab, rituximab, abatacept, anakinra, and tocizumab. In a preferred embodiment, said anti-TNFa agent is infliximab. Singh et al., Arthritis & Rheumathology, 2016, 68 (1), 1-16 provides a manual for the treatment of RA from the American College of Rheumatology. In particular, Table 1 provides a listing of treatments for RA that includes anti-TNFα agents.
    In a particular embodiment, said inflammatory disease is an inflammatory rheumatic disease. Preferably, said inflammatory rheumatic disease is selected from the group consisting of arthritis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sji: igren syndrome, juvenile rheumatoid arthritis, and spondyloarthropathy.
    In another particular embodiment, said inflammatory disease is selected from the group consisting of inflammatory bowel disease (for example, Crohn's disease or ulcerative colitis), psoriasis, arthritis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sji: igren syndrome, arthritis. Juvenile rheumatoid disease, spondyloarthropathy, uveitis, and Behcet's disease. In a preferred embodiment, said inflammatory disease is rheumatoid arthritis.
    A method for the prognosis and / or determination of the severity of a subject suffering from an inflammatory disease
    In a further aspect, the invention refers to an in vitro method for determining the severity and / or prognosis of the disease in a subject having an inflammatory disease, said method comprising step a) defined above.
    Likewise, it refers to an in vitro method for obtaining useful data to determine the severity and / or prognosis of the disease in a subject who has or is suspected of having an inflammatory disease, said method comprising step a) defined above.
    For each disease or group of inflammatory diseases, criteria have been established by the clinical community to determine the severity or degree of progression and / or activity of the disease that are well known to a person skilled in the art. As described above, the DAS28 index and the ACRJEULAR criteria are commonly used to determine the degree of severity of RA. For example, patients with severe disease are typically considered those who present one or more, preferably all, of the following characteristics: positivity for rheumatoid factor (RF) and / or antibodies against citrullinated protein antigens (ACPA), presence of erosions and resistance (eg DAS28 ~ 3.2 or intolerance) to at least one disease modifying antirheumatic drug (DMARDs) DMARDs agents are well known in the art and include eg methotrexate, leflunomide, hydroxychloroquine and sulfasalazine. One skilled in the art will understand that a greater severity or severity of the disease is associated with a worse prognosis.
    According to the data of Example 2, a correlation has been found between the severity of the disease and the presence of the (G) allele, in particular the 1473T variant is associated with increased erosion, especially in ACPA positive patients, and a lower response to anti-TNFa agents (Le., infliximab). Thus, in a particular embodiment of the method of the invention, the presence of the (G) allele in at least one of the alleles in the locus of SNP rs 11466657, preferably in both alleles, is indicative of an increased severity of the inflammatory disease .
    Also, the invention provides a method for treating a subject having an inflammatory disease, wherein said method comprises step a) defined above; and also includes:
    b) administering to said subject an anti-TNFa agent when allele (A) is
    present in at least one of the alleles at the SNP locus rs11466657,
    preferably when allele (A) is present in both alleles.
    In a preferred embodiment, the subject is ACPA positive. In a more preferred embodiment, said subject is an ACPA positive female.
    Furthermore, the present invention relates to an anti-TNFa agent for use in a subject who has or is suspected of having an inflammatory disease, wherein said subject has the (A) allele in at least one of the alleles in the locus. SNP rs11466657, preferably wherein said subject is homozygous for allele (A).
    The methods of the present invention may further comprise the determination of
    other biomarkers associated with said inflammatory disease, preferably, said
    biomarkers are selected from the group consisting of anti-protein antibodies
    citrullinated (ACPA), rheumatoid factor (RF) antibodies, anti-Iectin antibodies
    S mannose binding (MBL), erythrocyte sedimentation rate (ESR), C-reactive protein
    (CRP), platelet count, hemoglobin levels and hematocrit. More details
    about such biomarkers are provided for example in Gupta B et al. J Autoimmun.
    2006.27: 125-133, Afzal Net al. Clin Lab. 2011. 57: 895-899, Takizawa Yet al. Ann rheum
    Dis. 2006. 65: 1013-1020, Vossenaar ER et al. Arthritis Res Ther. 2004. 6: R142-R150 and
    10 EP0175270 which are incorporated herein by reference.
    Rheumatoid factor (RF) is the autoantibody that was first identified in arthritis
    rheumatoid. It is defined as an antibody against the Fc portion of IgG. RF and IgG bind
    to form immune complexes that contribute to disease. On the other hand,
    lS Antibodies to citrullinated protein antigens (ACPA) are autoantibodies that are
    They target peptides and proteins that are citrullinated. The ACPAs are present in the
    most patients with rheumatoid arthritis.
    In a preferred embodiment of the methods of the invention, the
    twenty anti-citrullinated protein antibodies (ACPA).
    Optionally, such methods may further comprise the determination of
    clinical parameters. Examples of signs and / or symptoms whose presence / absence may
    determined are: morning stiffness, presence of arthritis in three or more joints,
    2S involvement of the joints of the hands, symmetric arthritis, rheumatoid nodules, and the
    radiographic changes (see Table 1 of Rindfleisch and Muller, and classification criteria
    ACRlEULAR (Ann Rheum Dis 2010; 69: 1580-1588).
    The method of the invention may further comprise storing the results
    30 obtained in data storage device. In one embodiment, said
    Data storage device is a sheet of paper. In one embodiment
    Preferably, said data storage device is a human-readable medium.
    computer. As used herein, "a computer-readable medium" can
    be any device that may include, store, communicate, propagate or transport the
    3S results of the determination of the method of the invention. The medium can be a
    electronic, magnetic, optical, electromagnetic, infrared system (or apparatus or device)
    or semiconductor or a propagation medium.
    The methods of the present invention can be implemented by a computer. Therefore, a further aspect of the invention relates to a computer-implemented method, wherein the method is any of the methods described herein or any combination thereof.
    Thus, any computer program capable of implementing any of the methods of the present invention or used to implement any of these methods
    or any combination thereof, is also part of the present invention. Likewise, any device or apparatus comprising or carrying a computer program capable of carrying out, or for the application of, any of the methods of the present invention or any combination thereof, is also included as part of the present specification. descriptive.
    Finally, the methods of the present invention can be applied with individuals of both sexes, that is, men or women, and at any age. The profile determined by the present invention is predictive and prognostic.
    Kit of the invention and uses thereof
    In a further aspect, the invention provides a kit to determine in a biological sample isolated from a subject the genotype of one or more polymorphisms of TLR10 that give rise to a variant of TLR10 that has lost the ability to inhibit NFKB, where said kit understands:
    - a reagent for determining the genotype of said polymorphisms; -optionally, instructions for the use of said reagent in the determination of the genotype of said polymorphisms in an isolated biological sample.
    In a preferred embodiment, the invention refers to a kit for determining the genotype of the SNP rs11466657 in a biological sample isolated from a subject, comprising:
    - a reagent for genotyping the SNP rs11466657; -optionally, instructions for the use of said reagent in the determination of the genotype of the SNP rs11466657 in an isolated biological sample.
    In a particular embodiment, said reagent is a primer and / or probe specific for SEO ID NO: 1 that amplifies a region comprising the SNP rs11466657. Typically, such a reagent includes a forward primer partially identical, preferably substantially identical, to an SEO ID NO: 1 fragment, AND a probe and / or reverse primer is partially complementary, preferably substantially complementary, to a SEO ID NO fragment: 1.
    Preferably, said primer and / or probe comprises or consists of a sequence selected from the group consisting of SEO ID NO: 3, SEO ID NO: 4 and sequences identical to any of the same by at least 75%.
    The oligonucleotide sequences with an identity of at least 75% mentioned in the present invention preferably have an identity of at least 80%, at least 85%, at least 90%, at least 95%, more preferably 96 %, 97%, 98%, 99% or 100% with the respective reference sequences. Furthermore, these sequences with an identity of at least 75% can have the same number of nucleotides, or have more or fewer nucleotides than the reference sequence.
    In a particular embodiment, said kit comprises reagents to carry out a real-time PCR reaction, which normally contains a DNA polymerase, such as TaO DNA polymerase, a buffer, magnesium, dNTPs, and optionally other agents (for example, agents stabilizers such as gelatin and bovine serum albumin). In addition, real-time PCR reaction mixes also contain reagents for real-time detection and quantification of amplification products as previously described herein.
    Particular embodiments and preferred features of this aspect of the invention have been defined in previous aspects.
    The invention also relates to the use of a kit, as described in the previous aspect, to predict the clinical response of a subject who has an inflammatory disease, to identify a subject who has an inflammatory disease at risk of not responding to a treatment with an anti-TNFa agent, to select a treatment for a subject having an inflammatory disease, or to determine the severity and / or the
    prognosis of an inflammatory disease in a subject, in a method according to the
    above aspects of the invention.
    It is contemplated that any embodiment discussed in this specification may
    S be implemented with respect to any method, kit and use of the invention described in the
    present document. It will be understood that the particular embodiments described in the
    This document is shown by way of illustration and not as limitations of the
    invention. The main features of this invention can be employed in various
    embodiments without departing from the scope of the invention. The experts in the art
    10 recognize, or be able to determine, using only routine experimentation,
    numerous equivalents to the specific procedures described herein
    document. Such equivalents are considered to be within the scope of this invention.
    and are covered by the claims.
    lS All publications and patent applications mentioned in the specification are
    indicative of the level of knowledge of those skilled in the art to which this
    invention. All publications and patent applications are incorporated herein
    document for reference to the same extent as if it were indicated that each publication or
    Individual patent application is specifically and individually incorporated by reference.
    twenty
    The use of the word "one" or "one" can mean "one", but it also agrees with the
    meaning of "one or more", "at least one" and "one or more than one". The use of the term "other"
    it can also refer to one or more. The use of the term "or" in the claims is used
    to mean "and / or" unless explicitly stated that it refers only to alternatives
    2S or that the alternatives are mutually exclusive.
    As used in this specification and claim (s), the
    words "comprising" (and any form of understanding, such as "comprehend" and
    "understands"), "that has" (and any way of having, such as "have" and "has"), "that
    30 includes "(and any form of include, such as" includes "and" include ") or" containing "(and
    any form of contain, such as "contains" and "contain") are inclusive or of
    open meaning and do not exclude additional, uncited elements or method steps. The
    The term "comprises" also encompasses and expressly discloses the terms "consists of
    in "and" consists essentially of. "As used herein, the term
    3S "consisting essentially of" limits the scope of a claim to materials or
    specified stages and those that do not materially affect the characteristic (s)
    basic (s) and novel (s) of the claimed invention. As used herein, the term "consisting of" excludes any element, step or component not specified in the claim except, for example, impurities normally associated with the element or limitation.
    The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items that precede the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, combinations containing repetitions of one or more elements or terms are expressly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there are normally no limits on the number of elements or terms in any combination, unless otherwise apparent from the context.
    As used herein, approximation words such as, without limitation, "approximately", "about", "approximately" refer to a condition which when so modified is understood to not necessarily be absolute or perfect but could be considered close enough to those of ordinary skill in the art to warrant designation that the condition is present. The degree to which the description can vary will depend on the extent to which a change can be instituted and on whether one of ordinary skill in the art can still recognize that the modified feature still has the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by an approximation word such as "approximately" may vary from the value set at ± 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%. Preferably the term "approximately" means exactly the indicated value (± 0%).
    The following examples serve to illustrate the present invention and should not be construed as limiting the scope thereof.
    EXAMPLES Example 1.-Material and methods 1.1 Samples
    Two cohorts of patients with RA have been included in this study, a first cohort of
    5,453 unselected patients with follow-up at the Marqués de Valdecilla University Hospital (Santander, Spain) and the La Paz University Hospital (Madrid, Spain), and a second of 1201 patients recruited by the Consortium of Inflammatory Diseases Mediated by the Immune System ( Spain) [Avila-Pedretti G, et al. , PLoS One 2015, 10 (4): e0122088]. Clinical information, including demographic data, characteristics of
    10 disease and treatment are summarized in Table 1. All patients were diagnosed according to the classification criteria of the American College of Rheumatology [Arnett FC, et al., Arthritis Rheum 1988, 31 (3): 315-324]. As a control population, 1702 healthy individuals of the same genetic background (ie a southern European population) were also genotyped [Julia A, et al., Gut 2013, 62 (10): 1440-1445]. All control individuals are
    15 had been screened for the presence of an autoimmune disease or a family history of autoimmune disorders, and were excluded if they were positive.
    Table 1. Main characteristics of two cohorts of patients with RA.
    Cohort I Cohort"
    (n = 453) (n = 1,201)
    Women (%) 73.176.8
    Average age (years) 65.6 ± 14.5a46.5 ± 14.5a
    Duration of follow-up (months) 123.8 ± 91, Sa171.6 ± 123.6a
    Extra-articular manifestations (%) 22.2
    Presence of joint damage (%) 100
    Positive for FR (%) 64.874.6
    Positive for ACPA (%) 62.274.1
    Number of previous treatments with DMARDs 2.2 ± 1.5a1.7 ± 1.5a
    Number of previous biological therapies 1.8 ± 1.2a0.6 ± O, 9a
    RF, rheumatoid factor; DMARDs, disease modifying antirheumatic drugs;ACPA, anti-citrullinated proteins antibodies.aMedium ± SD.
    20 1.2 Cell lines
    K562 (ATCC, Middlesex, UK) and U937 (ATCC Middlesex, UK) cells were maintained in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Lonza, Verviers, Belgium). The medium was replaced every 2-3 days.
    1.3 Sequencing analysis of the TLR10 gene Coding exons and flanking regions of the TLR10 gene were sequenced in 66 selected RA patients with severe disease. Patients with severe disease were considered those with positivity for rheumatoid factor (RF) and / or antibodies against citrullinated protein antigens (ACPA), presence of erosions and resistance to at least one disease-modifying antirheumatic drug (OMAROs) and 30 healthy controls by next generation sequencing (NGS). The RF was measured by nephelometry (BNII, Siemens Healthcare) and the cut-off value was 22 IU / ml. ACPAs were measured using a commercial ELISA test (CCP2, Eurodiagnostica) and were considered positive above 50 U / ml. Those who presented a lack of response in the OAS28 index (OAS28 ~ 3.2) or intolerance were classified as resistant to OMAROS.
    The AON libraries were processed for hybrid enrichment using a SeqCap EZ design (Roche Nimblegen, Basel, Switzerland) containing the coding sequences for TLR10. Next, double barcode libraries were sequenced using a MiSeq NGS platform (11Iumina, Madison, WI). For Sanger sequencing, DNA was collected from whole blood using the DNA kit OIAamp (Oiagen, Hamburg, Germany) and amplified with primers for human TLR10 (SEO ID NO: 3): 5'-CATGGCCAGAAACTGTGGTC-3 ' and (SEO ID NO: 4): 5'-ACCATCCAACCATCATGACC-3 '. Sequence analysis of the amplified fragment was carried out using a genetic analyzer (Applied Biosystems, Foster City, CA).
    1.4 Single SNP analysis The 1473T variant of TLR10 (rs11466657; http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=11466657) was analyzed in an independent cohort of 1201 patients with AR and 1493 healthy controls using the TaqMan® genotyping platform (Assay Id C_25643390_30, Life Technologies, Carlsbad, CA). All end-point fluorescence readings and PCR reaction were performed using an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). The conditions of the PCR reaction were as follows: 50 o C for 2 min and 95 o C for 10 min, followed by 40 cycles of 92 o C for 15 s and 60 o C for 1 min. The genotyping error was estimated by genotyping the 20% of the samples in duplicate (error <1%) [Lopez-Lasanta M, Julia A, Maymo J, Fernandez-Gutierrez B, Urena-Garnica 1, Blanco FJ, Canete JD, Alperi-Lopez M, Olive A, Corominas H et al: Variation at Interleukin-6 Receptor Gene Is Associated to Joint Damage in Rheumatoid Arthritis. Arthritis Res Ther 2015, 17: 242].
    1.5 Transfections to perform reporter gene assays TLR10 cDNA (NM_030956; http://www.ncbLnlm.nih.gov/nuccore/NM_030956.3; SEO ID NO: 1) was cloned into pCMV6 (Origene, Rockville, MD) . The 1473T variant was generated by site-directed mutagenesis using the Ouick Change Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with the following primers: SEO ID NO: 5 5'-GGCCTTACGAGAACTAAATACTGCATTTAATTTTCTAACTGATC-3 'and SEO ID NO: 6 5 '-ATCAGTTAGAAAATTAAATGCAGTATTTAGTTCTCGTAAGGCC-3'. The modified graft was sequenced to verify the mutation. K562 and U937 cells were cotransfected with 2 f.lg of wild-type or mutant TLR10 constructs, 1 f.lg of reporter plasmid pBVI-Luc, containing six tandem repeats of the NFKB recognition sites within the promoter region bound to the luciferase gene [Inohara N, et al., J Biol Chem 2001, 276 (4): 2551-2554] and 0.2 f.lg of pRSV- ~ -gal using Lipofectamine 2000 (SigmaAldrich, St Louis, MO).
    1.6 Reporter gene assays After 24 h of transfection, K562 and U937 cells were incubated with 10 ng / ml and 20 ng / ml tumor necrosis factor-alpha (TNFa) in the presence or absence of 200 f.lg / ml of infliximab and after 24 h the cell extracts were prepared and analyzed for relative luciferase activity using a Dual-Light reporter gene assay system (Applied Biosystems). Results were normalized to determine transfection efficiency with values obtained with pRSV- ~ -gal.
    1.7 NFKB target gene expression analysis To assess the expression of individual genes, complementary DNA was generated and amplified using primers for TNFa. human SEO ID NO: 7 (5'-CAATGGCGTGGAGCTGAGAG-3 'and SEO ID NO: 8 5'-GATGGAGTTGAAGGTAGTTTCGTG-3'). Quantitative real-time PCR was performed on a 7000 Sequence Detection System (Applied Biosystems). The ratio of the abundance of differentiation markers to that of-actin transcripts as 2n was calculated, where n is the threshold cycle value of 3-actin minus the threshold cycle value of the corresponding mRNA and was normalized by the value of the sample with the lowest expression level of these genes. The specificity of the desired PCR products was determined with melting curve analysis.
    5'-GGCTGATGGTGTGGGTGAGG-3 '), CCL2SEOIDNO:9
    (5'-CTCGCTCAGCCAGATGCAA T-3 ' andSEOIDNO:10
    5 '-GTCTTCGGAGTTTGGGTTTGC-3'), TRAILSEOIDNO:eleven
    (5 '-GAGCTGAAGCAGATGCAGGAC-3' andSEOIDNO:12
    5 '-TGACGGAGTTGCCACTTGACT-3'), and3-actinSEOIDNO:13
    (5 '-GCTGCCTCAACACCTCAAC-3' andSEOIDNO:14
    Real-time PCR was carried out using the Applied Biosystems 7000 Real-Time PCR system (Bio-Rad). Each PCR reaction contained 12.5 / 11 SYBR GREEN PCR Master Mix (Applied Byosystems), 400 nM of each primer, and diluted 1/11 cONA (100 ng) in a total volume of 25/11. The reactions were carried out in a 96-well reaction plate under the following conditions: 2 min at 60 oC, 10 min at 95 oC, followed by 40 cycles
    15 s at 95 oC, 3 s and 1 min at 60 oC.
    The delta Ct (~ Ct) method was used for data analysis of the PCR matrix. The normalized value (~ Ct) for each gene of interest (GOl) was calculated by subtracting the mean Ct of the two constitutive expression genes (Uhousekeeping genes ") from the Ct of each GOL. Then, the double delta Ct (MCt) for each GOl was calculated by subtracting the mean ~ Ct of each GOl in the control group from the ACt of each GOl. The factor of change (Ufold-change ") of each GOl compared to the control group was calculated as ZlillCI of the log1Q2-tc ,.
    1.8 Statistical analysis All statistical analyzes were performed using the SPSS 20 program (IBM, Armonk, New York) and the R statistical software (version 3.2.0). Differences in quantitative variables between groups of patients were compared with the Mann-Whitney U test, and the chi-square statistic was used for categorical variables. Statistical significance between groups was performed in in vitro analysis using the two-sided Student's t test to
    independent data. The level of significance was established at p <0.05. Genetic association analyzes were performed using linear and logistic regression models. As previously described, the covariates in these genetic analyzes included the years of disease evolution and the baseline disease activity score (DAS28) for 5 the association of the rs11466657 genotype with the level of joint damage [Lopez-Lasanta M , Julia
    A. Maymo J, Fernandez-Gutierrez B, Urena-Garnica 1, Blanco FJ, Canete JO, Alperi-Lopez M, Olive A, Corominas H et al: Variation at Interleukin-6 Receptor Gene Is Associated to Joint Damage in Rheumatoid Arthritis. Arthritis Res Ther 2015, 17: 242], and the change in DAS28 at 12 weeks [Acosta-Colman 1, Palau N, Tornero J, Fernandez-Nebro A, Blanco
    10 F, Gonzalez-Alvaro 1, Canete JO, Maymo J, Ballina J, Fernandez-Gutierrez B et al: Gwas Replication Study Confirms the Association of Pde3a-SIc01 c1 with Anti-Tnf Therapy Response in Rheumatoid Arthritis. Pharmacogenomics 2013, 14 (7): 727-734], respectively. 15 Example 2.- The 1473T variant is associated with the severity of the disease and response to treatment in patients with RA
    The role of TLR10 is controversial and its association with RA has hardly been studied. In order to evaluate whether the TLR10 variants contribute to modifying the development of the
    20 disease in RA patients, the coding exons of the TLR10 gene were sequenced in 66 selected RA patients and 30 healthy controls. After filtering for bases that had at least 30X sequence coverage, sixteen variants were identified (see Table 2 below).
    Table 2. Sixteen allelic variants of the TLR10 gene found by NGS in control and RA populations. REF, reference allele; AL T, alternative allele; MAF, minor allele frequency; (+) harmful change; (-) no harmful change; nr, no registration.
    SNP ID REFALTMAFvalue ofChangeClassPredictedPrediction
    reference PFrom aafunctionalonthrough
    Mediant SNPs30
    eSIFT
    rs10776482 TOG0.290.23060809SILENTnrnr
    rs4129008 CT0.010.5652R799QPASSWORD
    Uncle
    SNP ID REFALTMAFvalue ofChangeClassPredictedPrediction
    reference PFrom aafunctionalonthrough
    Mediant SNPs3D
    eSIFT
    rs4129009 TC0.2710.18891775VPASSWORD
    TIDO
    rs10776483 TOG0.3020.3296H724SILENTnrnr
    rs11466658 GTO0.0260.1599R525WPASSWORD
    TIDO
    rs11466657 TOG0.0890.043271473TPASSWORD++
    TIDO
    rs11096955 TG0.4220.072961369LPASSWORD
    TIDO
    rs11096956 CTO0.2920.3918P344SILENTnrnr
    rs11466653 TOG0.0260.5824M326TPASSWORDnr
    TIDO
    rs11466652 TC0.130.1403K303SILENTnrnr
    rs11466651 CT0.0260.5824V2981PASSWORD
    TIDO
    rs11096957 TG0.4380.015N241HPASSWORD
    TIDO
    rs11466650 TOG0.00050.137L 167PPASSWORD++
    TIDO
    rs11466649 CTO0.0260.5824A163SPASSWORD
    TIDO
    rs10856837 CT0.0260.5824T37SILENTnrnr
    rs10856838 TOT0.1460.5813113SILENTnrnr
    Then, a selection was made based on the allele frequencies in both patient and control populations and the predicted functional impact on the protein using the SIFT programs (Nq PC and Henikoff S, Nucleic Acids Res. 2003, 31 (13): 38125 4) AND SNPs3D (Yue et al., BMC Bioinformatics. 2006, 7: 166). Most of the nonsense changes found were located in the LRR domains (Figure 1A). As a result of the selection procedure, variant 1473T (Table 2) was identified for further association studies. First, a population of 453 patients was studied and it was found that the genotype frequencies (86.6% AA, 11.9% AG, 1.5% GG) were not significantly different from those of a population of 209 controls (84.5% AA, 14.3% AG, 1.2% GG). Afterwards, several clinical findings were analyzed in the
    patient population associated with the severity of the disease, including the presence of typical AR autoantibodies (RF and ACPA), extra-articular manifestations and the need for specific biological treatments and surgery. All of them indicated a trend correlation between severity and the GG genotype that was not observed with the AA genotype (Figure 2A). To reinforce this observation, a larger cohort of 1201 RA patients and 1493 healthy controls were studied. As previously observed, the genotype distribution was found to be similar in both patient and control populations (Figure 1B), which is consistent with the genotypes described for the European population in the HapMap database (Nature. 2005, 437 (7063): 1299-320; Figure 1C). It is interesting, as shown in Figure 2B, that the 1473T variant is highly associated with erosive disease in RA positive for ACPA (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to treatment with infliximab measured by the change in the DAS28 index (p = 0.012 in the total cohort) as well as by the EULAR response criteria (p = 0.025 in the total cohort), indicating that this variant selects a group of patients with a more serious illness. Example 3.-The 1473T variant modifies the regulatory capacity of TLR10 NFKB
    Based on a 3D structure model using the Jmol viewer (Nepomnyachiy S1, et al., Structure. 2015 May 5; 23 (5): 941-8; Figure 3A), position 473 is within a beta chain in the LRR18 domain and it is occupied by an isoleucine, a highly hydrophobic amino acid, hidden within the core of the protein. In the TLR10 variant, isoleucine is substituted for threonine, a polar amino acid that can participate in hydrogen bonds and is usually located on the surface of the protein. The LRR domains provide an outstanding framework for achieving various protein interactions. Therefore, this structural change decreases hydrophobic contacts and can alter functionally relevant protein-protein interactions. Members of the TLR family are known to be regulators of NFKB activity, a key pathway in inflammation. To experimentally demonstrate the functional consequences of the 1473T variant on NFKB activation, the codon alteration nucleotide was introduced into a construct containing TLR10 cDNA by site-directed mutagenesis and the ability of this variant to modify transcriptional activity was studied. by NFKB. As shown in Figures 3B and 3C, wild-type TLR10 down-regulates the transcriptional activity of NFKB under both non-stimulated and TNFα-stimulated conditions in the K562 and U937 hematopoietic cell lines. However, the 1473T substitution blocks the inhibitory capacity of TLR1 O. In order to translate the reduced response to
    infliximab in patients to the in vitro model, cells were stimulated with TNFa in the presence or absence of infliximab. Consequently, cells expressing the 1473T variant had a higher level of NFKB activity that remained after infliximab treatment compared to cells expressing the wild-type variant (3D Figures 5 and 3E). This result was then confirmed by analyzing the expression of NFKB target genes. The expression levels of CCL2, TRAIL and TNFa were down-regulated in the presence of wild-type TLR10 after stimulation of the NFKB pathway with TNFa. In line with previous results, NFKB target genes were downregulated when cells were transfected with the variant-containing construct and
    10 cultured with TNFa (Figure 4A-E). The expression of CCL2 also confirmed that the variant generates a lower response to infliximab (Figure 4F).
    In conclusion, the 1473T variant leads to a reduced ability of TLR10 to inhibit NFKB activation in response to inflammatory stimuli. This effect could be explained
    15 because the amino acid change decreases the hydrophobicity of an LRR domain, which can alter the interaction with TLR proteins necessary for TLR10 signaling [Govindaraj RG, et al. , PLoS One 2010, 5 (9): e12713].
    权利要求:
    Claims (22)
    [1]
    1. In vitro method for predicting the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFa agent, wherein said method comprises:
    a) determining in a biological sample isolated from said subject the genotype of the single nucleotide polymorphism (SNP) rs11466657;
    where the presence of the allele (G) in at least one of the alleles in the locus of the SNPrs11466657 indicates an increased risk of not responding to the anti-TNFa agent; and
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [2]
    2. In vitro method to identify a subject, who has or is suspected of having an inflammatory disease, who presents a higher risk of not responding to treatment with an anti-TNFa agent, wherein said method comprises step a) according to claim 1;
    where the presence of the (G) allele in at least one of the alleles at the SNP locus rs11466657 indicates an increased risk of not responding to the anti-TNFa agent; and
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [3]
    3. The method according to any one of claims 1 or 2, wherein the presence of the (G) allele in both alleles indicates an increased risk of not responding to the anti-TNF agent.
    [4]
    Four. In vitro method for selecting a treatment for a subject, who has or is suspected of having an inflammatory disease, wherein said method comprises step a) according to claim 1; And it also includes:
    b) selecting an anti-TNFa agent when allele (A) is present in both alleles in the locus of SNP rs11466657;
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [5]
    5. In vitro method for determining the severity and / or prognosis of the disease in a subject having an inflammatory disease, wherein said method comprises step a) according to claim 1;
    where the presence of the allele (G) in at least one of the alleles in the locus of SNP rs11466657 is indicative of severe disease; and
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [6]
    6. The method according to claim 5 wherein the presence of allele (G) in both alleles is indicative of severe disease.
    [7]
    7. In vitro method to obtain useful data to determine the clinical response of a subject, who has or is suspected of having an inflammatory disease, to an anti-TNFa agent, the severity and / or prognosis of said disease where said method comprises the stage a) according to claim 1;
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [8]
    8. The method according to any of claims 1 to 7, wherein the subject is positive for anti-citrullinated peptide antibodies (ACPA).
    [9]
    9. The method according to any of claims 1 to 8, wherein the biological sample is a blood sample.
    [10]
    10. The method according to any one of claims 1 to 9, wherein the genotype of SNP rs11466657 is determined by direct sequencing or quantitative PCR.
    [11 ]
    eleven . The method according to any one of claims 1 to 10, wherein said method further comprises storing the results of the method on a data storage device.
    [12]
    12. Computer-implemented method, wherein the method is as defined in any one of claims 1 to 11.
    [13]
    13. The method according to any of claims 1 to 12, wherein said anti-TNFa agent. is selected from the group consisting of etanercept, adalimimab, infliximab, golimumab, certolizumab, rituximab, abatacept, anakinra and tocizumab ..
    [14]
    14. The method according to any of claims 1 to 12, wherein said anti-TNFa agent. it is infliximab.
    [15]
    fifteen. Use of infliximab in the manufacture of a drug for the treatment of an inflammatory disease in a subject, who has or is suspected of having said inflammatory disease, in which said subject has the (A) allele in both alleles in the rs1 SNP locus 1466657,
    wherein said inflammatory disease is selected from the group consisting of inflammatory bowel disease, psoriasis, psoriasic arthritis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, juvenile rheumatoid arthritis, spondyloarthropathy, uveitis, and Behcet's disease.
    [16]
    16. The method according to any one of claims 1 to 140 the use of infliximab according to claim 15, wherein said inflammatory disease is rheumatoid arthritis.
    [17]
    17. The method according to any of claims 1 to 14 or 16.0 the use of infliximab according to any of claims 15 to 16, wherein said subject is a human subject.
    [18]
    18. Kit to determine in a biological sample isolated from a subject the genotype of
    SNP rs11466657, comprising: - a reagent for determining the genotype of SNP rs11466657; -optionally, instructions for the use of said reagent in the
    determination of the genotype of the SNP rs11466657 in an isolated biological sample.
    [19]
    19. The kit according to claim 18, wherein said reagent is a primer and / or probe specific for SEO ID NO: 1 that amplifies a region comprising the SNP rs11466657.
    [20]
    twenty. The kit according to claim 19 wherein said specific primer and / or probe is selected from the group consisting of SEO ID NO: 3, SEO ID NO: 4, and sequences identical to any of them by at least 75%.
    [21 ]
    twenty-one . Use of a kit according to any of claims 18 to 20 to predict the clinical response of a subject having an inflammatory disease, to identify a subject having an inflammatory disease at risk of not responding to treatment with an anti-inflammatory agent. TNFa., To select a treatment for a subject having an inflammatory disease, and / or to determine the severity and / or prognosis of an inflammatory disease in a subject.
    TO)
    B) Population of this studyAAGenotype AGGG
    HC RA 0.880 0.8810.116 0.1170.004 0.002
    C) HapMap populationAAGenotype AGGG
    CEU HCB JPT YRI TSI 0.92 1.00 1.00 1.00 0.830.08 0.00 0.00 0.00 0.130.00 0.00 0.00 0.00 0.04
    FIG. 1
    TO)
    -1 -0.5 O 0.5 1.5 2 2.5 3 Bela
    FIG. 2
    1200, ...
    C)
    K562 U937
    *
    ~ 800 ~
    ~
    .3
    ." it
    : 2 400
    ~
    t;
    EC
    e wt mut TLR10
    D)
    AND)K562
    + + + + TNFa + + Inniximab
    FIG. 3
    + + Inftiximab
    A) B) C)
    [2]
    2.0 ~ U937. ~ 2.0 "" U937 ~ ~
    - '
    > ~ fW ... 1-.
    ~ "
    j !: 1.0 ~ .. 1.0 ..... r1: L ... E
    AND
    ~ 0.5 ~ 0.5
    elel
    TNFa ~ = -.... ±. = -.... ±. TNFa e wt mut e wt mut D) TLR10 TLR10 F) K562 •
    nl7
    ti 1.0 u ..
    ...
    E 0.5
    z
    '" the
    ~~ = -.... ±. ~ IFX
    - + ~ ~ TNFa
    wt mut wt mute wt mut TLR10 TLR10TLR10
    - + - + ~ TNFa
    e wt mut TLR10
    FIG.4
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    ES2656587B1|2018-12-11|Method to predict the clinical response to a treatment with anti-inflammatory agents
    同族专利:
    公开号 | 公开日
    WO2018020060A1|2018-02-01|
    ES2656587B1|2018-12-11|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20090297563A1|2004-10-27|2009-12-03|Anders Borglum|Diagnosis And Treatment of Immune-Related Diseases|
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