# Patent

## Title
Fibrosis susceptibility gene and uses thereof

## Abstract

The present invention discloses the identification of a fibrosis susceptibility gene locus, the CTGF gene locus, which can be used for detecting predisposition to, diagnosis and prognosis of fibrosis as well as for the screening of therapeutically active drugs. The invention resides, in particular, in a method which comprises detecting in a sample from the subject the presence of an alteration in the CTGF gene locus, the presence of said alteration being indicative of the presence or predisposition to fibrosis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the national stage entry under 35 USC 371 for PCT/EP2010/052048, filed Feb. 18, 2010, which claims the benefit of the Feb. 19, 2009 priority date of European Application No. 09305159.7. The contents of both the foregoing applications are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates generally to the fields of genetics and medicine. The present invention discloses in particular the identification of a human fibrosis susceptibility gene, which can be used for the diagnosis or prognosis of fibrosis or for the detection of predisposition to fibrosis, occurring in hepatic diseases, in cirrhosis, cutaneous keloid, obesity and any fibrotic disease. The invention more particularly discloses certain alleles of the CTGF gene on chromosome 6 related to susceptibility to fibrosis and representing novel targets for the screening of therapeutically active drugs. The present invention relates more specifically to particular mutations in the CTGF gene and expression products, as well as to diagnostic tools and kits based on these mutations. The invention can be also used for the prevention and/or treatment of fibrosis occurring in all the fibrotic human diseases.
BACKGROUND OF THE INVENTION
Fibrosis is an excessive growth 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 hepatic inflammations of various aetiologies such as in Hepatitis Virus and Schistosome infections. It was shown previously that certain subjects infected by Schistosomes are slow fibrosers whereas others are rapid fibrosers and that this depends in part on a major gene located on Chr 6q22-q23 (Dessein et al., 1999; Mohamed-Ali et al., 1999).
Schistosomiasis is caused by helminths that develop in the vascular system of their hosts and lay eggs that are for some of them carried over to the liver where they trigger inflammation in the periportal space. Since worms live for years in their human host, chronic liver inflammation associated with much tissue destruction is common in infected subjects. Tissue repair requires the deposit of extracellular matrix protein (ECMP) in the damaged tissues that are later on turned over and replaced by normal hepatocytes. In some patients ECMP accumulate in the periportal space forming fibrosis deposits that reduce blood flow causing varicose veins, ascites. After months or years of chronic or repeated injury, fibrosis becomes permanent and irreversible. Subjects die of the consequences of fibrosis.
In South countries, it is estimated that 5 to 10% of the 350 millions of infected subjects may develop severe hepatic fibrosis. There is no good marker allowing to predict and follow hepatic fibrosis progression in Schistosome infected subjects.
Diagnosis of hepatic fibrosis is mostly based on liver biopsy, elastometry (Boursier et al., 2008; Macias et al., 2008) and ultrasound analysis (Richter et al., 2001; Lambertucci et al., 2004; King et al., 2003).
Biopsies are obtained via percutanous, transjugular, radiographically-guided fine-needle or laparoscopic route, depending upon the clinical setting. Histopathological examination enables the clinician to grade the severity of necroinflammation and stage the extent of fibrosis. The Metavir scoring system attributes a score to the stages of fibrosis on a 1-4 scale as follows: F0=no fibrosis, F1=portal fibrosis without septa, F2=portal fibrosis and few septae, F3=numerous septae without cirrhosis, F4=cirrhosis (Bedossa et al., 1996). Liver biopsy is an invasive and costly procedure, and samples only a small portion of the liver. Thus it cannot afford a global assessment of hepatic fibrosis, and is subject to sampling variation and inter- and intra-observer error. In addition, liver biopsy is associated with significant morbidity of 3% and a mortality rate of 0.03% (Garcia-Tsao et al., 1993). Potential complications include local hematoma, infection and pain related to the biopsy.
Noninvasive tests (i.e., serologic markers, elastometry, ultrasound analysis) are also used but are not yet ready for routine clinical use.
Panels of blood markers have been tested mostly in patients with chronic hepatitis C or cirrhosis due to viral hepatitis C. These studies revealed that serum markers can rule on or rule out fibrosis in approximately 35% of patients (Sebastiani et al., 2006). However, when looking at patients individually, these markers could not reliably differentiate between the various stages of fibrosis. A more recent study incorporated three panels of serum markers to devise an algorithmic approach that improved diagnostic accuracy (Parkes et al., 2006). The three panels evaluated were the APRI (aspartate transaminase to platelet ratio index), the Forms' index (platelets, gammaglutamyltranspeptidase, cholesterol) and the Fibrotest (GGT, haptoglobin, bilirubin, apolipoprotein A, alpha-2-macroglobulin). An algorithm consisting of the APRI followed by the Fibrotest boosted the diagnostic accuracy of fibrosis to above 90%. This group estimated that 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 highly active hepatic inflammation.
Fibroscan is another innovative approach to staging hepatic fibrosis, which is based on elastography, which provides rapid measurement of mean hepatic tissue stiffness (Ziol et al., 2005). A probe is employed to transmit a vibration of low frequency and amplitude into the liver. This vibration wave triggers an elastic shear wave, whose velocity through the liver is directly proportional to tissuestiffness measured in kilopascals (kPa). Sensitivity of the Fibroscan technique ranged from 79 to 95%, and specificity from 78 to 95%, compared to the liver biopsy. However, the limitations of this technique are associated with attenuation of elastic waves in fluid or adipose tissue, which would impair assessment of fibrosis in patients. In addition, Fibroscan is an extremely expensive instrument.
However, no efficient method exists to prognose the fibrosis progression and the treatment efficiency.
A large number of molecules have been tested for treatment of hepatic fibrosis. For example, corticosteroids have been used to suppress hepatic inflammation in autoimmune and alcoholic hepatitis (Czaja et al., 2003). Ursodeoxycholic acid has been proven to increase survival in PBC patients by binding bile acids, and thus also decreasing hepatic inflammation (Poupon et al., 1997). Neutralizing inflammatory cytokines with specific receptor antagonists (TNFalpha, IL-1 receptor antagonists) and prostaglandin E have been tested in murine models, but not yet in humans (Bruck et al., 1997).
Another attractive target in curtailing hepatic fibrosis is the downregulation of hepatic stellate cell activation. Interferon gamma is used in combination with ribavirin for therapy of hepatitis C infection. It is postulated that the antifibrotic effects of the interferons may be partially related to downregulation of stellate cell activation. This mechanism could explain the improvement in fibrosis described in patients with viral hepatitis C who do not have a virologic response to interferon alpha (Poynard et al., 1998).
Trials of antioxidants (nacetylcysteine, alpha-tocopherol) are currently underway in humans. Angiotensin II receptors are upregulated in stellate cell activation, thus angiotensin converting enzyme inhibitors and angiotensin receptor blockers have demonstrated antifibrotic activity in vitro and in animals. This has yet to be replicated in humans (Jonsson et al., 2001). Promoting matrix degradation through matrix metalloproteinases is an antifibrotic strategy shown to be beneficial in a murine model (Iimuro et al., 2003). Specific apoptosis of hepatic stellate cells is another interesting theoretical idea, but has not yet been investigated (Gressner et al., 1998). Treatments aimed at reversing the fibrosis are usually too toxic for long-term use (i.e., corticosteroids, penicillamine) or have no proven efficacy (i.e., colchicine).
In conclusion, efficient and well-tolerated antifibrotic drugs are currently lacking and current treatment of fibrosis is limited to withdrawal of the noxious agent.
It has been previously reported that fibrosis development is markedly influenced by a major locus on Chr 6q23 (Dessein et al., 1999). It has been also proposed that CTGF gene contributes to increasing fibrosis by synergizing with various pro-fibrogenic growth factors (Leask et al., 2006) including PGDF, VEGF and the master fibrogenic molecule TGF-β. More specifically CTGF acts as a TGF-β downstream modulator (Leask et al., 2006; Leask et al., 2003). It increases TGF-β binding to its receptor, interferes with the negative Smad-7 feedback loop on TGF-β. (Wahab et al., 2005); it inhibits receptor binding of the principal TGF-β antagonist BPM-7 (Abreu et al., 2002). An important consequence of CTGF action on TGF-β is the stimulation of the trans-differentiation of hepatic stellate cells and other parenchymal cells into ECMP producing myofibroblasts which is involved in progressive fibrotic process (Kalluri et al., 2003; Neilson et al., 2005). CTGF is also thought to increase ECMP networking trough its binding capacities to fibronectin domains on ECMP (Gressner et al., 2007; Yoshida et al., 2007). CTGF is produced by a variety of cells including hepatocytes (Kobayashi et al., 2005; Gressner et al., 2007), hepatic stellate cells, myofibroblasts and endothelial cells (Gressnet et al., 2008). An overexpression of CTGF transcripts have been reported in different tissues including liver (Rachfal et al., 2003) affected by fibrosis of different aetiological origin. Experimental work in rats has shown that inhibiting CTGF by siRNAs prevents or reduces tissue fibrosis (Li et al., 2006; George et al., 2007).
However, the genetic factors that control the human fibrosis susceptibility were not identified as well as their effect on fibrosis progression.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a new genetic approach for fibrosis prognosis and treatment. The present invention now discloses the identification of a human fibrosis susceptibility gene locus, the CTGF gene locus (CCN2), which can be used for detecting predisposition to, diagnosis and prognosis of fibrosis, especially of hepatic fibrosis, as well as for the screening of therapeutically active drugs. The invention resides, in particular, in a method which comprises detecting in a sample from the subject the presence of an alteration in the CTGF gene locus (CCN2), the presence of said alteration being indicative of the presence or predisposition to fibrosis.
A particular object of this invention resides in an in vitro method of detecting predisposition to or diagnosis and/or prognosis of fibrosis occurring in a subject, the method comprising detecting the presence of an alteration in the CTGF gene or polypeptide in a sample from the subject, the presence of said alteration being indicative of the presence of fibrosis or the predisposition to fibrosis. A particular object of this invention resides in a method for assessment (prediction) of the progression of fibrosis.
In a preferred embodiment, said alteration is located within 20 kb, upstream the start codon of the CTGF gene and 20 kb, downstream the 3′UTR of the CTGF gene.
Preferably, the alteration lies in the surrounding sequences of 15.3 kb region, upstream the starting codon of the CTGF gene and 14.1 kb region, downstream 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 several single nucleotide polymorphism(s) SNP(s) or a haplotype of SNPs associated with fibrosis. Preferably, said single nucleotide polymorphisms are SNPs flanking CTGF gene, which are allelic variants lying close to the CTGF gene.
The method of the invention allows for detection and prognosis of fibrosis which occurs in a human fibrotic disease selected from hepatic diseases fibrosis, cirrhosis, cutaneous keloid, hypertrophic scars and obesity. Especially, the hepatic fibrosis may be caused by hepatic A virus, hepatic B virus, hepatic C virus (HCV), Schistosoma japonicum (S. japonicum) or Schistosoma mansoni (S. mansoni) infection.
In a particular embodiment, the method comprises detecting the presence of SNP rs9402373 and/or rs6918698 in the CTGF gene locus in a biological sample of a subject infected with HCV, wherein the presence of an allele C at position rs9402373 and/or allele G at position rs6918698 is indicative of a risk of developing a hepatic fibrosis or of the development of a hepatic fibrosis, or of a poor prognostic of hepatic fibrosis in the subject.
Preferably, the alteration in the CTGF gene locus is determined by performing a selective hydridization assay, a sequencing assay, a microsequencing assay, and/or an allele-specific amplification assay.
In another aspect of the invention, said alteration in the CTGF gene is determined by restriction enzyme digestion, the detection of at least one said SNP being an indication of fibrosis.
This invention also relates to a method for selecting a therapeutic compound for a subject that has or is predisposed to develop fibrosis, said method comprising contacting a test compound with a CTGF polypeptide or gene or a fragment thereof and determining the ability of said test compound to enhance or reduce biological activity or function of a pathway related to the CTGF gene.



LEGEND TO THE FIGURES
FIG. 1: Correlation bins in the CTGF (=CCN2) gene locus in fishermen.
Thirty three markers were genotyped on 70 unrelated subjects as described in Materials and Methods. Correlations (r2 values) between SNPs were determined using Haploview software. The darkest colors indicate the strongest correlations. Correlation bins (r2>0.05) were as follows SNPs rs9321314, rs6940184, rs12523697, rs9493149 and rs12529636 were in bin 1; SNPs rs12527705 and rs12526196 in bin 2; SNPs rs9399005, rs6918698, and deletion/insertion (D/I) rs3037970 in bin 3; SNP rs2151532 and SNP rs1931002 in bin 4; SNPs rs9493150, rs928501, rs11966728, rs12198610 and a new insertion/deletion (132319925-/GAAA) were in bin 5; SNPs rs7747601, rs7768619 and rs9402373 in bin 6; SNPs rs12527379 and rs2095252 in bin 7.
Sequencing CCN2 (3.2 kb) and 15.3 kb upstream the starting codon and 14.1 kb in the 3′UTR region was done on 8 cases and 2 controls. It revealed 61 SNPs among whose 50 had been described before.
Since the inventors were looking for a gene with major effects they focused the study on 53 SNPs with a MAF>20%. 33 SNPs with a MAF>20% were genotyped on 70 fishermen. Correlations (r2 values) between SNPs were determined using Haploview software. Bottom figure shows r2 values×100. The darkest colors indicate the strongest correlations. Above this representation, a physical map with the relative position of each marker is also provided. These markers are grouped in seven correlation (r2=0.5) bins (I to VII).
SNPs rs9321314, rs6940184, rs12523697, rs9493149 and rs12529636 were in bin 1; SNPs rs12527705 and rs12526196 in bin 2; SNPs rs9399005, rs6918698 and D/I rs3037970 in bin 3; SNP rs2151532 and SNP rs1931002 in bin 4; SNPs rs9493150, rs928501, rs11966728, rs12198610 and a new insertion/deletion (132319925-/GAAA) were in bin 5; SNPs rs7747601, rs7768619 and rs9402373 in bin 6; SNPs rs12527379 and rs2095252 in bin 7.
Twenty two SNPs were further genotyped in the whole fisherman sample (201 controls and 99 cases). The results of the univariate analysis are shown on the top of FIG. 1 and in Tables 1 and 4. SNPs associated with hepatic fibrosis were SNP rs12527705 (p=0.02, OR=2.3) and SNP rs12526196 (p=0.02, or =2.2) in bin II, SNP rs9399005 (p=0.02, OR=2.2), SNP rs6918698 (p=0.02, OR=2) and D/I rs3037970 (p=0.003, OR=2.6) in bin III, SNP rs1931002 (p=0.004, OR=2.3) and rs2151532 (p=0.02, OR=1.98) in bin IV and SNP rs9402373 (p=0.015, OR=2) in bin VI. The analysis was adjusted on gender (p<0.001) exposure (fishing years: p<0.001, born on boat: p<0.01) and number of treatments (p<0.01).
In the present analysis, D/I rs3037970 excluded rs6918698 in a multivariate analysis testing simultaneously both SNPs. Likewise SNP rs1931002 excluded SNP rs2151532; SNP rs1256196 excluded SNP rs12527705 and SNP rs9399005. This indicated that SNPs or deletions rs12526196, rs30337970, rs1931002, rs9402373 had the strongest association with hepatic fibrosis (HF). When all four SNPs were tested in the same regression model (lower part Table 4), SNPs rs12526196 (p=0.007, OR=3), rs9402373 (p=0.002, OR=2.8) and rs1931002 (p=0.002, OR=2.8) showed independent associated with HF. An haplotype 1002C, 6196T, present in 53.9% controls and 67.5 cases was associated with HF (p<0.005). The inventors also tested a phenotype that associated advanced HF with evidence of portal hypertension. They found (154 controls and 151 cases) rs9402373 (p=0.005, OR=2.6) and rs3037970 (p=0.05, OR=2) were associated with that phenotype, SNP rs1256196 showed a trend for an association with this more severe disease phenotype (p=0.12). Gender (p=0.001) entered the model as covariate. This suggested that both rs9402373 and rs3037970 could have a more important contribution to severe disease.
FIG. 2A: Electrophoretic mobility shift assay reveals allele specific binding of nuclear factors with SNPs rs12526196 and rs9402373.
EMSA were performed according to Materials and Methods using nuclear extracts of a stimulated human hepatocyte cell line (HEPG2). The 12526196T allele bound nuclear factors (complex 1) with a higher affinity than the C allele. SNP rs9402373C allele bound nuclear factors (complex 2) that were not bound by the G allele. No allele specific binding was observed with SNP rs12527705 and SNP rs1931002.
FIG. 2B: Competitive electrophoretic mobility shift assay for rs9402373 polymorphism. Competitive reactions were done with 100 or 200 fmol of unbiotinylated rs9402373G and C probe. Competitive reactions performed with unbiotinylated rs9402373C probe at concentration (200 fmol) but not with the unbiotinylated rs9402373G has competed the binding of the biotinylated rs9402373C probe.
FIG. 3 shows the meta analysis of the association of SNP rs9402373 with hepatic fibrosis in HCV or Schistosome infected subjects.



DETAILED DESCRIPTION OF THE INVENTION
This invention identifies the most critical steps in fibrosis development and provides valuable genetic markers to predict disease progression in fibrosis, especially in hepatic fibrosis.
Early detection of fibrosis and regular monitoring of fibrosis, would allow for initiation of anti-fibrotic therapies capable of halting and even reversing this process. This would in turn prevent progression to human fibrosis disease, for example hepatic fibrosis or hepatic cirrhosis, and the morbidity and mortality this condition entails. The development of these various early fibrosis detection techniques bodes well for the future care of patients with liver disease.
The inventors have now identified the major gene associated with human fibrosis. They have shown that fibrosis in two Chinese, one Sudanese and one Brazilian cohort infected with Schistosoma japonicum and with Schistosoma mansoni respectively is markedly dependent on allelic variants lying in the CTGF gene. Two of these variants affect nuclear factor binding.
Various nucleic acid samples from individuals with fibrosis were submitted to a particular analysis process. Four populations with hepatic fibrosis were examined, inter alia a population of fishermen of the Dong Ting lake in central China infected with Schistosoma japonicum, a population of farmers living in Hunan province also infected with Schistosoma japonicum. 
Two additional populations infected with Schistosoma mansoni were also included (a Sudanese and a Brazilian population). This analysis process led to the identification of particular single nucleotide polymorphism(s) SNP(s) in said populations that are overrepresented in subjects having hepatic fibrosis.
The inventors first examined fibrosis in Fishermen of the Dong Ting lake in central China near Yueyang city. Analysis of the various factors that influence the development of fibrosis in the fishermen population has shown that gender, exposure to infected waters and anti-schistosome treatments with Praziquantel were significantly associated with the risk of fibrosis. The testing of several covariates has shown that the “the number of years fishing” and “being born on a fishing boat” were the best covariates to measure exposure. The “treatment” covariate is the number of Praziquantel treatments over the last twenty years.
Using these covariates, the inventors tested on a population sample (N=300) whether SNPs (three per gene) in 12 candidate genes (two genes in the 6q23 region (IFNGR1, CTGF) and ten genes out of this region) were associated with the risk of fibrosis. One SNP rs9399005 near the CTGF gene yield a suggestive association (p=0.02) with fibrosis.
Then, the inventors sequenced the 3.2 kb of the CTGF gene and 15.3 kb upstream the starting codon and 14.1 kb in the 3′UTR region in 8 cases and 2 controls (fisherman population). 61 SNPs were recorded, among whose 50 had been described before. Fifty three SNPs (53 SNPs) had a MAF>20%. Since the inventors were looking for a gene with major effects, they selected SNPs with a MAF>20%. 33 out of 53 SNPs were genotyped on 70 fishermen.
They found seven correlation bins (r2=0.5), using data from the Hap Map project, that included 5, 2, 3, 2, 5, 3, 2 SNPs in bins I to VII respectively (FIG. 1). Finally, 22 SNPs or genetic variations were genotyped for further analysis. These 22 were added to the three SNPs tested in the early steps.
On these 25 SNPs or genetic variations (22+3) of the CTGF gene locus, 8 genetic variations were identified as being correlated to hepatic fibrosis in human subjects (rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, 1931002, rs2151532 and rs9402373). Thus, a particular object of this invention resides in a method of detecting predisposition to and/or prognosis of fibrosis, the method comprising detecting the presence of one or several SNPs or alteration selected in the group consisting of rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, 1931002, rs2151532 and rs9402373.
Among these SNPs, C allele of rs9402373 or T allele of rs12526196 specifically binds nuclear factors SRY, Lyf-1, CdxA, CP2, Ik-2, or Nkx-2.
SNPs rs6918698 and rs9402373 were shown to be associated with hepatis fibrosis in HCV infected subjects. In particular, allele C of rs9402373 was shown to be deleterious, i.e. it is strongly correlated with severe hepatic fibrosis.
Although the experimental data gathered by the inventors did not allow to confirm association of certain alleles, depending on the tested population, the present invention is not limited to the particular SNPs that were found significantly correlated with fibrosis in all tested populations. Indeed, several reasons could account for the failure in confirming significant correlation in some populations, including an insufficient cohort, the incomplete assessment of confounding variables, a lower frequency of the SNPs in said populations, etc.
DEFINITIONS
Within the context of this invention, “fibrosis” designates all types of human fibrosis occurring in all the fibrotic human diseases, for example in hepatic diseases, cirrhosis, cutaneous keloid, hypertrophic scars, sclerodermia, obesity and any fibrotic disease.
Within the context of this invention, “hepatic fibrosis” or “HF” designates all types of fibrosis occurring in a liver, tissue thereof or any part of tissue thereof. Hepatic fibrosis occurs especially in response to an injury. Hepatic fibrosis can be the common response to chronic liver injury, ultimately leading to cirrhosis and its complications, portal hypertension, liver failure, and hepatocellular carcinoma. Hepatic fibrosis is overly exuberant wound healing in which excessive connective tissue builds up in the liver. The extracellular matrix is either overproduced, degraded deficiently, or both. The trigger is chronic injury, especially if there is an inflammatory component. Various types of chronic liver injury can cause fibrosis, such as chemical fibrosis (CCl4), bacterial (i.e., brucellosis), parasitic (i.e., bilharziosis/schistosomiasis caused by Schistosoma species; or echinococcosis infections) or viral (i.e., hepatitis caused by hepatic A virus (HAV), hepatic B virus (HBC) or hepatic C virus (HCV) infections).
Within the context of this invention, “cutaneous keloid” is an excessive growth of scar tissue on the skin. More particularly, keloids and hypertrophic scars (HSc) are dermal fibroproliferative disorders unique to humans that occur following trauma, inflammation, surgery, burns and sometimes spontaneously. These are characterized by excessive deposition of collagen in the dermis and the subcutaneous tissues. Contrary to the fine line scar characteristics of normal wound repair, the exuberant scarring of keloid and HSc results typically in disfigurement, contractures, pruritis and pain. Keloids occur in individuals with a familial disposition among the Blacks, Hispanics and Orientals. Unlike HSc, the keloid scars enlarge and extend beyond the margins of the original wound and rarely regress. 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 extra cellular matrix (ECM) proteins and remodelling of the newly synthesized matrix. Biologically, keloids are fibrotic tissue characterized by a collection of atypical fibroblasts with excessive deposition of extracellular matrix components, especially collagen, fibronectin, elastin, and proteoglycans. Generally, keloids contain relatively acellular centers and thick, abundant collagen bundles that form nodules in the deep dermal portion of the lesion. The release and activation of growth factors during the inflammatory phase of healing are pre-requisites for the scar processes, including angiogenesis, reepithelialization, recruitment and proliferation of fibroblasts and matrix deposition. Then, abnormal production of activity of the regulating cytokine including CTGF, could contribute to the development of keloids.
Within the context of this invention, “the CTGF gene locus” (Connection Tissue Growth Factor), also called CCN2 gene locus, designates all sequences or products in a cell or organism, including CTGF coding sequences, CTGF non-coding sequences (e.g., introns), CTGF regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator, etc.), all corresponding expression products, such as CTGF RNAs (e.g., mRNAs) and CTGF polypeptides (e.g., a pre-protein and a mature protein); as well as surrounding sequences of 20 kb region, preferably 15.3 kb region, upstream the starting codon of the CTGF gene and 20 kb region, preferably 14.1 kb region, downstream the untranslated region (3′UTR). For example, the CTGF locus comprises surrounding sequences comprising the 61 SNPs identified by sequencing (FIG. 1) and in particular 8 SNPs of Table 1. However in a particular embodiment most alterations are not in the promoter sequence.
Within the context of the present invention, the term “prognosis” includes the detection, monitoring, dosing, comparison, etc., at various stages, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth. Prognosis typically includes the assessment (prediction) of the progression of fibrosis and the characterization of a subject to define most appropriate treatment (pharmaco-genetics), etc. The present invention provides prognostic methods to determine the speed of the progression of fibrosis or an associated disorder resulting from a mutation or a polymorphism in the CTGF gene locus. Prognosis, which analyzes and predicts response to a treatment or drug, or side effects to a treatment or drug, aims at determining whether an individual should be treated with a particular treatment drug. For example, if the prognosis indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the prognostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. Clinical drug trials represent another application for the CTGF gene locus SNPs. One or more CTGF SNPs indicative of response to a drug or to side effects to a drug may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favourably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
Alterations
The alteration may be determined at the level of the CTGF DNA, RNA or polypeptide. Optionally, the detection is performed by sequencing all or part of the CTGF gene locus or by selective hybridization or amplification of all or part of the CTGF gene locus. More preferably a CTGF gene locus specific amplification is carried out before the alteration identification step. An alteration in the CTGF gene locus may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up 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 occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene locus. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene locus. Rearrangement includes inversion of sequences. The CTGF gene locus alteration may result in the creation of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing or processing, product instability, truncated polypeptide production, etc. The alteration may result in the production of a CTGF polypeptide with altered function, stability, targeting or structure. The alteration may also cause a reduction in protein expression or, alternatively, an increase in said production.
In a preferred embodiment, said alteration is a mutation, an insertion or a deletion of one or more bases. In a particular embodiment of the method according to the present invention, the alteration in the CTGF gene locus is selected from a point mutation, a deletion and an insertion in the CTGF gene or corresponding expression product, more preferably a point mutation and a deletion. The alteration may be determined at the level of the CTGF DNA, RNA or polypeptide.
A particular object of this invention is a method of detecting predisposition to and/or prognosis of fibrosis, the method comprising detecting the presence of one or several alterations or SNPs selected in the group consisting of rs9402373, rs1931002, rs12526196, rs1257379, rs12527705, rs9399005, rs6918698, rs3037970, rs2151532, rs9321314, rs9321315, rs6910279, rs6940184, rs12523697, rs9493149, rs12529636, rs6917644, rs9493150, rs928501, rs11966728, rs7747601, rs12198610, 132319925 D/I, rs9483364, rs2095252, rs6926879, rs34118837, rs1931003, rs12191459, rs12206863, rs7768619, rs10872386 and rs2327184.
The 132319925 D/I (deletion/insertion) polymorphism is a new polymorphism that was never described before. To be able to identify this polymorphism the inventors gave as name the position according to the coordinate system and D/I to indicate that this polymorphism induces a deletion or an insertion. This polymorphism can be also written 132319925-/GAAA.
Preferably, said alteration(s) or SNP(s) associated with fibrosis is(are) selected from the group consisting of rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, rs1931002, rs2151532 and rs9402373. More preferably, said rs9402373C and rs12526196T alleles preferentially bind nuclear factors.
These alterations/SNPs are reported in the following Table 1.







TABLE 1







Fibrosis-associated alterations in the CTGF


gene locus










Nucleotide





position in





genomic
Alteration/




sequence of
SNP
Poly-
Sequence


chromosome
reference
morphism
reference





132304944
rs12527705
A/T
SEQ ID NO: 1


 


132305169
rs12526196
C/T
SEQ ID NO: 2


 


132310657
rs9399005
C/T
SEQ ID NO: 3


 


132314950
rs6918698
C/G
SEQ ID NO: 4


 


132316891
rs3037970
-/TAAAA
SEQ ID NO: 5




(D/I)



 


132320175
rs1931002
A/G
SEQ ID NO: 6


 


132316423
rs2151532
C/T
SEQ ID NO: 7


 


132319124
rs9402373
C/G
SEQ ID NO: 8


 


132321533
rs12527379
A/G
SEQ ID NO: 9


 


132294634
rs9321314
A/G
SEQ ID NO: 10


 


132294969
rs9321315
A/T
SEQ ID NO: 11


 


132295757
rs6910279
A/G
SEQ ID NO: 12


 


132299137
rs6940184
G/T
SEQ ID NO: 13


 


132299717
rs12523697
C/T
SEQ ID NO: 14


 


132300452
rs9493149
A/G
SEQ ID NO: 15


 


132301414
rs12529636
A/G
SEQ ID NO: 16


 


132307358
rs6917644
A/G
SEQ ID NO: 17


 


132315684
rs9493150
C/G
SEQ ID NO: 18


 


132317130
rs928501
A/C
SEQ ID NO: 19


 


132318298
rs11966728
C/T
SEQ ID NO: 20


 


132318597
rs7747601
A/G
SEQ ID NO: 21


 


132318713
rs12198610
A/G
SEQ ID NO: 22


 


132323491
rs9483364
A/G
SEQ ID NO: 23


 


132325281
rs2095252
A/G
SEQ ID NO: 24


 


132304054
rs6926879
A/C
SEQ ID NO: 25


 


132307157
rs1931003
A/G
SEQ ID NO: 26


 


132309432
rs12191459
C/T
SEQ ID NO: 27


 


132310735
rs12206863
A/C
SEQ ID NO: 28


 


132319026
rs7768619
C/T
SEQ ID NO: 29


 


132323557
rs10872386
C/T
SEQ ID NO: 30


 


132324717
rs2327184
A/G
SEQ ID NO: 31


 


132304021
rs34118837
C/G
SEQ ID NO: 32


 


132319925
-(promoteur 5)
-/GAAA





(D/I)









Alterations in the CTGF gene may be detected by determining the presence of an altered CTGF RNA expression. 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 quantity of RNA, etc. These may be detected by various techniques known in the art, including by sequencing all or part of the CTGF RNA or by selective hybridisation or selective amplification of all or part of said RNA, for instance.
In a further variant, the method comprises detecting the presence of an altered CTGF polypeptide expression. Altered CTGF polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of CTGF polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies), for instance.
As indicated above, various techniques known in the art may be used to detect or quantify altered CTGF gene or RNA expression or sequence, including sequencing, hybridisation, amplification and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Some of these approaches (e.g., SSCA and CGGE) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments may then be sequenced to confirm the alteration. Some others are based on specific hybridization between nucleic acids from the subject and a probe specific for wild-type or altered CTGF gene or RNA. The probe may be in suspension or immobilized on a substrate. The probe is typically labelled to facilitate detection of hybrids. Some of these approaches are particularly suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, more preferably of a specific antibody.
In a preferred embodiment, the method comprises detecting the presence of an altered CTGF gene expression profile in a sample from the subject. As indicated above, this can be accomplished more preferably by sequencing, selective hybridisation and/or selective amplification of nucleic acids present in said sample.
Sequencing
Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete CTGF gene locus or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious 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. Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. 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 from the CTGF gene locus are able to specifically hybridize with a portion of the CTGF gene locus that flank a target region of said locus, said target region being altered in certain subjects having fibrosis or associated disorders. Examples of such target regions are provided in Table 2. Another particular object of this invention resides in a nucleic acid primer useful for amplifying sequences from the CTGF gene or locus including surrounding regions. Such primers are preferably complementary to, and hybridize specifically to nucleic acid sequences in the CTGF gene locus. Particular primers are able to specifically hybridize with a portion of the CTGF gene locus that flank a target region of said locus, said target region being altered in certain subjects having fibrosis or associated disorders. Primers that can be used to amplify CTGF target region comprising SNPs as identified in Table 2 may be designed based on their sequence or on the genomic sequence of CTGF.
The invention also relates to a nucleic acid primer, said primer being complementary to and hybridizing specifically to a portion of a CTGF gene locus coding sequence (e.g., gene or RNA) altered in certain subjects having fibrosis or associated disorders. In this regard, particular primers of this invention are specific for altered sequences in a CTGF gene locus or RNA. By using such primers, the detection of an amplification product indicates the presence of an alteration in the CTGF gene locus. In contrast, the absence of amplification product indicates that the specific alteration is not present in the sample. The invention also concerns the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to fibrosis or an associated disorder in a subject or in a method of assessing the response of a subject to a treatment of fibrosis or an associated disorder.
Selective Hybridization
Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s). A particular detection technique involves the use of a nucleic acid probe specific for wild-type or altered CTGF gene or RNA, followed by the detection of the presence of a hybrid. The probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies). The probe is typically labeled to facilitate detection of hybrids. In this regard, a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered CTGF gene locus, and assessing the formation of an hybrid. In a particular preferred embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for wild type CTGF gene locus and for various altered forms thereof. In this embodiment, it is possible to detect directly the presence of various forms of alterations in the CTGF gene locus in the sample. Also, various samples from various subjects may be treated in parallel.
Within the context of this invention, a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridization with a (target portion of a) CTGF gene or RNA, and which is suitable for detecting polynucleotide polymorphisms associated with CTGF alleles which predispose to or are associated with fibrosis. Probes are preferably perfectly complementary to the CTGF gene, RNA, or target portion thereof. Probes typically comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. It should be understood that longer probes may be used as well. A preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a CTGF gene locus or RNA that carries an alteration.
The method of the invention employs a nucleic acid probe specific for an altered (e.g., a mutated) CTGF gene or RNA, i.e., a nucleic acid probe that specifically hybridizes to said altered CTGF gene or RNA and essentially does not hybridize to a CTGF gene or RNA lacking said alteration. Specificity indicates that hybridization to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that certain mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridization.
Particular examples of such probes are nucleic acid sequences complementary to a target portion of the genomic region including the CTGF gene locus or RNA carrying a point mutation as listed in Table 1 above. More particularly, the probes can comprise a sequence selected from the group consisting of SEQ ID NO 1 to 8 or a fragment thereof comprising the SNP or a complementary sequence thereof.
The sequence of the probes can be derived from the sequences of the CTGF gene and RNA as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe. Such chemical modifications may be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labelling, etc. The invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to fibrosis or an associated disorder in a subject or in a method of assessing the response of a subject to a treatment of fibrosis or an associated disorder.
Specific Ligand Binding
As indicated above, alteration in the CTGF gene locus may also be detected by screening for alteration(s) in CTGF polypeptide sequence or expression levels. In this regard, contacting the sample with a ligand specific for a CTGF polypeptide and determining the formation of a complex is also described. Different types of ligands may be used, such as specific antibodies. In a specific embodiment, the sample is contacted with an antibody specific for a CTGF polypeptide and the formation of an immune complex is determined. Various methods for detecting an immune complex can be used, such as ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Within the context of this invention, an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab′2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, poly-functional antibodies, etc. An antibody specific for a CTGF polypeptide designates an antibody that selectively binds a CTGF polypeptide, i.e., an antibody raised against a CTGF polypeptide or an epitope-containing fragment thereof. Although non-specific binding towards other antigens may occur, binding to the target CTGF polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding.
It is also disclosed a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence of an alteration in the CTGF gene locus or polypeptide, in the CTGF gene or polypeptide expression, and/or in CTGF activity. Said diagnostic kit comprises any primer, any pair of primers, any nucleic acid probe and/or any ligand, preferably antibody, described in the present invention. Said diagnostic kit can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction.
Linkage Disequilibirum
Once a first SNP has been identified in a genomic region of interest, more particularly in CTGF gene locus, other additional SNPs in linkage disequilibrium with this first SNP can be identified. Indeed, any SNP in linkage disequilibrium with a first SNP associated with fibrosis or an associated disorder will be associated with this trait. Therefore, once the association has been demonstrated between a given SNP and fibrosis, the discovery of additional SNPs associated with this trait can be of great interest in order to increase the density of SNPs in this particular region. Identification of additional SNPs in linkage disequilibrium with a given SNP involves: (a) amplifying a fragment from the genomic region comprising or surrounding a first SNP from a plurality of individuals; (b) identifying of second SNP in the genomic region harboring or surrounding said first SNP; (c) conducting a linkage disequilibrium analysis between said first SNP and second SNP; and (d) selecting said second SNP as being in linkage disequilibrium with said first marker. Sub-combinations comprising steps (b) and (c) are also contemplated. These SNPs in linkage disequilibrium can also be used in the methods according to the present invention, and more particularly in the diagnosic methods according to the present invention.
Causal Mutation
Mutations in the CTGF gene locus which are responsible for fibrosis may be identified by comparing the sequences of the CTGF gene locus from patients presenting fibrosis or an associated disorder and control individuals. Based on the identified association of SNPs of CTGF, the identified locus can be scanned for mutations. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the CTGF gene locus are scanned for mutations. Preferably, patients presenting fibrosis carry the mutation shown to be associated with fibrosis and control individuals do not carry the mutation or allele associated with fibrosis. It might also be possible that patients presenting fibrosis carry the mutation shown to be associated with fibrosis with a higher frequency than control individuals. The method used to detect such mutations generally comprises the following steps: amplification of a region of the CTGF gene locus comprising a SNP or a group of SNPs associated with fibrosis from DNA samples of the CTGF gene locus from patients presenting fibrosis and control individuals; sequencing of the amplified region; comparison of DNA sequences of the CTGF gene from patients presenting fibrosis or an associated disorder and control individuals; determination of mutations specific to patients presenting fibrosis.
Drug Screening
New methods for the screening of drug candidates or leads are also described. These methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems, in animals, etc. A particular object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a CTGF gene or polypeptide according to the present invention and determining the ability of said test compound to bind said CTGF gene or polypeptide. Binding to said gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to fibrosis in a subject. In a preferred embodiment, the method comprises contacting in vitro a test compound with a CTGF polypeptide or a fragment thereof according to the present invention and determining the ability of said test compound to bind said CTGF polypeptide or fragment. The fragment preferably comprises a binding site of the CTGF polypeptide. Preferably, said CTGF gene or polypeptide or a fragment thereof is an altered or mutated CTGF gene or polypeptide or a fragment thereof comprising the alteration or mutation. A particular object of this invention resides in a method of selecting compounds active on fibrosis, said method comprising contacting in vitro a test compound with a CTGF polypeptide according to the present invention or binding site-containing fragment thereof and determining the ability of said test compound to bind said CTGF polypeptide or fragment thereof. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation. The method for the screening of drug candidates comprises contacting a recombinant host cell expressing a CTGF polypeptide according to the present invention with a test compound, and determining the ability of said test compound to bind said CTGF and to modulate the activity of CTGF polypeptide. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation. The determination of binding may be performed by various techniques, such as by labelling of the test compound, by competition with a labelled reference ligand, etc. The method of selecting biologically active compounds also comprises contacting in vitro a test compound with a CTGF polypeptide and determining the ability of said test compound to modulate the activity of said CTGF polypeptide. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation.
The method of selecting biologically active compounds for a subject that has or is predisposed to develop fibrosis, also comprises contacting in vitro a test compound with a CTGF gene according to the present invention and determining the ability of said test compound to modulate the expression of said CTGF gene. Preferably, said CTGF gene or a fragment thereof is an altered or mutated CTGF gene or a fragment thereof comprising the alteration or mutation.
The method of screening, selecting or identifying active compounds, particularly compounds active on fibrosis, also comprises contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a CTGF gene promoter, and selecting the test compounds that modulate (e.g. activate or inhibit) expression of the reporter gene. Preferably, said CTGF gene promoter or a fragment thereof is an altered or mutated CTGF gene promoter or a fragment thereof comprising the alteration or mutation.
The above screening assays may be performed in any suitable device, such as plates, tubes, dishes, flasks, etc. Typically, the assay is performed in multi-wells plates. Several test compounds can be assayed in parallel. Furthermore, the test compound may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., in isolated or in mixture with other substances. The compounds may be all or part of a combinatorial library of products, for instance.
Pharmaceutical Compositions, Therapy
It is also described a pharmaceutical composition comprising (i) a CTGF polypeptide or a fragment thereof, a nucleic acid encoding a CTGF polypeptide or a fragment thereof, a vector or a recombinant host cell as described above and (ii) a pharmaceutically acceptable carrier or vehicle. The invention also relates to a method of treating or preventing fibrosis or an associated disorder in a subject, the method comprising administering to said subject a functional (e.g., wild-type) CTGF polypeptide or a nucleic acid encoding the CTGF gene locus. It is also described a method of treating or preventing fibrosis in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a CTGF gene locus or protein according to the present invention. Said compound can be an agonist or an antagonist of CTGF, an antisense or a RNAi of CTGF, an antibody or a fragment or a derivative thereof specific to a CTGF polypeptide. In a particular embodiment of the method, the modulation is an inhibition. In another particular embodiment of the method, the modulation is an activation.
It is also disclosed the use of a functional CTGF polypeptide, a nucleic acid encoding the CTGF gene locus, or a compound that modulates expression or activity of a CTGF gene or protein according to the present invention, in the manufacture of a pharmaceutical composition for treating or preventing fibrosis in a subject. Said compound can be an agonist or an antagonist of CTGF, an antisense or an RNAi of CTGF, an antibody or a fragment or a derivative thereof specific to a CTGF polypeptide. In a particular embodiment of the method, the modulation is an inhibition. In another particular embodiment of the method, the modulation is an activation.
The present invention demonstrates the correlation between fibrosis and the CTGF gene locus. The invention thus provides a novel target of therapeutic intervention. Various approaches can be contemplated to restore or modulate the CTGF activity or function in a subject, particularly those carrying an altered CTGF gene locus. Supplying wild-type function to such subjects is expected to suppress phenotypic expression of fibrosis in a pathological cell or organism. The supply of such function can be accomplished through gene or protein therapy, or by administering compounds that modulate or mimic CTGF polypeptide activity (e.g., agonists as identified in the above screening assays).
The wild-type CTGF gene or a functional part thereof may be introduced into the cells of the subject in need thereof using a vector as described above. The vector may be a viral vector or a plasmid. The gene may also be introduced as naked DNA. The gene may be provided so as to integrate into the genome of the recipient host' cells, or to remain extra-chromosomal. Integration may occur randomly or at precisely defined sites, such as through homologous recombination. In particular, a functional copy of the CTGF gene may be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, cationic lipid-mediated transfection, etc. Gene therapy may be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional CTGF polypeptide.
Other molecules with CTGF activity (e.g., peptides, drugs, CTGF agonists, or organic compounds) may also be used to restore functional CTGF activity in a subject or to suppress the deleterious phenotype in a cell. Restoration of functional CTGF gene locus function in a cell may be used to prevent the development of fibrosis or to reduce progression of said disease.
Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application.
Materials and Methods
Statistical Analysis
Multivariate logistic regression was used to analyse the relationship between the probability of an individual developing fibrosis and genetic variants including the main covariates known to affect disease progression in subjects infected with schistosomes. The statistical SPSS software (version 10.0) was used for this analysis. Age and gender were tested in the regression models and kept when they showed an association (p<0.05) with disease. Since the cohorts were matched for gender and age, these covariates had little effects on the association between genetic variants and disease. The number of Praziquantel treatments, infection with HBV and exposure to infection were included in the regression models when these covariates could be evaluated accurately as in the Chinese fishermen (exposure, number of treatments) or in the Chinese farmers (HBV infection, place of birth).
DNA Extraction
Aliquots of 5 to 15 ml of blood were collected on sodium citrate and kept at −20° C. DNA was extracted using the standard salting out method (Sambrook et al., 1989). Some subjects refused bleeding. In this case, buccal cell samples were collected using foam-tipped applicators and applied to indicating FTA1 cards following the protocol described by Whatman (http://www.whatman.co.uk/). Place the indicating FTA1 card on a clean, dry, flat surface. Label the FTA1 card with a unique identifying name or number. Remove one foam-tipped applicator from the protective packaging. Holding the plastic handle of the applicator, place the foam tip in the mouth and rub one side of the foam tip on the inside of the cheek for 30 s. Repeat using the opposite side of the foam tip for the other cheek. Run the foam tip along the gum-line and fold of the cheek and under the tongue, soaking up as much saliva as possible. Remove the applicator from the mouth. Carefully lift the paper cover of the indicating FTA1 card and press the flat and circular foam applicator tip with the sample area. Without lifting the foam tip from the card. Squeeze the tip using a side to side motion (908 in each direction) three times to completely saturate the sample area. The sample area will turn white upon transfer of the sample. Position the FTA1 card for drying on a clean surface. Allow the card to dry at least 1 h at room temperature. Afterwards the punches are washed three times in 200 μl of FTA purification buffer and in TE buffer successfully.
DNA Amplification
All the DNA purified from FTA card were pre amplified before genotyping. Polymerase chain reactions (whole genome amplifications) were conducted in 50 μl reactions containing one punch of biological sample (FTA1-bound buccal cell DNA) or 100 ng of genomic DNA, 1.5 OD of 15-base totally degenerate random primer (Genetix, Paris, France), 200 mM dNTPs, 5 mM MgCl2, 5 ml of 10×PCR buffer and 0.5 unit of high fidelity Taq DNA polymerase (BIOTAQ DNA Polymerase, Bioline London, England). Samples were amplified in a multiblock thermocycler as follows: a pre-denaturation step 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 step of 5 min at 72° C.
Sequencing
Purified PCR products were sequenced using ABI Prism BigDye Terminator cycle sequencing system (PE Applied Biosystems, Foster City, U.S.A.) on ABI Prism automatic sequencer. Sequencing reactions were performed on both strands Sequencing by GATC biotech (GATC, Marseille France). The sequencing primers are described in Table 2. Sequencing CCN2 (3.2 kb) and 15.3 kb upstream the starting codon and 14.1 kb in the 3′UTR region in eight cases and two controls revealed 61 SNPs (FIG. 1) among whose 50 had been described before.
Polymorphism Genotyping by PCR 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 fluorescently-labelled hybridisation probe in a total volume of 5 μl. RT-PCR was conducted in an ABI Prism Sequence Detection System 7900 (Applied Biosystems, Lafayette USA) using the following conditions: 50° C. for 2 min, 95° C. for 10 min and 40 cycles of amplification (95° C. denaturation for 15 s, 60° C. annealing/extension for 1 min). The following SNPs or alterations were genotyped by this methodology: rs9321314, rs9321315, rs6910279, rs6940184, rs12523697, rs9493149 rs12529636, rs12527705, rs12526196, rs6917644, rs9399005, rs6918698, rs9493150, rs2151532, rs3037970, rs928501, rs11966728, rs7747601, rs12198610, rs9402373, 132319925 D/I, rs1931002, rs12527379, rs9483364, rs2095252.
Polymorphism Genotyping by PCR and Restriction Enzyme Digestion
Polymorphisms (rs6926879, rs34118837, rs1931003, 12191459, rs12206863, 7768619, rs10872386 and rs2327184) were genotyped by restriction enzyme analysis under standard conditions described by the enzyme manufacturers (Euromedex, Mundolsheim, France or New England Biolabs, Beverly, USA). Polymerase chain reaction (PCR) amplifications were carried out on a robocycler gradient 96 (Stratagene, La Jolla, U.S.A.) according to standard protocol. Each digestion was resolved on either agarose or acrylamide gels, stained with ethidium bromide and visualized by UV. Primers are described in Table 2.
Nuclear Extract Preparation
Nuclear extracts were prepared from human hepatocyte cell line (HEPG2) stimulated for one hour with dexamethasone (1 mM) since hepatocytes produce CTGF in hepatic fibrosis (HF) Kobayashi et al., 2005, Gressner et al., 2007) together with hepatic stellate cells, and endothelial cells and myo-fibroblasts (Gressner et al., 2008). The extracts were prepared with the nuclear and cytoplasmic extraction reagents from Pierce (NE-PER; Pierce, Rockford, Ill., USA).
Electrophoretic Mobility Shift Assay (EMSA)
Complementary single-stranded oligonucleotides were commercially synthetised to span approximately 10 bp on either side of the variant nucleotide, as follows:









rs9402373C



(SEQ ID NO: 33)



GCTCTCAAAACTAAGCCCAACTC



 



rs9402373G



(SEQ ID NO: 34)



GAGTTGGGCTTAGTTTTGAGAGC



 



rs12527705A



(SEQ ID NO: 35)



GTAATATAGAAGATGGGTCTA



 



rs12527705T



(SEQ ID NO: 36)



GTAATATAGATGATGGGTCTA



 



rs12526196C



(SEQ ID NO: 37)



GAATATACAACGAATATGGGC



 



rs12526196T



(SEQ ID NO: 38)



GAATATACAATGAATATGGGC



 



rs1931002A



(SEQ ID NO: 39)



TGGATGATTCAAACAACTTGG



 



rs1931002G



(SEQ ID NO: 40)



TGGATGATTCGAACAACTTGG






Complementary strands were annealed by placing reactions (oligonucleotide sens and oligonucleotide antisens) in boiling water for 10 min and allowing to cool to room temperature. Binding reactions were set up with LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, Ill., USA). Aliquots of 20 fmol of complementary DNA were incubated at room temperature for 20 min with 4 mg of nuclear extract in 10 mM Tris, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2, 50 ng/μl poly d(I-C), 0.05% NP-40, pH 7.5. Then reactions were loaded onto an 8% non-denaturing polyacrylamide gel and run for 150 min at 110V. Free DNA and DNA/protein complexes were transferred to nylon N+ membrane by capillary action. Binding was detected according to manufacturer's instructions (Pierce, Rockford, Ill., USA).







TABLE 2







Primers used to prepare templates for sequencing










Position according
PCR


Primer sequences
to the
product


(forward and reverse)
coordinate system.
size













AAGGGGCAAGAGACCAAAGT
(SEQ ID NO: 41)
132325212-132325194
831 bp


CACCCTGCCATTTCATAGAAC
(SEQ ID NO: 42)
132324384-132324404



 


ACCAGGGTGATGGTGCTAAA
(SEQ ID NO: 43)
132324557-132324538
783 bp


AATGACCATGAAAGGGCTTG
(SEQ ID NO: 44)
132323775-132323794



 


TGCCCCCATATGTACAGAAA
(SEQ ID NO: 45)
132323861-132323842
844 bp


GGGACATTTTGCAAGGGTTA
(SEQ ID NO: 46)
132323018-132323037



 


TTGCTAGAAAAAGGCTGTCAA
(SEQ ID NO: 47)
132323290-132323270
827 bp


CAGCCCAATATTCCACCAAG
(SEQ ID NO: 48)
132322464-132322483



 


GCCAAGTTTATTTTGGCAGGT
(SEQ ID NO: 49)
132322614-132322593
826 bp


TTGGTTCTTCTTGATTGTGGTTT
(SEQ ID NO: 50)
132321788-132321810



 


GCAAAGCAACATTGGTTCAA
(SEQ ID NO: 51)
132321921-132321912
844 bp


GGTATGCTTTGGGAGGCTTA
(SEQ ID NO: 52)
132321088-132321107



 


TCTCAGCCGAAACAAGACTG
(SEQ ID NO: 53)
132321195-132321176
756 bp


TTTAGCGGGAACGCTTCTTA
(SEQ ID NO: 54)
132320440-132320459



 


ACCGTGAAAGGGCTTTGTAA
(SEQ ID NO: 55)
132320556-132320537
808 bp


TGCTCAGCTTATTTTTGTCACC
(SEQ ID NO: 56)
132319749-132319770



 


CCCTAACTCTCCCCATCCTC
(SEQ ID NO: 57)
132319847-132319828
823 bp


ATGTGCAGCTCAAGGAGACA
(SEQ ID NO: 58)
132319044-132319025



 


TTGATTTTCTCTGAGCTCTCACC
(SEQ ID NO: 59)
132319238-132319216
812 bp


TAGGAATGAGCCTGGTGGTC
(SEQ ID NO: 60)
132318427-132318446



 


GGTGTCCCCAAATCACACAT
(SEQ ID NO: 61)
132318639-132318620
838 bp


TATGGGCATCTGGACAGTGA
(SEQ ID NO: 62)
132317802-132317821



 


ACGCCCTCTCCTTAACCTTC
(SEQ ID NO: 63)
132318031-132318012
838 bp


AGCCCCTTTGATTGACAGC
(SEQ ID NO: 64)
132317194-132317212



 


GTTAATTCCTCATTTTACCACGA
(SEQ ID NO: 65)
132317353-132317331
647 bp


AGTCCCTCGAAGTCTCAAAAA
(SEQ ID NO: 66)
132316706-132316726



 


GCACAGAAACTTTTTCTTTCCTG
(SEQ ID NO: 67)
132316835-132316813
625 bp


AAGGGTAGGGATGTGCAGTG
(SEQ ID NO: 68)
132316211-132316230



 


CAGGCATGAAGATGGTGGTA
(SEQ ID NO: 69)
132316364-132316345
679 bp


GGTGCAAAGATCGGCTTTAG
(SEQ ID NO: 70)
132315687-132315706



 


TCTTCTTGGCTATTTTTCAACAGA
(SEQ ID NO: 71)
132315803-132315780
739 bp


GGGGTTCACCTAGGAGCATT
(SEQ ID NO: 72)
132315065-132315084



 


CAAGACAAACCAAATCCAATCC
(SEQ ID NO: 73)
132315178-132315157
706 bp


TATCCTCTTCGCACCACTCC
(SEQ ID NO: 74)
132314474-132314493



 


GTGGACAGAACAGGGCAAAC
(SEQ ID NO: 75)
132314570-132314551
637 bp


GCTTACCCGGCTGCAGAG
(SEQ ID NO: 76)
132313934-132313951



 


ACAGCCCCGAGACGACAG
(SEQ ID NO: 77)
132314127-132314110
635 bp


GCAGCAGCTGGAGAAAGAAA
(SEQ ID NO: 78)
132313593-132313512



 


AAAAAGAAACCGCTCGGACT
(SEQ ID NO: 79)
 132313535-1323135016
602 bp


CCAGGCAGTTGGCTCTAATC
(SEQ ID NO: 80)
132312934-132312953



 


TCAGGGTCGTGATTCTCTCC
(SEQ ID NO: 81)
132313102-132313083
746 bp


TGGAGATTTTGGGAGTACGG
(SEQ ID NO: 82)
132312357-132312376



 


TCAAACTTCCTCCCCTCAAA
(SEQ ID NO: 83)
132312529-132312510
704 bp


TGCTCCTAAAGCCACACCTT
(SEQ ID NO: 84)
132311826-132311845



 


GGAAAAGATTCCCACCCAAT
(SEQ ID NO: 85)
 132311978-1323119459
659 bp


CCATTTCATGCTTTGAACGA
(SEQ ID NO: 86)
132311320-132311339



 


TTGTGTGTGTGTGTGTGTGTGT
(SEQ ID NO: 87)
132311497-132311476
806 bp


AATGCCAAAAGGACCAAGTG
(SEQ ID NO: 88)
132310673-132310692



 


CAGACTTGCAGGCATACACA
(SEQ ID NO: 89)
132310821-132310802
844 bp


TGGTTTGTGGTGTGGACAGT
(SEQ ID NO: 90)
132309978-132309997



 


CCACATGAGGATAGCTGAGGA
(SEQ ID NO: 91)
132310141-132310121
794 bp


GCTTTGTTGGAGTGCTAGGC
(SEQ ID NO: 92)
132309348-132309367



 


CCAAATCATCAACCACCGTA
(SEQ ID NO: 93)
132309544-132309525
828 bp


TTTCTGTAACCTGGCTTATTTCA
(SEQ ID NO: 94)
132308717-132308739



 


TCTGCACCTCCATGTTCATT
(SEQ ID NO: 95)
132308929-132308910
845 bp


TTTGTAGGGATTTGGCTTCA
(SEQ ID NO: 96)
132308085-132308104



 


ATTGAGTGGGTCAGGGACAA
(SEQ ID NO: 97)
132308323-132308304
806 bp


CCCTTTTCTTTAAACTCCAGCA
(SEQ ID NO: 98)
132307518-132307539



 


TGGACAGGCAAAGAAAATCC
(SEQ ID NO: 99)
132307722-132307703
833 bp


TGCAAATAAACAGGCAGAAATG
(SEQ ID NO: 100)
132306889-132306911



 


GCCCTTGCTGAGGAACCTAC
(SEQ ID NO: 101)
132307075-132307056
846 bp


ACACATTTCTTGCCCCAGAC
(SEQ ID NO: 102)
132306230-132306250



 


TCCCCCTACATCTGCCTACA
(SEQ ID NO: 103)
132306391-132306072
839 bp


AGCCTGTCTTCTGAGGCACT
(SEQ ID NO: 104)
132305563-132305572



 


GGGGGACAGAGAAAGACTCC
(SEQ ID NO: 105)
132305749-132305730
803 bp


TGGGTCTACATGTCTATTGCTTTG
(SEQ ID NO: 106)
132304957-132304970



 


GGGAATGCCCATATTCATTG
(SEQ ID NO: 107)
132305185-132305166
814 bp


TGGAAACCCAAGTTCTTCTGA
(SEQ ID NO: 108)
132304372-132304392



 


CCCGCAAAGTTGTTCTTTGT
(SEQ ID NO: 109)
132304558-132304539
775 bp


CCATGACACAGCCCTCAAG
(SEQ ID NO: 110)
132303384-132303402



 


GCCTCTATCCCACGTTTTGA
(SEQ ID NO: 111)
132304022-132304003
843 bp


TGTCCTTTGGAGGGACATAGA
(SEQ ID NO: 112)
132303180-132303200



 


TATACACGTGCCATGGTGGT
(SEQ ID NO: 113)
132303452-132303433
792 bp


TGACTTAATGAATAAGCCTGCTG
(SEQ ID NO: 114)
132302661-132302683



 


GCACCATATAAATGTGAGATTGGA
(SEQ ID NO: 115)
132302898-132302875
823 bp


AAAAGAAGCTGAATTTGCTTTAAAAT
(SEQ ID NO: 116)
132302086-132302101



 


AGCAGGATACTGACAGGCAAA
(SEQ ID NO: 117)
132302324-132302304
839 bp


TCATTTAAAAATAAATCCCTCTGGA
(SEQ ID NO: 118)
132301486-132301510



 


TGTTTTCCATTTTTCAATCCAA
(SEQ ID NO: 119)
132301639-132301618
772 bp


CCAGGGTCCCATTCCTAGTT
(SEQ ID NO: 120)
132300868-132300887



 


GAGGCTTGTGGAGCATTAGC
(SEQ ID NO: 121)
132301074-132301055
829 bp


AGCATGGGTTTCCATAGCAG
(SEQ ID NO: 122)
132300246-132300265



 


TGTTTGGGATTGAGGTCCTT
(SEQ ID NO: 123)
132300436-132300417
812 bp


TGTGCAGTTCAAACCCATGT
(SEQ ID NO: 124)
132299625-132299644



 


TGTGGTATTGGGTTGCCATT
(SEQ ID NO: 125)
132299746-132299727
843 bp


TTCTCCCTACAGGTCCCAGA
(SEQ ID NO: 126)
132298904-132298923
















TABLE 3







Primers for genotyping by restriction enzyme analysis.














Position according
PCR




Poly-
Primer sequences
to the
product
Restriction



morphism
(forward and reverse)
coordinate system.
size
Enzyme
Expected profils





rs6926879
ATGCACTACCACACTAGGCTGA
132304175-132304154
314 bp
BseYI
C/C genotype: 164 + 122 +



(SEQ ID NO: 127)



28 bp



AGCAGCATGAGACATCAATCAC
132303883-132303862


G/G genotype: 192 + 122 bp



(SEQ ID NO: 128)






 


rs34118837
ATGCACTACCACACTAGGCTGA
132304175-132304154
314 bp
BseYI
C/C genotype: 164 + 122 +



(SEQ ID NO: 129)



28 bp



AGCAGCATGAGACATCAATCAC
132303883-132303862


G/G genotype: 192 + 122 bp



(SEQ ID NO: 130)






 


rs1931003
TGATTCTTGAAATCAAACCTTG
132307324-132307301
273 bp
NlaIII
G/G genotype: 273 bp



AA







(SEQ ID NO: 131)







GCTAGTAGGTTCCTCAGCAAGG
132307073-132307052


A/A genotype: 170 + 103 bp



(SEQ ID NO: 132)






 


rs12191459
ATGGCAATGCACACTTTCAC
132309590-132309571
243 bp
BveI
C/C genotype: 150 + 93 bp



(SEQ ID NO: 133)







GCTTTGTTGGAGTGCTAGGC
132309367-132309348


T/T genotype: 243 bp



(SEQ ID NO: 134)






 


rs12206863
CTTGCAGGCATACACACCAC
132310798-132310817
162 bp
BsmAI
A/A genotype: 107 + 55 bp



(SEQ ID NO: 135)







AACGGCCAGAGAGGTACAAA
132310656-132310637


C/C genotype: 88 + 55 +



(SEQ ID NO: 136)



19 bp


 


rs7768619
TGAGAGCCACTGAAGAATGG
132319120-132319101
224 bp
KspAI
T/T genotype: 224 bp



(SEQ ID NO: 137)







TAGGTGGAGCCTAGGGGACT
132318916-132318897


C/C genotype: 127 + 97 bp



(SEQ ID NO: 138)






 


rs10872386
GGGGACATTTTCCAGACACA
132323619-132323600
229 bp
BanI
C/C genotype: 102 + 74 +



(SEQ ID NO: 139)



63 bp



TGTCATCAAATTGCCACAGG
132323410-132323391


T/T genotype: 165 + 74 bp



(SEQ ID NO: 140)






 


rs2327184
AGCGAGACTCCGTCTCAAAA
132324882-132324863
225 bp
BsuRI
A/A genotype: 225 bp



(SEQ ID NO: 141)







CTTTGCTTTCCGCTGTGATT
132324677-132324658


G/G genotype: 164 + 61 bp



(SEQ ID NO: 142)









EXAMPLES
Example 1
Association Between SNPs in the CTGF Gene Locus with Severe Hepatic Fibrosis (HF) in Two Chinese Populations' Samples
Data are provided for two independent Chinese samples (fishermen and farmers living in region endemic for S. japonicum). Disease phenotype included advanced hepatic fibrosis (fishermen) as described in Materials and Methods, or ascites and previous bleedings (farmers).
Association between genotypes and hepatic fibrosis (HF) phenotypes (described in Material and Methods) have been tested first using univariate analysis (upper part of the Table 4) and second by multivariate analysis (lower part of the Table 4) including SNPs or alterations rs12526196, rs1931002, rs3037970 and rs9402373 that showed the strongest associations when tested again SNPs from the same bin. Bins are correlation (r2>0.5) groups, genotype is the aggravating genotype, OR=Odd ratios, CI=Confidence interval of OR.
Fisherman sample: n=300, 99 cases and 201 controls; covariates: number of years fishing, being born on boat, gender, number of Praziquantel treatments. Chinese farmer sample: n=294, 113 controls and 181 cases; covariates place of birth is endemic or not endemic, cured or active HBV infection (p=0.05, OR=2.38).
Analysis of the various factors that influence the development of fibrosis in the fishermen population has shown that gender, exposure to infected waters and anti-schistosome treatments with Praziquantel were significantly associated with the risk of fibrosis. The testing of several covariates has shown that “the number of years fishing” and “being born on a fishing boat” were the best covariates to measure exposure. The “treatment” covariate is the number of Praziquantel treatments over the last twenty years.
Study Samples
Chinese fishermen were recruited (fisherman sample) on boats during a large field study, whereas farmers (farmer sample) were recruited from hospital records (cases) directly from their farms or villages (controls). Fishermen live on boat and or on small islands and fish since many years. They were much exposed to infection whereas most farmers have been infected a long time ago since parasite transmission in fields has been interrupted 15 to 20 years ago. Fishermen and farmers originate from different geographical regions. Fishermen are mostly from Jiangshu province and a few of them from Hubei and Jiangxi. Farmers have been living in Hunan province for several generations and for some of them were coming from the mountain area.
Hepatic fibrosis (HF) was evaluated accordingly to the WHO guidelines modified as indicated in (Arnaud et al, 2008). The fisherman sample comprised 300 subjects (201 controls, 99 cases). Cases exhibited either severe CentF (central fibrosis: CLH, D, E or F) or severe ParF (parenchymal fibrosis: GNH or GW) or both CLM and GNM. Controls had milder disease: CentFib≦CLL and ParF≦GNL. Covariates that could affect hepatic fibrosis in fishermen were exposure and the number of Praziquantel treatments. Exposure included two covariates: the number of years fishing and being born on a boat. The Chinese farmer sample included 294 subjects (113 controls and 181 cases). Cases had severe HF (as defined for the fishermen cases) and ascites and/or varicose veins. HBV and HCV infections could be evaluated in this sample; 3 subjects were infected with HCV and were not included, whereas >60% had an active or cured HBV infection. HBV infection was a significant covariate in the analysis (p<0.03), exposure and the number of treatments could not be evaluated accurately in Chinese farmers.
The inventors selected several SNPs for genotyping, two SNPs per bin and any SNPs in or outside the bins that could have a functional effect as determined by in silico analysis (see FIG. 1). These SNPs were then genotyped in a larger cohort of 450 individuals of the Chinese Fisherman cohort.
SNPs showing either positive or suggestive association with fibrosis are presented in Table 4. The correction factor to be applied to these data is <20 (see Materials and Methods). Since treatments could not be recorded accurately for all subjects, we show separately the data obtained with the 450 subjects for whom we knew exposure and with the 380 subjects for whom both exposure and treatments were known.
The inventors have first examined HF in fishermen of the Dong Ting Lake in central China that is highly endemic for S. japonicum. Covariates that influenced the development of HF were gender, exposure and anti-schistosome treatments. Using these covariates, we tested on 201 controls and 99 cases whether SNPs (3 SNPs per gene) in CCN2 and IFNGR1 were associated with HF. SNP rs9399005 close to CCN2 showed an association with HF (p=0.02). Sequencing CCN2 (3.2 kb) and 15.3 Kb upstream the starting codon and 14.1 Kb in the 3′UTR region revealed 61 SNPs (FIG. 1) among which 50 had been described before. The 53 SNPs with a MAF>20% were grouped in seven correlation (r2=0.5) bins (I to VII) (FIG. 1). We selected 22 SNPs for further genotyping, at least two SNPs per bin and SNPs (n=4) outside the bins that could have a functional effect as assessed by in silico analysis. SNPs associated with HF (Table 4) were SNP rs12527705 (p=0.02, OR=2.3) and SNP rs12526196 (p=0.02, or =2.2) in bin II, SNP rs9399005 (p=0.02, OR=2.2), SNP rs6918698 (p=0.02, OR=2) and D/I rs3037970 (p=0.003, OR=2.6) in bin III, SNP rs1931002 (p=0.004, OR=2.3) and rs21551532 (p=0.02, OR=1.98) in bin IV and SNP rs9402373 (p=0.015, OR=2) in bin VI. The analysis was adjusted on gender (p<0.001) exposure (fishing years: p<0.001, born on boat: p<0.01) and number of treatments (p<0.01). Deletion/insertion (D/I) rs3037970 excluded rs6918698 in a multivariate analysis testing simultaneously both SNPs. Likewise SNP rs1931002 excluded SNP rs21551532; SNP rs1256196 excluded SNP rs12527705 and SNP rs9399005. This indicated that SNPs rs12526196, rs30337970, rs1931002, rs9402373 had the strongest association with HF. When all four SNPs were tested in the same regression model (lower part Table 4), SNPs rs12526196 (p=0.007, OR=3), rs9402373 (p=0.002, OR=2.8) and rs1931002 (p=0.002, OR=2.8) showed independent associated with HF. An haplotype 1002C, 6196T, present in 53.9% controls and 67.5 cases was associated with HF (p<0.005). The inventors also tested a phenotype that associated advanced HF with evidence of portal hypertension. They found (154 controls and 151 cases) SNP rs9402373 (p=0.005, OR=2.6) and D/I rs3037970 (p=0.05, OR=2) were associated with that phenotype, SNP rs1256196 showed a trend for an association with this more severe disease phenotype (p=0.12). Gender (p=0.001) entered the model as covariate. This suggested that both rs9402373 and D/I rs3037970 could have a more important contribution to severe fibrosis.
The inventors sought replication of these results in an additional independent sample of Chinese farmers who were infected by S. japonicum while working in fields and were recruited from the records of local hospitals. The SNPs that had showed some evidence of an association with HF in the fisherman sample were genotyped in the farmer sample (113 controls, 181 cases). An univariate analysis showed associations between severe HF and SNP rs9402373 (p=0.003, OR=2.23) and SNP rs1256196 (p=0.02, OR=1.85). Other SNPs were not associated (p>0.1). Multivariate analysis indicated that both SNPs rs9402373 (p=0.03, OR=2.26) and rs1256196 (p=0.02, OR=1.88) were independently associated with severe fibrosis. Covariates were birth place (endemic or not endemic, p=0.03) and infection with HVB (p=0.05).







TABLE 4







Chinese samples




















SNP or



Controls %
Cases %
OR
95% CI
p
Controls %
Cases %
OR
95% CI
p












alteration
Position
Bins
Genotype
Chinese Fishermen
Chinese Farmers























12527705
132304944
II
AA
69.1
83.7
2.26
1.13-4.5 
0.02
65.1
72
1.67
0.98-2.84
0.06


12526196
132305169
II
TT
67.9
82.5
2.21
1.13-4.4 
0.02
61.6
71.8
1.85
1.1-3.1
0.02


9399005
132310657
III
CC
17.2
26.3
2.2
 1.1-4.29
0.02




NS


6918698
132314950
III
CC
26.6
35.1
2.0
1.1-3.7
0.02




NS


3037970
132316891
III
—/—
27.3
38.8
2.59
1.4-4.8
0.003




NS


1931002
132320175
IV
CC
52.5
66.7
2.32
 1.3-4.13
0.004




NS


21551532
132316423
IV
AA
55.4
65
1.98
1.12-3.5 
0.02




NS


9402373
132319124
VI
CC
50.3
60.6
2.04
1.15-3.54
0.015
25
43.1
2.23
1.32-3.76
0.003


12526196

II
TT


3.0
1.4-6.6
0.007


1.88
1.13-3.19
0.018


9402373

VI
CC


2.8
1.5-5.4
0.002


2.26
1.33-3.83
0.03


1931002

IV
CC


2.81
1.47-5.37
0.002









Example 2
Association Between SNPs in the CTGF Gene Locus with Severe Hepatic Fibrosis (HF) in Sudaneses and Brazilians Infected with Schistosoma mansoni 
The two principal schistosome strains that cause HF are S. japonicum in Asia, and S. mansoni in Africa and in South America. We have investigated whether HF in S. mansoni endemic region of Sudan and Brazil was also affected by CTGF allelic variants. CTGF polymorphisms that were associated with HF in Chinese fishermen (Table 4) were genotyped in both samples. The phenotypes were either severe HF associated with portal hypertension in Sudanese or significant secondary portal branch thickening in Brazilians who were much less affected than Sudanese since transmission was lower in the Brazilians than in the Sudanese sample.
Associations between genotypes and HF phenotypes (described in Materials and Methods) have been tested first using univariate analysis (upper part of Table 5) and second by multivariate analysis (lower part of Table 5) including SNPs rs12526196, rs 12527705 and rs9402373. Genotype is the aggravating genotype, OR=Odd ratios, CI=Confidence interval of OR.
Sudanese farmers: 314: 219 controls and 95 cases. Brazilians 133: 60 cases and 73 controls.
Study Samples
The Sudanese sample (314 subjects, 95 cases and 219 controls) was recruited among farmers living in villages of the Wad Medani region of Sudan (Dessein et al., 1999). Severe phenotypes associated HF (grade≦C) with evidence of portal hypertension as in (Dessein et al., 1999). All subjects were much exposed to infection with S. mansoni and had received no or few Praziquantel treatments. However, exposure and the number of treatments could not be accurately evaluated on this sample.
The Brazilian sample (133 subjects, 73 controls and 60 cases) was recruited in a village of North East Brazil among adult subjects with frequent contacts with a river populated with S. mansoni infested snails. Infection was much less, as assessed by schistosome egg excretion in teenagers, in Brazilians than in the Chinese and Sudanese. Severe HF with portal hypertension was observed in less than 2% of the population. All subjects showing clear evidence of periportal thickening of the secondary portal branches were cases, whereas the other subjects with similar contacts with the river were controls.
The inventors have investigated whether hepatic fibrosis in Brazilians infected with S. mansoni is also affected by CTGF allelic variants. The Brazilian cohort has been recruited for this study and has never been genotyped before. We have genotyped CTGF polymorphisms that yield a clear or a suggestive association with fibrosis in Chinese fishermen. In this association study cases were subjects with evidence of thickening of the wall of the secondary branches of the portal vein. The only confounding variable was age of the patients. All subjects with evidence of exposure to infection for many years were selected.
The inventors have investigated next whether HF caused by S. mansoni in endemic subjects in Sudan and Brazil was also affected by CTGF allelic variants. CTGF polymorphisms associated with HF in Chinese fishermen were genotyped in both samples. The phenotypes were either severe HF associated with portal hypertension in Sudanese or significant secondary portal branch thickening in Brazilians who were much less affected than Sudanese.
Conclusions:
Univariate analysis (Table 5 left) in Sudanese (219 controls, 95 cases) showed that SNPs rs9402373 (p=0.02, OR=2.7), rs1256196 (p=0.044, OR=3.2) and rs12527705 (p=0.05, OR=3) were associated with severe HF. The aggravating genotypes were identical in Sudanese and Chinese (Tables 4 and 5). Multivariate analysis indicated SNP rs9402373 (p=0.03, OR=2.7) and SNP rs1256196 (p=0.03, OR=3.5) were independently associated with the HF without additional covariates.
Univariate analysis of the genotyping data of the Brazilians (73 controls, 60 cases) showed (Table 5 right) associations between SNP rs9402373 (p=0.02, OR=2.57) and SNP rs6918698 (p=0.008, OR=3) with HF without additional covariates. The genotypes associated with disease for both SNPs were the same as in Chinese and Sudanese (Tables 4 and 5). Multivariate analysis indicated SNPs rs9402373 and rs6918698 were independently (p<0.001, OR>4) associated with HF in Brazilians.
In silico analysis had suggested that the allelic variants SNPs rs9402373 and rs12526196 might bind differently nuclear factors. We have demonstrated this with EMSA: the rs9402373C allele bound nuclear factors that were not bound by the G allele (FIG. 2A). This binding was competed with specific unbiotinylated probe (FIG. 2B). EMSA also showed a greater binding affinity of nuclear factors to the rs12526196T allele (FIG. 2A). EMSA did not reveal allele specific binding for rs12527705 and rs1931002 polymorphisms (FIG. 2A).
Then SNP rs9402373 was associated with HF in all four samples tested. This association was obtained in Chinese and Sudanese, using a strict fibrosis phenotype or a more severe phenotype (HF+evidence of portal blood hypertension). SNP rs1256196 was also independently associated with HF in Chinese and Sudanese. Our EMSA data indicate that allelic variants of SNP rs1256196 and SNP rs9402373 bind differently nuclear factor suggesting that they could affect the regulation of gene transcription or the stability of transcripts.
HBV infection was a significant covariate in the analysis of Chinese farmers indicating that HBV likely contributed significantly to severe disease. This also suggests that SNP rs1256196 and SNP rs9402373 might also modulate HF caused by HBV.
In conclusion the present study shows the association of polymorphisms with fibrosis, and identifies most critical steps in disease development indicating new therapeutical targets. It also provides valuable markers of HF progression.








TABLE 5







Sudanese sample
Brazilian sample




















SNP or

Controls
Cases





Controls
Cases





alteration
Genotype
%
%
OR
95% CI
P
SNP
Genotype
%
%
OR
95% CI
P























12527705
AA
82.1
90.5
3
1-9.1
0.05
12527705





NS


12526196
TT
80.8
90.5
3.2
1-9.7
0.44
12526196





NS


3037970
—/—




NS
3037970
—/—
80.5
90.2


0.12 


6918698
CC




NS
6918698
CC + CG
63
83.9
3.05
1.33-6.98
0.008


9402373
CC
80
89.8
2.75
1.17-6.5  
0.02
9402373
CC
60.3
80.0
2.57
1.18-5.64
0.018


12526196
TT


3.5
1.1-10.9 
0.03
6918698
CC


4.92
2.0-12 
5 × 10−4


9402373
CC


2.76
1.2-6.7  
 0.026
9402373
CC


4.25
 1.79-10.08
9 × 10−4









Example 3
Association Between SNPs in the CTGF Gene Locus with Hepatic Fibrosis (HF) in HCV Infected Subjects
Analysis of CTGF Genetic Variants in French Subjects Infected with HCV.
Example 1 describes SNP correlation bins (r2>0.8) in the CTGF encoding gene in a sample of Chinese population. These were defined over the entire gene and 10 Kb of the 3′ and 5′ flanking region. Only SNPs with a minor allele frequency >20% had been analyzed. There were seven bins of which four (groups II, III, IV and VI) showed some association with hepatic fibrosis caused by schistosome eggs in Chinese fishermen infected by Schistosoma japonicum. In Example 3, the inventors genotyped two SNPs in each of these four bins and an additional SNP rs9399005 (that showed suggestive associations with schistosomal fibrosis) in a French sample infected with HCV and determined whether any of them were associated with advanced hepatic fibrosis. They found evidence for association in bins III and VI but not in bins II and IV. SNP rs6918698 (p=0.03) and SNP rs3037970 (p=0.04) in bin IV, were associated with hepatic fibrosis. The CG and GG genotypes of SNP rs6918698 were associated with disease aggravation (OR=2.1, CI=1.1-4.0).
Bin VI only contains SNP rs9402373 that was also (p=0.007) associated with hepatic fibrosis in the French sample. Disease was aggravated by the CC genotype (OR=2.54; 1.3-4.96). Interestingly, the subjects homozygous C/C showed an average four-fold increase of the risk of severe hepatic fibrosis as compared to homozygous GG (OR=4.1 CI=1.1-14). Multivariate logistic regression analysis that included SNP rs6918698 and SNP rs9402373 indicated that the latter was the most strongly associated with hepatic fibrosis. Nevertheless in the presence of SNP rs9402373, SNP rs6918698 still showed a trend for an association with hepatic disease suggesting that SNP rs9402373 could not account for the full association of SNP rs6918698 with disease. Thus the inventors concluded both SNPs are independently associated with fibrosis.
CTGF Genetic Variants are Also Associated with Hepatic Fibrosis in Brazilians Infected with HCV.
The inventors then sought the confirmation of the above described findings by testing SNPs from the same four bins in Brazilians infected with HCV. They found that SNP rs6918698 (p=0.0004) was clearly associated with hepatic fibrosis. SNP rs3037970 showed a trend (p=0.07) for an association and SNP rs6918698 accounted for SNP rs3037970 association. The SNP rs6918698 aggravating genotype was G/G (OR 2.94; 1.5-7.7). As in the French sample, the inventors observed that SNP rs9402373 in bin VI, was strongly (p=0.003) associated with hepatic fibrosis (OR=2.94, 1.45-5.97) in the Brazilian sample. Of note, in this sample as in the French sample, C/C aggravated disease. Since subjects carrying the G/G genotype were few, the inventors could not compare the risk of severe fibrosis between homozygous subjects. Finally the inventors also showed a trend (p=0.06) for an association of SNPrs9399005 with hepatic fibrosis.
Logistic regression analysis showed that both SNP rs9402373 (p=0.029) and SNP rs6918698 (p=0.03) contributed independently to hepatic fibrosis with comparable relative risks (OR=2.4-2.5) and that the association of SNP rs9399005 with fibrosis was lost in the presence of the two other SNPs suggesting that the association of this SNP with hepatic fibrosis likely is entirely accounted for by its correlation with SNP rs9402373 and SNP rs6918698. It can not totally be excluded, however, that SNP rs9399005 could also be independently associated with hepatic fibrosis.







TABLE 6





SNPs rs 6918698 and rs9402373 are associated with hepatic fibrosis in HCV infected subjects

















French Sample




















GENOTYPE
CONTROLS
CASES

95%



Analysis
SNP
Position
Bins
(aggravating)2
%
%
OR
CI
P





Univariate
1257705
132304944
II





NS >











1.4



12526196
132305169
II
CT*




NS >











1.3



9399005
132310657

CC




NS >











0.3



3037970
132316891
III
Del, del (French)
67.8 (78)
79.5 (89)
1.9
1.03-3.6
0.04






Del, del, del/wt






(Brazilians)



6918698
132314950
III
GG + CG
68.4 (78)
81.7 (89))
2.1
1.1-4 
0.025






(French)






GG (Brazilians)



1931002
132320175
IV





NS >











0.5



2151532
132316423
IV





NS >











0.5



9402373
132319124
VI
CC
51.3 (61)
70.8 (46)
2.54
 1.3-4.96
0.007



12527379


AG + AA
57.1 (64)
  67 (69)
1.7
 0.94-2.97
0.08


Multivariate
6918698


C/G + G/G


1.54
0.77-3.1
0.22






(french)






G/G (Brazilians)



9402373


C/C


2.1
1.15-4  
0.017












Brazilian Sample






















GENOTYPE
CONTROLS
CASES

95%




Analysis
SNP
Position
Bins
(aggravating)2
%
%
OR
CI
P







Univariate
1257705
132304944
II





NS >












1.4




12526196
132305169
II
CT*




NS >












1.3




9399005
132310657

CC
57.4 (39)
72.4 (92)
1.9
0.97-3.7
0.06




3037970
132316891
III
Del, del (French)
  61 (41)
  75 (93)
1.9
0.95-3.9
0.07







Del, del, del/wt







(Brazilians)




6918698
132314950
III
GG + CG
16.4 (11)
34.4 (44)
3.3
 1.5-7.7
0.004







(French)







GG (Brazilians)




1931002
132320175
IV





NS >












0.2




2151532
132316423
IV





NS >












0.2




9402373
132319124
VI
CC
58.8 (40)
79.7 (102)
2.5
1.1-5 
0.003




12527379


AG + AA
38.2 (26)
50.0 (64)
1.8
0.94-3.5
 .08



Multivariate
6918698


C/G + G/G


2.5
 1.1-5.9
0.03







(french)







G/G (Brazilians)




9402373


C/C


2.4
1.1-5 
0.029







French (n = 222) and Brazilians (n = 196) cohorts, infected with HCV: Covariates Sexe p < 10−3






The inventors then performed a Meta analysis of the data obtained with SNP rs9402373 in French and Brazilians infected with HCV.
Such an analysis was not performed with SNP rs6918698 because the fibrosis associated genotypes were different in French (G/G+C/G) and in Brazilians (G/G). The inventors found that SNP rs9402373 CC genotype was associated (p=0.00005, OR=2.72; 1.67-4.43) with hepatic fibrosis caused by HCV.
Since the association of this same CC genotype of SNP rs9402373 was shown with hepatic fibrosis in the above examples, the inventors also performed a Meta analysis of the studies testing the association of this SNP with HCV induced HF (two studies) and schistosomal induced HF (4 studies). The results of such a Meta analysis are presented in FIG. 3 and show that CC of the SNP rs9402373 is strongly associated (p<10-9) with severe hepatic fibrosis (OR=2.47; 1.8-3.25) caused by different pathogens HCV, S. mansoni and S. japonicum) in populations (Chinese, Sudanese, Brazilian and French) with markedly different genetic background.
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## Claims

1. A method for determining an increased risk of hepatic fibrosis in a human subject, the method comprising:
(a) obtaining a sample from the human subject;
(b) detecting a CC genotype at rs9402373 in the sample using a probe consisting of SEQ ID NO: 33 or SEQ ID NO: 34; and
(c) identifying the human subject having a CC genotype at rs9402373 as having an increased risk of hepatic fibrosis as compared to a human subject having a GG genotype at rs9402373.

2. The method of claim 1, wherein the hepatic fibrosis is caused by hepatitis A virus infection.

3. The method of claim 1, wherein the hepatic fibrosis is caused by hepatitis B virus infection.

4. The method of claim 1, wherein the hepatic fibrosis is caused by hepatitis C virus infection.

5. The method of claim 1, wherein the hepatic fibrosis is caused by Schistosoma japonicum infection.

6. The method of claim 1, wherein the hepatic fibrosis is caused by Schistosoma mansoni infection.

7. A method of determining whether a subject is a candidate for a treatment for hepatic fibrosis, the method comprising:
(a) obtaining a sample from a human subject;
(b) detecting a CC genotype at rs9402373 in the sample using a probe consisting of SEQ ID NO: 33 or SEQ ID NO: 34;
(c) identifying the human subject having a CC genotype at rs9402373 as having an increased risk for hepatic fibrosis as compared to a human subject having a GG genotype at rs9402373; and
(d) determining that the subject with a CC genotype is a candidate for the treatment for hepatic fibrosis.

8. The method of claim 7, wherein the hepatic fibrosis is caused by hepatitis A virus infection.

9. The method of claim 7, wherein the hepatic fibrosis is caused by hepatitis B virus infection.

10. The method of claim 7, wherein the hepatic fibrosis is caused by hepatitis C virus infection.

11. The method of claim 7, wherein the hepatic fibrosis is caused by Schistosoma japonicum infection.

12. The method of claim 7, wherein the hepatic fibrosis is caused by Schistosoma mansoni infection.

13. A method for determining an increased risk of hepatic fibrosis in a human subject, the method comprising:
(a) obtaining a sample from the human subject;
(b) detecting a CC genotype at rs9402373 in the sample using a fluorescently labeled probe consisting of SEQ ID NO: 33 or SEQ ID NO: 34; and
(c) identifying the human subject having a CC genotype at rs9402373 as having an increased risk of hepatic fibrosis as compared to a human subject having a GG genotype at rs9402373.

14. The method of claim 13, wherein the hepatic fibrosis is caused by hepatitis A virus infection.

15. The method of claim 13, wherein the hepatic fibrosis is caused by hepatitis B virus infection.

16. The method of claim 13, wherein the hepatic fibrosis is caused by hepatitis C virus infection.

17. The method of claim 13, wherein the hepatic fibrosis is caused by Schistosoma japonicum infection.

18. The method of claim 13, wherein the hepatic fibrosis is caused by Schistosoma mansoni infection.