ES2731634T3 - IL22RA2 gene for fibrosis sensitivity and its uses - Google Patents

Abstract

An in vitro method for determining a risk of developing liver fibrosis or cirrhosis in a subject infected with hepatitis viruses or parasites, the method comprising detecting the presence of a single nucleotide polymorphism (SNP) at the IL22RA2 gene locus in a sample biological of said subject, wherein said SNP is selected from the group consisting of rs6570136, rs7774663, rs11154915 and rs2064501, where the presence of a G allele with respect to SNP rs6570136, a T allele with respect to SNP rs7774663, a T allele with respect to SNP rs11154915 and / or a CC genotype with respect to SNP rs2064501 is indicative of developing liver fibrosis or cirrhosis.

Description

DESCRIPTION

IL22RA2 gene for fibrosis sensitivity and its uses

Field of the Invention

The present invention relates generally to the fields of genetics and medicine. The present invention describes in particular the identification of a human sensitivity gene, which can be used for the diagnosis or prognosis of an abnormal deposit of extracellular matrix proteins (ECMP) in tissues, which could cause fibrosis, or for the detection of the predisposition to said abnormal deposit of ECMP or fibrosis, which occurs in liver diseases, in cirrhosis, skin keloids, obesity and any fibrotic disease and also in other tissues such as the heart, vessels or brain. The invention more specifically describes some alleles of the IL22RA2 gene on chromosome 6 related to fibrosis sensitivity and that represent new targets for the selection of therapeutically active drugs. The present invention relates more specifically to specific mutations in the IL22RA2 gene. The expression products of the IL22RA2 gene, as well as diagnostic tools and equipment based on these mutations, are also described.

Background of the invention

The accumulation of extracellular matrix proteins in tissue can have detrimental effects. Abnormal ECMP deposition in the tissue can cause tissue fibrosis.

Fibrosis is an overgrowth of fibrous connective tissue in an organ, any part or tissue thereof, for example in a liver, any part or tissue thereof, especially in response to an injury.

Abnormal fibrosis occurs in chronic liver inflammations of various etiologies, such as hepatitis virus and schistosome infections. It was previously shown that some subjects infected with schistosomes are slow fibrous, while others are fast fibrous and that this depends in part on a major gene located in Chr 6q22-q23 (Dessein et al., 1999; Mohamed-Ali et al., 1999). International patent application WO2010 / 094740 identifies CTGF (CCN2) as a fibrosis sensitivity gene in this region. Schistosomiasis is caused by helminths that develop in the vascular system of their hosts and lay eggs that, in some cases, are transferred to the liver, where they cause inflammation in the periportal space. Since worms live for years in their human host, chronic inflammation of the liver associated with the destruction of many tissues is common in infected subjects. Tissue repair requires ECMP deposition in damaged tissues that are then turned over and replaced by normal hepatocytes. In some patients, ECMP accumulate in the periportal space forming deposits of fibrosis that reduce blood flow causing varicose veins, ascites. After months or years of chronic or repeated lesions, the fibrosis becomes permanent and irreversible. Subjects die of the consequences of fibrosis.

In the countries of the South, it is estimated that between 5 and 10% of the 350 million infected subjects may develop severe hepatic fibrosis. There is no good marker that allows to predict and follow the evolution of liver fibrosis in subjects infected with schistosome.

The diagnosis of liver fibrosis is mainly based on liver biopsy, elastometry and ultrasonic analysis.

Biopsies are obtained percutaneously, transjugularly, with a thin needle guided radiographically or laparoscopically, depending on the clinical setting. Histopathological examination allows the doctor to assess the severity of necroinflammation and determine the degree of fibrosis. The Metavir scoring system attributes a score to the stages of fibrosis on a scale of 1 to 4 as follows: F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = portal fibrosis and few septa, F3 = numerous septa without cirrhosis, F4 = cirrhosis (Bedossa et al., 1996). Liver biopsy is an invasive and expensive procedure, and it samples only a small portion of the liver. Therefore, it cannot provide an overall evaluation of liver fibrosis and is subject to a variation in sampling and an inter and intraobserver error. In addition, liver biopsy is associated with a significant morbidity of 3% and a mortality rate of 0.03%. Possible complications include local hematoma, infection and pain related to the biopsy.

Non-invasive tests (ie, serological markers, elastometry, ultrasound analysis) are also used but are not yet ready for routine clinical use.

Groups of blood markers have been analyzed mainly in patients with chronic hepatitis C or cirrhosis due to viral hepatitis C. These studies showed that serum markers can control or rule out fibrosis in approximately 35% of patients (Sebastiani et al., 2006). However, when observing the patients individually, these markers could not reliably differentiate between the different stages of fibrosis. A more recent study incorporated three groups of serum markers to design an algorithmic approach that improves diagnostic accuracy (Parkes et al., 2006). The three groups evaluated were APRI (ratio of aspartate transaminase to platelets), the Forns index (platelets, gammaglutamyltranspeptidase, cholesterol) and Fibrotest (GGT, haptoglobin, bilirubin, apolipoprotein A, alpha-2-macroglobulin). An algorithm consisting of APRI followed by Fibrotest increased the diagnostic accuracy of fibrosis to more than 90%. This group estimated that the use of this algorithm could obviate the need for up to 50% of liver biopsies. However, the individual stages of fibrosis are not distinguishable using this algorithm. The limitation of these serum markers is the possibility of false positives when there is a very active liver inflammation.

Fibroscan is another method for staging liver fibrosis, which is based on elastography, which provides a rapid measurement of the average stiffness of liver tissue (Ziol et al., 2005). A probe is used to transmit a low frequency and amplitude vibration in the liver. This vibration wave triggers an elastic shear wave, whose velocity through the liver is directly proportional to the tissue stiffness measured in kilopascals (kPa). The sensitivity of the Fibroscan technique ranged between 79 and 95%, and the specificity between 78 and 95%, compared to liver biopsy. However, the limitations of this technique are related to the attenuation of elastic waves in liquid or adipose tissue, which would impair the evaluation of fibrosis in patients. In addition, Fibroscan is a very expensive instrument.

The current welfare standard (SOC) for the eradication of HCV from the liver consists of treatment with pegylated interferon type I (PegIFN) and synthetic nucleoside ribavirin (RBV) (Fried MW et al .; N. Engl. J. Med. 2002; 347 (13): 975-82; EASL Clinical Practice Guideline: Management of hepatitis C virus infection, J. Hepatol. 2011; 55: 245-264). However, this conventional treatment has limited and unpredictable efficacy, an extensive toxicity profile that frequently leads to treatment discontinuation and is very expensive. Less than half of individuals infected with HCV chronically of genotype 1 and 4 respond to long-term treatment (48 weeks) of conventional treatment (PegIFN / RBV) (Testino G. et al .; Hepatogastroenterology 2011; 58 ( 106): 536-8).

Therefore, there is a need for a method to select patients who have better opportunities to respond to a treatment to optimize treatment, avoid side effects for those who do not respond to treatment and reduce treatment costs.

Overall, there is still a need for an effective method to predict the evolution of fibrosis and the effectiveness of treatment.

Compendium of the invention

The object of the present invention is to provide a new genetic approach to the prognosis and treatment of fibrosis. The present invention now describes the identification of another locus of the human fibrosis sensitivity gene, the IL22RA2 gene locus, which can be used to detect predisposition to, diagnosis and prognosis of an abnormal ECMP deposit, especially fibrosis, especially liver fibrosis. , as well as for the selection of therapeutically active drugs. The invention is, in particular, in a method comprising detecting in a sample of the subject the presence of an alteration in the locus of the IL22RA2 gene, the presence of said alteration being indicative of the presence or predisposition to an abnormal ECMP deposit or fibrosis.

A specific object of this invention is in an in vitro method of detecting the predisposition to or diagnosis and / or prognosis of an abnormal ECMP deposition or fibrosis that occurs in a subject, the method comprising detecting the presence of an alteration in the IL22RA2 gene or polypeptide in a sample of the subject, the presence of said alteration being indicative of the presence of an abnormal ECMP deposit or fibrosis or the predisposition to an abnormal ECMP deposit or fibrosis. A specific object of this invention resides in a method for the evaluation (prediction) of the evolution of an abnormal deposit of ECMP or fibrosis.

In a preferred embodiment, said alteration is located within 500 kb, preferably 100 kb, preferably 20 kb, upstream of the start codon of the IL22RA2 gene and within 500 kb, preferably 100 kb, preferably 20 kb, downstream of 3 ' UTR of the IL22RA2 gene.

Preferably, the alteration is found in the surrounding sequences of the 10 kb region, upstream of the start codon of the IL22RA2 gene and the 10 kb region, downstream of the untranslated region (3'UTR).

In another preferred embodiment, said alteration is a mutation, an insertion or a deletion of one or more bases. In a more preferred embodiment, said alteration is one or more polymorphisms of a single nucleotide SNP or a haplotype of SNP related to fibrosis. Preferably, said single nucleotide polymorphisms are SNPs flanking the IL22RA2 gene, which are allelic variants close to the IL22RA2 gene.

The method of the invention allows the detection and prognosis of fibrosis that appears in a human fibrotic disease selected from liver diseases, fibrosis, cirrhosis, skin keloid, hypertrophic scars and obesity, alcoholism or drug hepatotoxicity. Especially, hepatic fibrosis can be caused by infection by liver virus A, liver virus B, liver virus C (HCV), Schistosoma japonicum ( S. japonicum) or Schistosoma mansoni ( S. mansoni).

In a specific embodiment, the method comprises genotyping SNP in the locus of the IL22RA2 gene in a biological sample of a subject, preferably infected with a hepatitis virus or parasite, where the presence of the GG genotype in SNP rs6570136, TT in SNP rs7774663, TT, CT in SNP rs11154915 and / or CC in SNP rs2064501, is indicative of a risk of developing an abnormal deposit of ECMP as a fibrosis or of the development of an abnormal deposit of ECMP, such as fibrosis, or of a disease prognosis of fibrosis in the subject. Fibrosis is more specifically liver fibrosis.

Alternatively, the method may comprise genotyping of any SNP in the coupling imbalance (DA) with those mentioned herein.

Preferably, the alteration in the locus of the IL22RA2 gene is determined by performing a selective hydration test, a sequencing test, a microsequencing test and / or a specific amplification test for alleles.

In another aspect of the invention, said alteration in the IL22RA2 gene is determined by digestion with restriction enzymes, the detection of at least one of said SNPs being an indication of fibrosis.

A method for selecting a therapeutic compound for a subject having 0 is predisposed to develop an abnormal ECMP deposition such as fibrosis is described herein, said method comprising contacting a compound under study with a IL22RA2 polypeptide or gene or one of its fragments and determine the ability of said compound under study to increase or reduce the biological activity or function of a path related to the IL22RA2 gene.

An in vitro method is also described herein to determine the probability that a patient affected with a viral infection responds to a treatment with an antiviral agent and / or an interferon, a method comprising determining the alteration in the locus of the IL22RA2 gene or in the protein expression of IL22RA2 or in the activity in a biological sample of the patient.

The method described herein comprises the genotyping of SNPs in the locus of the IL22RA2 gene in a biological sample of a subject, wherein the presence of a TT genotype with respect to SNP rs11154915, an AG or GG genotype with respect to SNP rs6570136, a CT genotype with respect to SNP rs2064501, and / or an AA genotype with respect to SNP rs1543509, is in favor of a patient's positive response to treatment. Alternatively, the method may comprise genotyping of any SNP in the coupling imbalance (DA) with those mentioned herein.

In a particular aspect, the treatment comprises an antiviral agent, optionally with an interferon.

Preferably said antiviral agent is a viral replication inhibitor, such as ribavirin.

Legends of the figures

Figures 1A and 1B show the contents of IL-22 and IL-17 in PBMC cultures of endemic subjects of S. japonicum. Data are obtained in 144 hours of rest and stimulated cultures with eggs of 19 references and 70 endemic subjects. Figure 1C shows FACS analysis of IL22 + cells from the blood of endemic subjects. The data is from a representative experiment of every 20.

Figure 2A shows that the contents of IL-22 in PBMC cultures vary with the number of treatments for schistosomiasis in the last ten years. The study subjects were over 30 years old and under 65 years old and had no active HBV infections (HBS Ag-)

Treatment against schistosomiasis: subjects have taken Praziquantel for the past ten years after the following regimens: never, 1 to 4 times, 5 to 10 times,> 10 times.

References are subjects who have not been exposed to S. japonicum and have never been treated with Praziquantel. Stars: Crops stimulated with S. japonicum eggs; White circles: Crop at rest: Number of subjects by groups: references (19), Treatments: no treatment (9), 1-4 (30), 5-10 (23), <10 (8) Figure 2B shows that IL-22 contents in PBMC cultures vary with the degree of liver fibrosis.

The study subjects were over 30 years old and under 65 years old and had no active HBV infections (HBS Ag-). References were subjects who had not been exposed to S. japonicum; Stars: Crops stimulated with S. japonicum eggs ; White circles: resting crops.

The degree of fibrosis was evaluated as described in Methods and they are mostly degrees of central fibrosis, only peripheral fibrosis was taken into account to divide subjects with mild central fibrosis into a group (CLL) without peripheral or mild fibrosis and a group (CLL, ^g N ^m ) with mild central fibrosis and advanced to severe peripheral fibrosis (GNM, GNH). Number of subjects per group: references (19), CLL (23), CLL GNM (27), CLM (10), CLH (7), D, E, F (3)

Figure 2C shows the contents of IL-22 in subjects with different degrees of liver fibrosis and different treatments. The study subjects were over 30 years old and under 65 years old and had no active HBV infections (HBS Ag-). References are subjects who have not been exposed to S. japonicum. Subjects have been treated 0 to 4 times (white circles) or more than 5 to 20 times (black circles). Number of subjects per group: the treatment groups were grouped as follows: 0 to 4 treatments and> 5 treatments in order to increase the number of subjects per point

References: 19; 0-4 treatments: 39; > 5 treatments: references: 31

Figure 3A shows the impact of schistosomiasis treatments on the contents of IL-22, IL-6, IL-1 or IL-23 in egg stimulated cultures. The study subjects were over 30 years old and under 65 years old and they had no active HBV infections (HBS Ag-). References were subjects who had not been exposed to S. japonicum. The PBMCs of the study subjects were stimulated with eggs and the cytokines were evaluated in the supernatants at 24 h (IL-1b, IL-23, IL-6) and at 144 h (IL-22). The contents of IL-6 were multiplied by 0.1. Figure 3B shows the contents of IL-22, IL-6, IL-1p or IL-23 in PBMC stimulated with reference eggs and subjects with varying degrees of liver fibrosis. Study and number of subjects in each group as in Figure 2A.

Figure 4 is a mapping that locates SNPs and correlation groups in IL22RA2.

Detailed description of the invention

This invention provides valuable genetic markers for predicting the evolution of the disease in fibrosis, especially in liver fibrosis, in humans.

The early detection of an abnormal accumulation of ECMP or fibrosis, and the regular monitoring of said accumulation or fibrosis, would allow the initiation of antifibrotic treatments capable of stopping and even reversing this process. This, in turn, would prevent the evolution to human fibrosis disease, for example, liver fibrosis or liver cirrhosis, and the morbidity and mortality associated with this condition. The development of these various techniques for early detection of fibrosis is a good omen for the future care of patients with liver diseases.

The inventors have now identified a gene related to human fibrosis. They have shown that fibrosis in Chinese, Sudanese and Brazilian cohorts infected with Schistosoma japonicum and Schistosoma mansoni, respectively, is remarkably dependent on the allelic variants found in the IL22RA2 gene. The IL22RA2 gene (for "interleukin-22 receptor alpha-2"), also called IL22R-BP, encodes a soluble form of the IL-22 receptor that competes for the binding of IL-22 to its receptor.

More specifically, the inventors conducted case control studies on independent samples of Chinese (exposed to S. japonicum), Sudanese and Brazilian (exposed to S. mansoni) living in endemic regions. Hepatic fibrosis (FH) was evaluated using ultrasound by at least two observers for each sample. All SNP Tag were analyzed in IL22RA2 (Frequency of minor alleles> 10%). To rule out whether SNPs in unbalanced coupling with associated SNPs could explain the observed associations, the inventors evaluated SNPs in the 500 Kb regions in 3 'and 5' of IL22RA2.

The invention thus provides a method for determining the risk of developing liver fibrosis or the development of liver fibrosis, or a poor prognosis of liver fibrosis in a subject, the method comprising detecting the presence of single nucleotide polymorphism alleles (SNPs). ) associated with the risk in the locus of the IL22RA2 gene in a sample of said subject.

The invention more specifically provides a method for determining the risk of developing liver fibrosis or the development of liver fibrosis, or a poor prognosis of liver fibrosis, the method comprising genotyping of a SNP in the locus of the IL22RA2 gene in a sample of said subject.

Another purpose of the present method is to provide a genetic approach to predict the response to the treatment of viral infection. The present method now describes the identification of an antiviral treatment response locus, the IL22RA2 gene locus, that can be used to predict the response to antiviral treatment of a patient suffering from a viral infection, especially HCV. In the present specification it is described in particular, in a method comprising detecting in a sample of the subject the presence of an alteration in the locus of the IL22RA2 gene, the presence of said alteration being indicative of the response to treatment, that is, indicative of a level of risk for the patient who does not respond to treatment.

The method allows prediction of the response to treatment with an antiviral agent such as ribavirin, and an interferon administered to a patient suffering from a viral infection, especially hepatitis C.

This aspect provides valuable markers to predict the response to antiviral treatment, especially in hepatitis C.

The early identification of subjects who respond and do not respond to antiviral treatment would allow the initiation of an individualized (personalized) treatment based on the genotype of the patients. This, in turn, would help doctors make a more informed decision and avoid unnecessary expenses and unnecessary side effects. The development of these various early prediction techniques is a good omen for the future care of patients with viral infection, especially hepatitis C.

The inventors have now identified a gene related to the response to an antiviral treatment. They have shown that the response to Ribavirin-IFN antiviral treatment in several HCV infected cohorts depends on the allelic variants found in the IL22RA2 gene.

Although the experimental data collected by the inventors did not confirm the association of certain alleles, depending on the population analyzed, the present invention is not limited to each SNP that was found significantly correlated with fibrosis in all populations analyzed. In fact, several reasons could explain the failure to confirm the significant correlation in some populations, including an insufficient cohort, incomplete evaluation of confounding variables, a lower frequency of SNPs in these populations, etc.

Definitions

In the context of this invention, the term "abnormal deposition of extracellular matrix proteins (ECMP)" refers to the components of the extracellular matrix (including laminin, EIIIA fibronectin, collagen I and IV, procollagen III, elastin, tenascin) that can accumulate in all types of human tissues. Such accumulation can be harmful, for example, when it occurs in the arteries, the heart or the brain. When the deposition is massive, the accumulation produces tissue fibrosis.

In the context of this invention, "fibrosis" designates all types of human fibrosis that occur in all human fibrotic diseases, for example in liver diseases, cirrhosis, skin keloid, hypertrophic scars, scleroderma, obesity and any fibrotic disease. In the context of this invention, "hepatic fibrosis" or "FH" designates all types of fibrosis that occur in a liver, its tissue or any part of its tissue. Liver fibrosis occurs especially in response to an injury. Liver fibrosis may be the normal response to a chronic liver injury, which ultimately leads to cirrhosis and its complications, portal hypertension, liver failure and hepatocellular carcinoma. Hepatic fibrosis is an overly exuberant wound healing in which excess connective tissue accumulates in the liver. The extracellular matrix is produced in excess, poorly degraded, or both. The trigger is a chronic injury, especially if there is an inflammatory component. Various types of chronic liver damage can cause fibrosis, such as chemical fibrosis (CCU), bacterial (i.e. brucellosis), parasitic (i.e. schistosomiasis caused by Schistosoma species ; or echinococcosis infections) or viral (i.e. hepatitis produced by liver virus A (HAV), liver virus B (HBV) or liver virus C (HCV). In the context of this invention, the "skin keloid" is an overgrowth of scar tissue in the skin. More specifically, keloids and hypertrophic scars (HSc) are fibroproliferative dermal disorders unique to humans that occur after trauma, inflammation, surgical intervention, burns and, sometimes, spontaneously. These are characterized by excessive deposition of collagen in the dermis and subcutaneous tissues. Contrary to the characteristics of the fine-line scar from normal wound repair, the exuberant healing of the keloids and the HSc generally produces disfigurement, contractures, pruritus and pain. Keloids occur in individuals with a familiar disposition among blacks, Hispanics and Orientals. Unlike HSc, keloid scars enlarge and extend beyond the margins of the original wound and rarely return. These disorders represent aberrations in the fundamental processes of wound healing, which include cell migration and proliferation, inflammation, increased synthesis and secretion of cytokines and extracellular matrix proteins (ECM) and remodeling of the freshly synthesized matrix. Biologically, keloids are fibrotic tissues characterized by a collection of atypical fibroblasts with excessive deposition of extracellular matrix components, especially collagen, fibronectin, elastin and proteoglycans. In general, keloids contain relatively acellular centers and thick and abundant bundles of collagen that form nodules in the deep dermal part of the lesion. The release and activation of growth factors during the inflammatory phase of healing are prerequisites for healing processes, including angiogenesis, reepithelialization, fibroblast uptake and proliferation and matrix deposition. Then, abnormal production of regulatory cytokine activity, including IL22RA2, could contribute to the development of keloids.

In the context of this invention, "the locus of the IL22RA2 gene" designates all sequences or products in a cell or organism, including IL22RA2 coding sequences, IL22RA2 non-coding sequences (eg, introns), regulatory sequences IL22RA2 that control transcription and / or translation (e.g., activator, enhancer, terminator, etc.), all corresponding expression products, such as IL22RA2 RNAs (e.g., mRNAs) and polypeptides of IL22RA2 (eg, a preprotein and a mature protein); as well as surrounding sequences of the 500 kb region, preferably 100 kb, preferably of the 20 kb region, upstream of the initiation codon of the IL22RA2 gene and of the 500 kb region, preferably 100 kb, preferably of the region 20 kb, downstream of the untranslated region (3'UTR). For example, the IL22RA2 locus comprises surrounding sequences that comprise the SNPs in Table 1.

In the context of the present invention, the term "prognosis" includes detection, monitoring, dosing, comparison, etc., in several stages, including initial, presymptomatic and late stages, in adults, children and premature infants. The prognosis generally includes the evaluation (prediction) of the evolution of fibrosis and the characterization of a subject to define the most appropriate treatment (pharmacogenetics), etc. The present invention provides prognostic methods to determine the rate of evolution of fibrosis or a related disorder resulting from a mutation or polymorphism in the locus of the IL22RA2 gene.

The "sample" can be any biological sample from a patient or subject, which contains nucleic acids or polypeptides. Examples of such samples include liquids, tissues, cell samples, organs, biopsies, etc. The most preferred samples are blood, plasma, saliva, urine, seminal fluid, etc. The sample can be collected according to conventional techniques and used directly for diagnosis or storage.

The "patient" can be any mammal, preferably a human being, regardless of age or sex. The patient may be infected with a virus, including a virus that is selected from the group consisting of Arenavirus family viruses (e.g., Lassa virus), Coronavirus (e.g., acute respiratory syndrome virus severe), Flaviviridae (e.g., hepatitis C or B virus), dengue virus, West Nile virus, yellow fever virus, Tick-borne encephalitis virus), Filovirus (e.g., Ebola , Marburg), Herpesviridae (eg, herpes simplex virus, Cytomegalovirus, Epstein-Barr virus, varicella zoster virus), Orthomyxoviridae (eg influenza A and B virus), Paramyxovirus (eg ., respiratory syncytial virus, para-influenza virus, ^p M ^v , measles), Poxviridae (eg, vaccine, smallpox), Rhabdoviridae (eg, vesicular stomatitis virus, viral hemorrhagic septicemia virus, rabies), Retroviridae (eg HIV and other retroviruses), Togaviridae (eg, Chikungunya, Sindbis, Semliki forest virus, Ross river virus, Eastern equine encephalitis virus). In a particular embodiment, the patient is infected with a hepatitis C virus, e.g. eg, hepatitis C virus genotype 1.

In a method to determine the probability that a patient affected with a viral infection responds to a treatment with an antiviral agent and / or an interferon, the term "viral infection" designates all types of human viral infection that can be treated with Ribavirin and / or IFN, for example, hepatitis C, hepatitis B, respiratory syncytial virus broncholitis (RSV), adenovirus disease, influenza and any human viral infection treated with Ribavirin and / or IFN.

In the context of this invention, "responding" refers to the phenotype of a patient responding to treatment with an antiviral agent, especially Ribavirin, and / or an IFN, that is, the viral load decreases, at least one of its symptoms. It relieves, or stops or slows the development of the disease.

In the context of this invention, "not responding" refers to the phenotype of a patient who does not respond to treatment with an antiviral agent, especially Ribavirin and / or an IFN, or who responds but relapses within one or two years. The lack of response to treatment refers to a viral load that does not decrease substantially and the patient's symptoms are not relieved, or the disease progresses. Recurrent patients respond to treatment for a short period of time, but their viral load and symptoms increase again within one or two years of the end of treatment.

The term "treatment" or "antiviral treatment" refers to the administration of an antiviral agent and / or interferons (IFN).

Preferably, the interferon is interferon gamma. However, other interferons are included, including interferon alfa 2B, pegylated interferon alpha, consensus interferon, interferon alfa 2A and interferon tau lymphoblastoid. In a preferred embodiment, the interferon is PEGylated interferon, such as PEGylated gamma interferon.

The "antiviral agent" can be any compound that interferes with the entry of the virus into a cell, or its replication, or that inhibits the activity of a viral protein. For example, it can be interfering RNA, complementary RNA, Imiqimod, Ribavirin, an inosine 5'-monophosphate dehydrogenase inhibitor, amantadine or rimantadine. More generally it can be a viral protease inhibitor.

When the virus is the HCV virus, the viral agent can be an inhibitor of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV input, HCV assembly, HCV or NS5A protein output of V ^h C.

In a preferred aspect, the interferon is gamma interferon, such as PEGylated gamma interferon. In another preferred aspect, interferon is alpha interferon, such as PEGylated alpha interferon. In a specific embodiment, the treatment comprises ribavirin and interferon gamma or alpha, preferably gamma interferon or PEGylated alpha. Alterations

The alteration can be determined in the DNA, RNA or polypeptide of IL22RA2. Optionally, the detection is performed by sequencing all or part of the locus of the IL22RA2 gene or by hybridization or selective amplification of all or part of the locus of the IL22RA2 gene. More preferably, a specific amplification of the IL22RA2 gene locus is carried out before the alteration identification step. An alteration in the locus of the IL22RA2 gene can be any form of mutation, deletion, rearrangement and / or insertions in the coding and / or non-coding region of the locus, alone or in various combinations. Mutations more specifically include point mutations. Eliminations may encompass any region of two or more residues in a coding or non-coding portion of the genetic locus, such as from two residues to the entire gene or locus. Typical deletions affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may also occur. The inserts may include the addition of one or more moieties in a coding or non-coding portion of the gene locus. The inserts may normally comprise an addition of between 1 and 50 base pairs in the gene locus. Reordering includes sequence inversion. The alteration of the locus of the IL22RA2 gene can lead to the creation of stop codons, mutations of reading frame changes, amino acid substitutions, splicing or processing of RNA, product instability, production of truncated polypeptides, etc. The alteration may result in the production of an IL22RA2 polypeptide with altered function, stability, orientation or structure. The alteration can also produce a reduction in the expression of the protein or, alternatively, an increase in said production.

In a preferred embodiment, said alteration is a mutation, an insertion or a removal of one or more bases. In a particular embodiment of the method according to the present invention, the alteration in the gene locus IL22RA2 is selected from a point mutation, a deletion and an insertion into the IL22RA2 gene or the corresponding expression product, more preferably a point mutation and a deletion. The alteration can be determined in the DNA, RNA or IL22RA2 polypeptide.

In a more preferred embodiment, the method comprises genotyping the IL22RA2 gene, to determine the presence of an SNP at any of the positions indicated in Table 1A.

Table 1A: SNPs associated with fibrosis in the locus of the IL22RA2 gene

Hapmap: European CEU Cohort, African YOR Cohort (Yorubas), Asian CHB Cohort (China)

Preferably, the method comprises genotyping a SNP selected in the group consisting of rs6570136, rs7774663, rs11154915 and rs2064501.

The presence of a G allele with respect to SNP rs6570136, more specifically of a GG genotype, is detrimental to the patient, that is, it is indicative that a patient is likely to develop an abnormal deposit of ECMP or fibrosis, especially liver fibrosis.

The presence of a T allele with respect to the SNP rs7774663, more specifically of a TT genotype, is detrimental to the patient, that is, it is indicative that a patient is likely to develop an abnormal deposit of ECMP or fibrosis, especially liver fibrosis.

The presence of a T allele with respect to SNP rs11154915, more specifically of a TT or CT genotype, is detrimental to the patient, that is, it is indicative that a patient is likely to develop an abnormal deposit of ECMP or fibrosis, especially fibrosis Hepatic

The presence of a C allele with respect to SNP rs2064501, more specifically of a CC genotype, is detrimental to the patient, that is, it is indicative that a patient is likely to develop an abnormal deposit of ECMP or fibrosis, especially liver fibrosis.

SNPs in the same groups are highly correlated (R2> 0.8) and have similar utility in the methods of the invention. SNPs in a strong coupling imbalance (which produces R2> 0.6) are also included. Another method of the invention may comprise determining if the patient comprises an unanswered genotype as defined in Table 1B.

The analysis was performed on 123 subjects (69 responding subjects and 54 responding subjects), all infected with HCV.

Table 1B: Alterations associated with the response to antiviral treatment at the locus of the IL22RA2 gene

Preferably, the method comprises genotyping a SNP selected in the group consisting of rs11154915, rs6570136, rs2064501 and rs1543509.

The presence of a TT genotype with respect to SNP rs11154915 is in favor of the positive response of the patient to antiviral treatment.

The presence of an AG or GG genotype with respect to SNP rs6570136 is in favor of the positive response of the patient to antiviral treatment.

The presence of a CT genotype with respect to SNP rs2064501 is in favor of the positive response of the patient to antiviral treatment.

The presence of an AA genotype with respect to SNP rs1543509 is in favor of the positive response of the patient to antiviral treatment.

SNPs in the same groups are highly correlated (R2> 0.8) and have similar utility in the methods of the invention. SNPs in coupling imbalance are also included.

The odd proportions associated with each genotype are indicated in Table 1B. They range between 1.9 and 4 when each SNP was evaluated alone; When all SNPs were evaluated together in the same multifactorial model (which takes into account the effects of confusion between SNPs), the ORs range between 1.9 and 13, and subjects carrying genotypes that respond to treatment for all four polymorphisms will be approximately 50 to 300 times more likely to respond to treatment than subjects who carry genotypes that do not respond to treatment for all genotypes.

Coupling imbalance (DA) is defined as the non-random association of alleles in different locus across the genome. The alleles in two or more locuses are in DA if their combination occurs more or less frequently than expected in the population.

When there is a causal locus in a region of DNA, due to AD, it is likely that one or more nearby SNPs are associated with the trait as well. Therefore, any SNP in strong DA (producing an R2> 0.6) with a first SNP related to an abnormal ECMP deposit will be associated with this trait.

The identification of additional SNPs in coupling imbalance with a given SNP implies: (a) amplification of a fragment of the genomic region comprising or surrounding a first SNP of a plurality of individuals; (b) the identification of second SNPs in the genomic region that hosts or surrounds said first SNP; (c) performing a coupling imbalance analysis between said first SNP and second SNP; and (d) select said second SNPs in coupling imbalance with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.

Methods to identify SNPs and to perform coupling imbalance analysis can be performed by one skilled in the art without undue experimentation using well known methods.

It is well known that many SNPs have alleles that show strong DA with other nearby SNP alleles and in regions of the genome with strong DA, a selection of uniformly spaced Np s, or those selected based on their DA with other SNPs (proxy SNPs or SNP Tag), can capture most of the genetic information of SNPs, which are not genotyped with only a slight loss of statistical power. In association studies, this DA region is adequately covered using few SNPs (the SNPs Tag) and a statistical association between an SNP and the phenotype under study means that the SNP is a causal variant or is in DA with a causal variant. The two most used metrics to measure DA are D 'and r2 and can be written in terms of frequencies between each other and allelic. It is a general consensus that a proxy SNP (or Tag) is defined as an SNP in DA (r2> 0.8) with one or more other SNPs. The genotype of the proxy SNP could predict the genotype of the other SNP through DA and vice versa. In particular, any SNP in DA with one of the SNPs used herein can be replaced by one or more proxy SNPs defined according to their DA as r 2> 0.8.

These SNPs in coupling imbalance can also be used in the methods according to the present invention, and more specifically in the diagnostic methods according to the present invention.

Alterations in the IL22RA2 gene can be detected by determining the presence of an altered expression of the IL22RA2 RNA. Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, the presence of an altered amount of RNA, etc. These can be detected by various known techniques, including by sequencing all or part of the IL22RA2 RNA or by selective hybridization or selective amplification of all or part of said RNA, for example. In a further variant, the method comprises detecting the presence of an expression of the altered IL22RA2 polypeptide. Expression of the altered IL22RA2 polypeptide includes the presence of an altered polypeptide sequence, the presence of an altered amount of IL22RA2 polypeptide, the presence of an altered tissue distribution, etc. These can be detected by various known techniques, including by sequencing and or binding to specific ligands (such as antibodies), for example.

As indicated above, several known techniques can be used to detect or quantify the altered IL22RA2 gene or RNA expression or sequence, including sequencing, hybridization, amplification and / or binding to specific ligands (such as antibodies). Other suitable methods include allele specific oligonucleotide (ASO), allele specific amplification, Southern blotting (for DNA), Northern blotting (for RNA), single stranded configuration analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), migration gel, fixed denaturing gel electrophoresis, heteroduplex analysis, RNase protection, cleavage of chemical imbalances, ELISA, radioimmunoassay (RIA) and immunoenzymatic assays (IEMA). Some of these methods (for example, SSCA and CGGE) are based on a change in the electrophoretic mobility of nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is observed by a change in the mobility of the gels. The fragments can then be sequenced to confirm the alteration. Some others are based on specific hybridization between the subject's nucleic acids and a probe specific for the natural or altered IL22RA2 gene or RNA. The probe may be suspended or immobilized on a substrate. The probe is usually marked to facilitate the detection of hybrids. Some of these methods are especially suitable for evaluating a polypeptide sequence or expression level, such as Northern blotting, ELISA and RIA. The latter require the use of a specific ligand for the polypeptide, more preferably a specific antibody.

In a further aspect, the method comprises detecting the presence of an expression profile of the altered IL22RA2 gene in a sample of the subject. As indicated above, this can be achieved more preferably by sequencing, selective hybridization and / or selective amplification of nucleic acids present in said sample. Sequencing

Sequencing can be carried out using well known techniques, using automatic sequencers. Sequencing can be performed in the locus of the complete IL22RA2 gene or, more preferably, in its specific domains, generally those known or suspected of carrying harmful mutations or other alterations.

Amplification

Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. The amplification can be performed according to various known techniques, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), chain shift amplification (SDA) and amplification based on the sequence of nucleic acid (NASBA). These techniques can be performed using reagents and protocols available in the market. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers to initiate the reaction. Nucleic acid primers useful for amplifying sequences of the IL22RA2 gene locus can specifically hybridize with a portion of the IL22RA2 gene locus that flanks a target region of said locus, said target region being altered in certain subjects with fibrosis or related disorders.

This invention makes use of nucleic acid primers useful for amplifying sequences of the IL22RA2 gene or locus, including surrounding regions. Such primers are preferably complementary to nucleic acid sequences and specifically hybridize with them in the locus of the IL22RA2 gene. Certain primers may specifically hybridize with a part of the IL22RA2 gene locus that flanks a target region of said locus, said target region being altered in some subjects with fibrosis or associated disorders.

Selective hybridization

Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect alterations in the nucleic acid sequence. A certain detection technique involves the use of a specific nucleic acid probe for the natural or altered IL22RA2 or RNA gene, followed by the detection of the presence of a hybrid. The probe may be suspended or immobilized on a substrate or support (as in a nucleic acid matrix or chip technologies). The probe is usually marked to facilitate the detection of hybrids. In this regard, a specific embodiment of this invention comprises contacting the subject's sample with a specific nucleic acid probe for an altered IL22RA2 gene locus, and assessing the formation of a hybrid. In a specific preferred embodiment, the method comprises simultaneously contacting the sample with a set of probes that are specific, respectively, for the locus of the natural IL22RA2 gene and for several altered forms thereof. In this embodiment, it is possible to directly detect the presence of various forms of alterations in the locus of the IL22RA2 gene in the sample. In addition, several samples of various subjects can be treated in parallel.

In the context of this invention, a probe refers to a polynucleotide sequence that is complementary to an (objective part of a) gene or RNA of IL22RA2 and capable of specific hybridization with it, and that is suitable for detecting polynucleotide polymorphisms associated with IL22RA2 alleles that predispose to or are associated with fibrosis. The probes are preferably perfectly complementary to the IL22RA2 gene, the RNA or the target part thereof.

The probes generally comprise single stranded nucleic acids between 8 and 1000 nucleotides in length, for example, between 10 and 800, more preferably between 15 and 700, generally between 20 and 500. It should be understood that longer probes can also be used. A preferred probe of this invention is a single stranded nucleic acid molecule between 8 and 500 nucleotides in length, which can specifically hybridize with a region of an IL22RA2 or RNA gene locus that carries an alteration.

The method of the invention employs a nucleic acid probe specific for an IL22RA2 gene or its altered RNA (e.g., mutated), that is, a nucleic acid probe that specifically hybridizes with said IL22RA2 or altered RNA gene and essentially does not hybridize with an IL22RA2 gene or its RNA lacking such alteration. Specificity indicates that hybridization with the target sequence generates a specific signal that can be distinguished from the signal generated by non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that some mismatch can be tolerated, provided that the specific signal can be distinguished from non-specific hybridization. Specific examples of such probes are the nucleic acid sequences complementary to an objective portion of the genomic region including the locus of the IL22RA2 gene or its RNA that carries a point mutation as listed in Table 1 above. The sequence of the probes can be derived from the sequences of the IL22RA2 gene and the RNA as provided in the present application. Nucleotide substitutions can be made, as well as chemical modifications of the probe. Such chemical modifications can be made to increase the stability of the hybrids (eg, interleaver groups) or to label the probe. Typical examples of markers include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labeling, etc. The invention also relates to the use of a nucleic acid probe as described above in a method to detect the presence or predisposition to fibrosis or a related disorder in a subject or in a method to evaluate a subject's response to a treatment. of fibrosis or a related disorder.

Specific ligand binding

As indicated above, alteration in the locus of the IL22RA2 gene can also be detected by selecting alterations in the IL22RA2 polypeptide sequence or expression levels. In this regard, the contact of the sample with a specific ligand for an IL22RA2 polypeptide and the determination of complex formation is also described. Different types of ligands can be used, such as specific antibodies. In a specific embodiment, the sample is contacted with an antibody specific for a IL22RA2 polypeptide and the formation of an immunocomplex is determined. Several methods can be used to detect an immunocomplex, such as ELISA, radioimmunoassay (RIA) and immunoenzymatic analysis (IEMA). In the context of this invention, an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof that have substantially the same antigenic specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives include single chain antibodies, humanized antibodies, multifunctional antibodies, etc. An antibody specific for an IL22RA2 polypeptide designates an antibody that selectively binds to an IL22RA2 polypeptide, that is, an antibody raised against an IL22RA2 polypeptide or a fragment containing an epitope thereof. Although non-specific binding to other antigens can occur, binding to the target IL22RA2 polypeptide occurs with greater affinity and can be reliably discriminated against non-specific binding.

Also described is a diagnostic kit comprising products and reagents for detecting in a sample of a subject the presence of an alteration in the locus or polypeptide of the IL22RA2 gene, in the expression of the IL22RA2 gene or polypeptide, and / or in the activity of IL22RA2. Said diagnostic equipment comprises any primer, any pair of primers, any nucleic acid probe and / or any ligand, preferably antibody, described in the present invention. Said diagnostic equipment may further comprise reagents and / or protocols for performing an hybridization, amplification or immunological reaction of antigen-antibody.

Drug detection

New methods for the detection of experimental or main drugs are also described. These methods include binding assays and / or functional assays, and can be performed in vitro, in cellular systems, in animals, etc. Also described herein is a method of selecting biologically active compounds, said method comprising in vitro contacting a compound under study with an IL22RA2 gene or polypeptide according to the present invention and determining the ability of said compound under study to bind to said IL22RA2 gene or polypeptide. Binding to said gene or polypeptide provides an indication of the ability of the compound to modulate the activity of said target and, therefore, to affect a route that leads to any abnormal ECMP deposition or fibrosis in a subject. In a preferred embodiment, the method comprises contacting in vitro a compound under study with an IL22RA2 gene or polypeptide or one of its fragments according to the present invention and determining the ability of said compound under study to bind to said IL22RA2 polypeptide or fragment. . The fragment preferably comprises a binding point of the IL22RA2 polypeptide. Preferably, said IL22RA2 gene or polypeptide or one of its fragments is an altered or mutated IL22RA2 gene or polypeptide or one of its fragments comprising the alteration or mutation. Also described herein is a method for selecting active compounds in any abnormal ECMP or fibrosis deposit, said method comprising in vitro contacting a compound under study with an IL22RA2 polypeptide according to the present invention or one of its fragments containing the point of attachment and determine the ability of said compound under study to bind to said IL22RA2 polypeptide or one of its fragments. Preferably, said IL22RA2 polypeptide or one of its fragments is an altered or mutated IL22RA2 polypeptide or one of its fragments comprising the alteration or mutation. The method for the identification of experimental drugs comprises contacting a biotechnological host cell that expresses an IL22RA2 polypeptide according to the present invention with a compound under study, and determining the ability of said compound under study to bind said IL22RA2 and modulate activity of the IL22RA2 polypeptide. Preferably, said IL22RA2 polypeptide or one of its fragments is an altered or mutated IL22RA2 polypeptide or one of its fragments comprising the alteration or mutation. Binding can be determined by various techniques, such as by labeling the compound under study, by competition with a labeled reference ligand, etc. The method of selecting biologically active compounds also comprising in vitro contacting a test compound with a polypeptide 22RA2 and determining the ability of said test compound to modulate the activity of said polypeptide 22RA2. Preferably, said IL22RA2 polypeptide or one of its fragments is an altered or mutated IL22RA2 polypeptide or one of its fragments comprising the alteration or mutation.

The method of selection, of biologically active compounds for a subject that is predisposed or is predisposed to develop any abnormal ECMP or fibrosis deposition, also comprises contacting in vitro a compound under study with an IL22RA2 gene according to the present invention and determining the ability of said compound under study to modulate the expression of said IL22RA2 gene. Preferably, said IL22RA2 gene or one of its fragments is an altered or mutated IL22RA2 gene or one of its fragments comprising the alteration or mutation.

The method of detecting, selecting or identifying biologically active compounds, especially active compounds in any abnormal ECMP or fibrosis deposit, also comprises contacting a compound under study with a biotechnological host cell comprising an indicator assembly, said indicator assembly comprising indicator gene under the control of an activator of the IL22RA2 gene, and select the compounds under study that modulate (e.g., activate or inhibit) the expression of the indicator gene. Preferably, said activator of the IL22RA2 gene or one of its fragments is an activator of the altered or mutated IL22RA2 gene or one of its fragments comprising the alteration or mutation.

The above detection tests can be performed on any suitable device, such as plates, tubes, plates, flasks, etc. Normally, the assay is performed in multiwell plates. Various compounds under study they can be analyzed in parallel. In addition, the compound under study can be of diverse origins, nature and composition. It can be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., isolated or mixed with other substances. The compounds may be all or part of a combinatorial library of products, for example.

Other aspects and advantages of the present invention will be described in the following experimental section, which should be considered illustrative and not restrictive of the scope of the present application.

Examples

Example 1: Production and modulation of IL-22 in human schistosome infections

Production of IL-22 in human schistosome infections

The inventors have compared the contents of IL-22 in PBMC cultures of 140 subjects exposed to S. japonicum infections with cultures of 20 references that had not previously been exposed to schistosome infections (Figure 1A) but lived in the same region in comparable living conditions. IL-22 was detected in resting cultures of subjects exposed at 72 and 144 hours and was significantly improved by the addition of schistosome eggs at time 0 of the culture. IL-22 was detected in reference resting cultures only at 144 hours and was not improved by the addition of eggs. The inventors detected significant amounts of IL-17A in 144-hour cultures, but the contents of IL-17 did not increase with egg stimulation (Figure 1B). Therefore, it is unlikely that IL-22 in the crops was produced by Th17. FACS analysis of the IL22 + cells in the blood of exposed patients showed that IL-22 is produced by CD3 + CD4 + and by CD3-CD4-, none of these cell populations produced IL-17 (Fig. 1C). The latter are probably NK cells. The percentage of CD3 + CD4 + IL17-IL22 + and CD3-CD4-IL17-IL22 T cells in reference and endemic subjects are shown in Figure 1C.

Modulation of IL-22 production is modified by schistosomiasis treatment and modulated according to liver fibrosis

The amounts of IL-22 in cultures stimulated with eggs varied markedly among the exposed subjects. Since treatments with Praziquantel against schistosomiasis destroy worms and suspend egg production until reinfection occurs, the inventors evaluated whether differences in treatments with Praziquantel could have modulated the production of IL-22. Some patients had been treated every year for at least 10 years, others had never been treated and others had received 1 to 10 treatments in the last 10 years. Figure 2A demonstrates that the number of treatments with Praziquantel had a significant impact on the production of IL-22 by the PBMCs of the subjects: IL-22 in egg-stimulated cultures increased significantly with the number of treatments in the last ten years ( p = 0.005, covariate in the regression model was gender p = 0.08); however, this effect was much less for subjects who had been treated at least once a year each year, probably because these subjects did not become infected again (see below). Therefore, the frequency of treatments with Praziquantel in the last ten years had a significant impact on the production of IL-22 by PBMC of exposed subjects.

The inventors then evaluated whether the contents of IL-22 in cultures were related to the degree of liver disease of the patient. Figure 2B shows that IL-22 was low in patients with mild liver disease, measured by degrees of liver fibrosis and constantly increased with increased degrees of fibrosis reaching maximum levels in subjects with advanced central periportal fibrosis. This suggests that there may be an increase in the production of IL-22 in response to / in relation to severe liver disease. However, surprisingly, the IL-22 of patients with very severe hepatic fibrosis (degrees of FH D, E, F) did not produce much IL-22.

The effect of the number of treatments with Praziquantel in this configuration is shown in Figure 2C. Praziquantel treatments impacted the IL-22 produced by the cells of all fibrosis groups. Three observations are the most relevant for the study: first, the increase in IL-22 production in advanced fibrosis (CLH) is observed in different Praziquantel regimens and is not due to differences in the frequency of treatment of these patients; secondly, treatments with Praziquantel improved the production of IL-22 in all cultures, but in cell cultures of subjects with degrees of severe fibrosis; Third, subjects with high Praziquantel regimens (> 10, at least once a year) are in the mild fibrosis group and explain the increase in IL-22 production observed in this group.

In summary, IL-22 is affected by two independent factors: treatments with Praziquantel and the degree of liver disease. In addition, subjects with very severe liver disease cannot produce much IL-22 and this did not improve with Praziquantel treatments, while the same treatments had a markedly increased effect on IL-22 production in subjects with mild liver fibrosis. to advanced.

IL-6 and possibly IL-1p are likely to be key regulators of IL-22 production in subjects with different degrees of liver fibrosis and different treatment regimens.

The inventors observed that the cytokines IL-6, IL-1p and IL-23 improved significantly (IL-23 (p = 3.10-6), IL-6 (p <10-6) and IL-1 p (p <10 -6) by stimulation with eggs in 24-hour PBMC cultures of exposed subjects (Figure 3A) To determine if any of these 3 cytokines could play a role in the modulation of IL-22 contents, a linear regression analysis was performed with IL-22 contents in egg-stimulated cultures, including these 3 cytokines, patient age and sex. This analysis demonstrated a very significant association (p = 0.0002) between IL-22 and IL-6 and a weak association (p = 0.06) with IL-1p. IL-23 was excluded from the regression model. This result is illustrated in Figures 3B, 3C showing the variations of IL-6, IL-1p and IL-23 with treatments with Praziquantel (Fig. 3B) and with hepatic fibrosis (Fig. 3C). Therefore, the connection between treatments with Praziquantel and IL-22 and the degrees of fibrosis and IL-22 is, at least in part, IL-6.

The results presented above are consistent with the view that the incorporation of a protective response of IL-22 increased with liver injury and that the most serious liver disease may result in part from the inability to incorporate such a response. To further test this hypothesis, the inventors looked for genetic tests that indicate that IL-22 was really crucial in the control of liver fibrosis and had a significant impact on liver disease.

Example 2: Polymorphisms in IL22RA2 encoding IL22 BP are related to liver fibrosis in two samples of Chinese fishermen and farmers living in an endemic area of S. japonicum

Materials and methods

Statistic analysis

Multifactorial logistic regression was used to analyze the relationship between the probability that an individual develops fibrosis and genetic variants, including the main covariates that are known to affect the evolution of the disease in subjects infected with schistosomes. For this analysis the statistical software SPSS (version 10.0) was used. Age, sex and exposure to infection were analyzed in the regression models and were maintained when they showed an association (p <0.05) with the disease. Since the cohorts were matched by sex and age, these covariates had little effect on the association between genetic variants and disease. HBV infection and infection exposure were included in the regression models when these covariates could be accurately assessed as in Chinese fishermen (exposure, number of treatments) or in Chinese farmers (HBV infection, place of birth) .

Extraction of DNA

Aliquots of 5 to 15 ml of blood were collected in sodium citrate and kept at -20 ° C. DNA was extracted using the standard salting method (Sambrook et al., 1989).

DNA amplification

All purified DNA from the FTA card was previously amplified before genotyping. Polymerase chain reactions (genome-wide amplifications) were performed in reactions of 50 pl containing a biological sample punch (buccal DNA attached to FTA1) or 100 ng genomic DNA, 1.5 OD of completely random primer Degenerate 15 bases (Genetix, Paris, France), 200 mM dNTP, 5 mM MgCh, 5 ml of 10x PCR buffer and 0.5 units of high fidelity DNA Taq DNA polymerase (BIOTAQ DNA Polymerase, Bioline, London, England). The samples were amplified in a multi-block thermocycler as follows: a previous denaturation stage of 3 min at 94 ° C, 50 cycles consisting of 1 min at 94 ° C, 2 min at 37 ° C, 1 min of ramp ( 37-55 ° C) and 4 min at 55 ° C. Final extension stage of 5 min at 72 ° C.

Sequencing

The purified PCR products were sequenced using the ABI Prism BigDye Terminator cycle sequencing system (PE Applied Biosystems, Foster City, USA) in the ABI Prism automatic sequencer. Sequencing reactions were performed on both chains. Sequencing by GATC biotech (GATC, Marseille, France). Genotyping of PCR polymorphisms with specific TaqMan probes

Allelic discrimination was assessed using TaqMan probe assays (Applied Biosystems, Lafayette USA). Each reaction contained 12.5 ng of genomic DNA, TaqMan Universal PCR Master Mix (Applied Biosystems, Lafayette, USA), 900 nM of each primer and 200 nM of each fluorescence-labeled hybridization probe in a total volume of 5 pl. RT-PCR was performed on an ABI Prism 7900 sequence detection system (Applied Biosystems, Lafayette, USA) using the following conditions: 50 ° C for 2 min, 95 ° C for 10 min and 40 amplification cycles (denaturation at 95 ° C for 15 s, annealing inhibition at 60 ° C / extension for 1 min).

Results

The inventors have selected SNPs in IL22RA2 (29.7 Kb) and 10 Kb in 3 'and 5' of the gene that had a Minor allele frequency in Chinese greater than 10% based on HapMap data. These SNPs were grouped into six correlation groups (r2 = 0.8) containing n = 10 (group I), 5 (II), 2 (III), 3 (IV), 13 (V) and 3 (VI), and 4 singletones that are placed as in figure 4. The inventors genotyped one or two SNPs of each group in a sample of Chinese fishermen (n = 268, 176 subjects with mild FH and 92 patients with severe FH) who have been fishing during the At least 20 years at Dong Ting Lake, where S. japonicum has been endemic for at least 40 years. They found that 3 SNPs belonging to two groups were related to FH. SNP rs6570136 GG (p = 0.007, OR = 2.7 (CI = 1.3-5.6)), rs7774663 TT (p = 0.006, OR = 2.5 (1.3-4.7)) both in group I and rs7749054 TT (p = 0.045, OR = 1.8 (1.1 -3.1) showed some relationship with FH (Table 2). The significant covariates introduced in the statistical model were gender (p <10-3, OR = 6.9 (2.8-17 )), exposure (number of years of fishing) (p = 0.05, OR = 1.02 (1-1.05)), splenectomy (p = 0.008, OR = 6.6 (1.6-27)). Multifactor analysis that tested two SNPs simultaneously in the same models in the presence of the same covariates indicated that the SNP rs 11154915 CT was associated (p = 0.04, OR = 1.9 (1-3.5) when tested in the presence of rs 6570136 (p = 0.007 OR = 2.9 (1.4 -6) Interestingly (see the study in Sudanese and Brazilians) SNP resistance 2064501 showed a tendency to association with FH that was not reduced when other SNPs were introduced into the regression model.

Finally, the association of rs7749054 was probably due to its LD with SNPs in group I, since the association of rs7749054 with FH was completely lost in the presence of SNP rs6570136 or SNP rs7774663.

Table 2. IL22RA2 SNPs related to liver fibrosis: analysis in Chinese fishermen

Sample of Chinese fisherman

SNP Position Group Genotype References Cases OR 95% p CI

%%

Analysis rs6570136 137536315 I GG 13.0 22.1 2.7 1.3 0.007 unifunctional 5.6

rs7774663 137552586 I TT 18.1 28.4 2.5 1.3 0.006

4.7 rs7749054 137542479 II TT 32.8 41.1 1.8 1.0 - 0.045

2.9 rs202563 137503185 III AA 38.7 45.8 0.3 rs276466 137508307 IV AG GG 24.3 30.2> 0.5 rs11154915 137524675 V CT (without TT) 97.2 99.1 0.16 rs2064501 137519516 VI CC TT 55.4 62.5 0.17 Analysis rs6570136 I GGun 2.9 1.4 - 6.07 pol rs 2.9 1.4 - 0.007 pol rs 2.9 1.4 - 0.007 pol rs 2.9 1.4 - 0.007 pol rs 2.9 1.4 - 0.007 pol rs 2.9 1.4 - 0.007 pol rs CT (without 1.9 1.0 - 0.04

TT) 3.5

(n = 268, 92 cases and 176 references)

The inventors then attempted to replicate these results in a second Chinese sample of farmers from an endemic region for S. japonicum. This sample differs from the fishermen sample because it was incorporated into an outpatient clinic hospital that cares for serious liver diseases, including ascites, varicose veins and cirrhosis. 92.2% of the incorporated patients lived in a region where S. japonicum was still endemic, 7.8% had been living in an endemic region of schistosome, but transmission in their region had been interrupted ten or fifteen years ago. A fraction (86.5%) of these subjects had evidence of prior HBV infection (20.9% AgHBS +) one patient had been infected with HCV. Therefore, liver disease in the majority of farmers in this second sample is probably due to infections both by schistosome and VBH.

All SNPs tested in the fishermen sample were genotyped in the farmers sample (298, 97 subjects with mild liver disease and 201 subjects with severe liver disease). Association with liver disease was observed with 4 SNPs from 3 different groups: SNP rs6570136 GG, GA (p = 0.009, OR = 2 (1.2-3.3)), rs7774663 TT, CT (p = 0.01, OR = 2 (1.2-3.3 )) both in group I; SNP rs 276466 GA (p = 0.01, OR = 2.2 (1.2-4)) in group IV; s Np rs1114915 CC, CT (p = 0.04, OR = 5.7 (1.1-29.6)) in group V (Table 3). Association trends were also observed for S ⁿ P rs202563 AA, GG (p = 0.06) in group III and SNP rs2064501 CT (p = 0.08). The covariates in these association tests were age (p = 0.05, OR = 1.03 (1-1.06), sex (p = 0.02, OR = 2.3 (1.1-4.8) and if the patient was living in an endemic region / non-endemic (p = 0.09, OR = 2) The multifactorial analysis performed in the SNPs of different groups indicated two possible statistical models: one model included the SNPs rs6570136 (or rs7774663) (p = .03, OR = 1.8 (1.1- 3)) in group I and SNP rs11154915 (p = 0.1, OR = 4 (0.8-21.2) in group V; the other model included SNP rs 276466 (p = 0.02, OR = 2 (1.1-3.8)) and SNP rs11154915 (p = 0.05, OR = 9.2 (1.1-80)) The analysis could not discriminate between these two models.

Table 3: IL22RA2 SNPs associated with liver fibrosis; Chinese farmer analysis

Chinese farmer sign

SNP Position Group Genotype References Cases OR 95% CI p%%

Analysis rs6570136 137536315 I GG AG 53.6 67.8 2 1.2-3.3 0.009 unifunctional

rs7774663 137552586 I CT TT 58.8 72.3 2 1.2-3.3 0.01 rs7749054 137542479 II GG 15.1 18.9 0.5 rs202563 137503185 III AA GG 46.9 57.3 1.6 0.98-2.7 0.06 rs276466 137508307 IV AG (without GG) 18.7 34.5 2.2 1.2-4.0 0.015 rs CT 93.8 99.3 5.7 1.1-29.6 0.036 rs2064501 137519516 VI CT 40.2 51.2 1.6 0.95-2.7 0.08 Analysis rs6570136 I GG AG 1.6 1-2.6 0.06 ^{polyfunctional} rs11154915 V CT TT 4.8 0.93-25 0.06 Model 1

(n = 298, 201 cases and 97 references)

Example 3: Extension of the association to populations of Sudan and Brazil exposed to Schistosoma mansoni

To assess whether our observation could be extended to subjects infected with S. mansoni, we tested the same SNPs in a sample from Sudan and in a sample from Brazil. Once again, a significant fraction of the subjects in the Sudanese sample who had also been infected with HBV in the Chinese farmer, while very few Brazilians had HBV infections.

The genotyping of the Sudanese sample (n = 202, 144 FH mild and 58 FH severe) showed that SNP rs6570136 GG (p = 0.01, OR = 3.1 (1.3-7.2)), rs7774663 TT, TC (p = 0.01, OR = 1.7 (1-3.1), rs11154915 TT (p = 0.05, OR = 6.2 (1 35.3)) showed associations with FH while an association trend was also detected for SNPrs7749054 TT (p = 0.07, OR = 2 (1-3.6 ) and for rs2064501 CC (p = 0.06, OR = 2.71-7.3).) See table 4.

Table 4. Extension of associations detected in Chinese to Sudanese infected with S.mansoni

Sudanese sample

SNP Position Group Genotype References Case OR 95% CI p%%

Analysis rs6570136 137536315 I GG 8.3 24.5 3.1 1.3 -7.2 0.006 unifuncional

rs7774663 137552586 I CC TT 45.5 66.7 1.7 1 -3.1 0.04 rs7749054 137542479 II TT 0.2 rs202563 137503185 III GG 21.8 34 1.6 0.9 -3.0 0.09 rs276466 137508307 IV AG GG 0.25 rs11154915 137524675 V TT 0.8 5.7 6.21 5.3 17 2.6 1.2 -5.7 0.02 Analysis rs6570136 I GG 10 3 -34 0.0002 ^{polyfunctional} Rs2064501 VI TT 3.4 1.2 -9.4 0.018 ^{Model 1} rs11154915 V TT 7.4 0.75 -74 0.09 (n = 189, 53 cases and 133 references)

Similarly, the typing of these same SNPs in the Brazilian sample (n = 161, 119 low FH and 42 severe FH) showed association with FH for SNP rs6570136 GG (p = 0.0001, OR = 6 (2.4-10 14.7)) , rs7774663 TT (p = 0.03, OR = 3 (1.4-6.8) and SNPrs7749054 TT (p = 0.03, OR = 2.8 (1.2-5.6), in addition to rs11154915 TT, CT showed a trend of association with FH (p = 0.14 , OR = 2.4 (0.9-6.5)).

See table 5:

Table 5. Replication of the association in Brazilians infected with S.mansoni

Brazilian show

SNP Position Group Genotype References Case OR 95% CI p%%

Analysis rs6570136 137536315 I GG 11.5 35.6 4.2 0.001 unifunctional

rs7774663 137552586 I TT 24.5 44.2 2.4 1.2 -5.1 0.02 rs7749054 137542479 II TT 45.5 67.4 2.5 1.2 -5.1 0.01 rs202563 137503185 III AG GG 73.5 86 0.13 rs276466 137508307 IV AG GG 26.5 41.9 2 0.9 -4.3 0.07 rs111549 CT37567515 0.9 -6 0.09 rs2064501 137519516 VI CT TT 52.8 66.7 0.15 Analysis rs6570136 I GG 24.8 3 -205 0.003 ^{polyfunctional} rs2064501 VI CT TT 10.1 1.1 -93 0.04 rs11154915 V CT TT 2.3 0.8 -6.9 0.14

The multifactorial analysis performed in the Sudanese sample confirmed the independent associations of SNP rs6570136, 20564501 and 11154915. Most notable was that the high OR related to rs11154915 was confirmed in the multifactorial model. In addition, subjects with aggravating genotypes for both SNPs had in both OR models for FH greater than 25. Multifactorial analysis showed that the association of SNP rs7749054 was lost in the presence of SNP rs6570136 confirming that this association was not independent of SNP rs6570136 In In summary, SNP rs6570136 GG and rs7774663 TT, which belong to the same correlation group, were associated with FH, in the four samples analyzed. SNPrs11154915 TT, CT and rs2064501 ^c C showed a tendency to association with FH in all samples; most importantly, these trends were confirmed by multifactor analysis that shows that these SNPs were acting independently of SNP rs6570136; taking into account these 2 SNPs increased the strength of the association of SNP rs6570136 with FH.

Example 4: Associations between SNPs in IL22RA2 and the response to anti-HCV treatment.

The inventors have performed their genetic analysis on 123 subjects (69 who responded to treatment with ribavirin IFN, 54 who did not respond to treatment) who had or have been infected with HCV genotypes 1 or 4. They tested at least one SNP in each of the 7 groups identified in IL22RA2 using Hapmap data. Unifactorial analysis showed associations with SNP rs2064501 (group VI, p = 0.013) and SNP rs1543509 (group VII, p = 0.012), but also suggested possible associations with SNPs rs7774663, rs6570136 (group I, p <0.13), SNP rs77449054 (group II), p = 0.1), SNP rs202563 (group III, p = 0.07), SNP rs28366 (group IV p = 0.15), and SNP rs2064501 (group VI, p = 0.2). This high number of SNPs in possible associations with the response to treatment could be due to correlations between the SNPs tested, and also suggested that different SNPs could have confounding effects on each. Then a multifactor analysis was performed step by step.

Tests of all SNPs two at a time indicated that the association of SNP rs202563 and rs6570136 with the response to treatment was equivalent, but that SNPs rs276466 and rs28366 were clearly excluded from the regression model by SNP rs6570136. All tests showed that the association of SNP rs1154915 with the response to treatment was improved by the presence of other SNPs (such as SNP rs6570136 or SNP rs2064501 or SNP rs1543509) in the regression model. The association of rs6570136 with the response to treatment was lost in the presence of the SNP rs2064501 or rs1543509, but this association was recovered when SNP rs11154915 was added to the model (3 SNPs in the model). This is due to the strong coupling imbalance between SNP rs11154915 and SNP rs6570136 (or any of the SNPs in group I), as a consequence, the SNP rs1154915 ^t T genotype that responds to treatment is 100% associated with genotypes that do not respond to treatment with group I. Therefore, the model must include at least 3 SNPs (rs11154915 (p = 0.03), rs7774663 (or rs6570136, p = 0.03) and SNP rs2064501 (p = 0.001). Finally SNP rs1543509 and SNPs of group I (p = 0.15) also enter this model.

In summary, the inventors have found that SNPs belonging to four different groups in IL22RA2 are independently associated with the response to treatment. It is important to note that these results that were obtained when testing the TagSNP can be extended to any SNP in the same groups. Then most, if not all SNPs in groups I, V, VI and VII can be expected to be a response of genetic markers to treatment with IFN ribavirin. It can also be seen that 3 of these identified groups were associated with the evolution of fibrosis (the SNPs in group VII have not yet been tested in fibrosis). Interestingly, most genotypes that aggravate liver fibrosis are associated with a better response to treatment.

References

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Claims (6)

1. An in vitro method for determining a risk of developing fibrosis or liver cirrhosis in a subject infected with hepatitis virus or parasites, the method comprising detecting the presence of a single nucleotide polymorphism (SNP) in the locus of the IL22RA2 gene in a biological sample of said subject, wherein said SNP is selected from the group consisting of rs6570136, rs7774663, rs11154915 and rs2064501, wherein the presence of a G allele with respect to SNP rs6570136, a T allele with respect to SNP rs7774663 , a T allele with respect to SNP rs11154915 and / or a CC genotype with respect to SNP rs2064501 is indicative of developing fibrosis or liver cirrhosis. 2. The method according to claim 1, wherein said subject is infected with liver virus A, liver virus B, liver virus C, Schistosoma japonicum or Schistosoma mansoni. 3. The method according to any of claims 1 or 2, wherein the presence of the SNP is detected by sequencing, selective hybridization and / or selective amplification or digestion by restriction enzymes. 4. The method according to any of claims 1 or 3, wherein the presence of GG genotype with respect to SNP rs6570136 is indicative of a risk of developing fibrosis or liver cirrhosis. 5. The method according to any of claims 1 or 3, wherein the presence of TT genotype with respect to SNP rs7774663 is indicative of a risk of developing fibrosis or liver cirrhosis. 6. The method according to any of claims 1 or 3, wherein the presence of TT or CT genotype with respect to SNP rs11154915 is indicative of a risk of developing fibrosis or liver cirrhosis.