WO2013150002A1

WO2013150002A1 - Methods and kits for determining if a subject is predisposed to fast progression of liver fibrosis

Info

Publication number: WO2013150002A1
Application number: PCT/EP2013/056895
Authority: WO
Inventors: Laurent Abel; Pierre-Yves Bochud
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre Hospitalier Universitaire Vaudois
Priority date: 2012-04-03
Filing date: 2013-04-02
Publication date: 2013-10-10

Abstract

The present invention thus relates to a method of determining if a subject is predisposed to fast progression of liver fibrosis, the method comprising testing for said subject the genotype of at least one single nucleotide polymorphism selected from the group consisting of rs16851720, rs4374383 and rs9380516 or any single nucleotide polymorphism in linkage disequilibrium with rs16851720, rs4374383 or rs9380516.

Description

METHODS AND KITS FOR DETERMINING IF A SUBJECT IS PREDISPOSED TO FAST PROGRESSION OF LIVER FIBROSIS

FIELD OF THE INVENTION:

The present invention relates to methods and kits for determining if a subject is predisposed to fast progression of liver fibrosis.

BACKGROUND OF THE INVENTION:

Liver fibrosis is the formation or development of excess fibrous connective tissue in the liver characterized by the increased production and decreased degradation of extracellular matrix materials. Liver fibrosis is common to liver diseases of many etiologies, including chronic viral hepatitis B and C, autoimmune liver disease, such as autoimmune hepatitis and primary biliary cirrhosis, alcoholic liver disease, nonalcoholic fatty liver disease, metabolic disorders, such as lipid, glycogen, or metal storage disorders, and drug-induced liver disease. The fibrosis exhibited in these disorders results from chronic insults to the liver from, for example, viral infection, alcohol, or drugs.

Hepatitis C (HCV), for example, is one of the leading causes of liver fibrosis. HCV is a single-stranded positive RNA virus, which belongs to the family of Flaviviridae, genus Hepacivirus. It is estimated that 170 million patients worldwide and about 1 % of the population in developed countries are chronically infected with hepatitis C virus (HCV). Approximately 85% of the infected subjects develop chronic hepatitis, and up to 20% progress to fibrosis and then to cirrhosis (which is end-stage severe liver fibrosis and is generally irreversible). HCV infection is the major cause of cirrhosis and hepatocellular carcinoma (HCC), and accounts for one third of liver transplantations. The interval between infection and the development of cirrhosis may exceed years but varies widely among subjects. Based on fibrosis progression rate, chronic HCV patients can be roughly divided into three groups: rapid, median, and slow fibrosers.

Previous studies have indicated that host factors may play a role in the progression of fibrosis, and these include age at infection, duration of infection, alcohol consumption, and gender. However, these host factors account for only 17% of the variability in fibrosis progression. Viral load or viral genotype has not shown significant correlation with fibrosis progression. Thus, other factors, such as host genetic factors, are likely to play an important role in determining the rate of fibrosis progression. Recent studies suggest that some genetic polymorphisms influence the progression of fibrosis in patients with HCV infection. For example, a number of candidate gene approaches have been used in attempts to identify variants influencing the development of liver disease in HCV-infected patients, but most of these studies produced results that were not consistently replicated (Osterreicher CH, Stickel F, Brenner DA. Genomics of liver fibrosis and cirrhosis. Semin Liver Dis 2007;27:28-43 ; Bataller R, North KE, Brenner DA. Genetic polymorphisms and the progression of liver fibrosis: a critical appraisal. Hepatology 2003;37:493-503). Two related studies (Huang H, Shiffman ML, Cheung RC, et al. Identification of two gene variants associated with risk of advanced fibrosis in patients with chronic hepatitis C. Gastroenterology 2006;130: 1679-87.; Huang H, Shiffman ML, Friedman S, et al. A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C. Hepatology 2007;46:297-306.) investigating -25,000 putative functional SNPs identified a panel of SNPs predicting the risk of developing cirrhosis; this panel requires validation in prospective studies. A recent study of 36 candidate genes, related to the fibrogenesis/fibro lysis process, identified a single cluster of variants of the IFNGR2 gene associated with progression to severe fibrosis (Nalpas B, Lavialle-Meziani R, Plancoulaine S, et al. Interferon gamma receptor 2 gene variants are associated with liver fibrosis in patients with chronic hepatitis C infection. Gut 2010;59: 1120-6.). The results of these studies are interesting, but their approach may have prevented identification of genes strongly associated with liver fibrosis present in parts of the genome not tested.

SUMMARY OF THE INVENTION:

The present invention thus relates to a method of determining if a subject is predisposed to fast progression of liver fibrosis, the method comprising testing for said subject the genotype of at least one single nucleotide polymorphism selected from the group consisting of rsl6851720, rs4374383 and rs9380516 or any single nucleotide polymorphism in linkage disequilibrium with rsl6851720, rs4374383 or rs9380516.

DETAILED DESCRIPTION OF THE INVENTION:

Clearance of hepatitis C virus (HCV) infection is modulated by IL28B polymorphisms, as shown by genome-wide association studies (GWAS). Only a fraction of patients with chronic infection develop liver fibrosis, a process that may also be controlled by human genetic factors. The inventors carried out a two-stage GWA study of liver fibrosis progression related to HCV infection. They studied well characterized HCV-infected patients of European descent who had liver biopsy before treatment and defined various liver fibrosis phenotypes on the basis of Metavir score, with and without taking the duration of HCV infection into account. GWAS was conducted on a filtered primary cohort of 1,161 patients, using 780,650 single nucleotide polymorphisms (SNPs). The inventors genotyped 96 SNPs with P-values<5xl0^~5 in an independent replication cohort of 962 patients. Finally, they assessed the most interesting replicated SNPs in an additional sample of 219 patients genotyped in a previous GWAS on HCV clearance. In the combined cohort of 2,342 HCV- infected patients, two SNPs, rsl6851720 in the total sample and rs4374383 in blood- transfused patients, provided genome-wide significant evidence of association with fibrosis progression (Pcombined=8.9xl0^~9 and 2. lxlO^"9, respectively). SNP rsl6851720 is located within RNF7, which acts as an antioxidant protecting against apoptosis. SNP rs4374383, together with another replicated SNP, rs9380516 (Pcombined=5.4xl0^~7), are linked to two functionally related genes, MERTK and TULP1, involved in phagocytosis of apoptotic cells by macrophages. Thus the GWAS identified several susceptibility loci for HCV-induced liver fibrosis related to the apoptosis pathway.

As used herein, the term "subject" includes both young and old human beings of both sexes. Preferably, this term encompasses subjects who are at risk to develop liver fibrosis, for example, subjects who are infected with hepatitis C virus, or with other hepatotoxic viruses (e.g., hepatitis B, D), subjects who suffer from hepatotoxicity due to consumption of more than 2 units of alcohol daily or hepatotoxic drugs, subjects having liver cancer, non alcoholic fatty liver disease (NAFLD), an autoimmune disease such as autoimmune hepatitis (AIH), primary biliari cirrhosis (PBC) and primary sclerosing cholangitis (PSC), a metabolic liver disease such as Hemochromatosis, Wilson's disease and alpha 1 anti trypsin and/or a disease with secondary involvement of the liver such as celiac disease or amyloidosis. Preferably, the subject of the present invention is a human being which is infected with the hepatitis C virus. As intended herein the expressions " hepatitis C virus-infected subject" refers to a subject who has a chronic hepatitis C virus (HCV) infection and notably relates to an subject in whom HCV R A can be detected in particular in blood, serum or plasma samples. Numerous methods, and in particular commercial methods, are known in the art for detecting HCV RNA. Typically, said subject may be infected by any of HCV genotypes (la, lb, 2, 3, 4, 5, 6...).

As used herein, the term "predisposed" when used with respect to fast progression of liver fibrosis refers to a subject which is more likely to develop fast progression of liver fibrosis than a non-predisposed subject. The phrase "fast progression of liver fibrosis" as used herein refers to the development of liver fibrosis within a time period which is shorter than expected according to the individual's age at the time of infection based on the Poynard's fibrosis progression model (Poynard et al, 2001. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J. Hepatol. 34: 730-9). For example, a normal rate of progression of liver fibrosis in an individual younger than 20 years of age is 40 years. On the other hand, individuals who are infected at the age of 40 or older will develop liver fibrosis following 10- 20 years from the time of infection. Thus, fast progressing liver fibrosis is defined herein as fibrosis which occurs over a time period which is at least 5 years shorter than expected, more preferably, at least 10 years, most preferably, at least 20 years shorter than expected according to the Poynard's fibrosis progression model.

In a particular embodiment, the subject is tested for 1, 2, 3 or even more SNP selected from the group consisting of rsl6851720, rs4374383 and rs9380516 and any neighbouring loci in linkage disequilibrium with rsl6851720, rs4374383 or rs9380516. Typically, the subject can be tested for rsl6851720, rs4374383 and rs9380516.

The term "Allele" has the meaning which is commonly known in the art, that is, an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated result in functional or dysfunctional (including non- existent) gene products.

The term "Single nucleotide polymorphism" or "SNP" means a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the human population. There are millions of SNPs in the human genome. Most commonly, these variations are found in the DNA between genes. When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene's function. SNPs are well known to one of skill in the art and are notably described in the NCBI database dbSNP (www.ncbi.nlm.nih.gov/SNP/). As used herein, the SNP that are concerned by the invention are described as follows (Table A):

Table A

The term "risk-allele" according to the invention refers to the allele which, when present in one or two copies in a subject results in increased propensity for developing liver fibrosis. The term "risk-allele," thus refers to an SNP or allele that is associated with high relative risk of developing liver fibrosis. According the invention the risk alleles are allele A for rsl6851720, allele G for rs4374383 and allele T for rs9380516.

Typically, the method is performed by determining the presence or absence, in a homozygous or heterozygous for of at least one risk allele. Preferably in the above-defined method, it is deduced that the subject has an increased risk to develop fast progression of liver fibrosis if said subject is homozygous or heterozygous for the risk allele for rs 16851720 and rs4374383. More particularly it is deduced that the subject who are homozygous for the risk allele for rs 16851720 has a higher risk to develop fast progression of liver fibrosis than a subject who is heterozygous for the risk allele. It is deduced that only a subject who is homozygous for the risk allele for rs9380516 has an increased risk to develop fast progression of liver fibrosis. The test results may be summarized in Table B: SNP Subject's genotype and results rsl6851720 • Homozygous for C (C/C) has the baseline risk of developing liver fibrosis

• As compared to C/C subjects, heterozygous (C/A) has an increased risk to develop fast progression of liver fibrosis

• Homozygous for A (A/ A) has an even more increased risk to develop fast progression of liver fibrosis than a heterozygous subject

rs4374383 • Homozygous for A (A/ A) has the baseline risk of developing liver fibrosis

• As compared to A/A subjects, heterozygous (A/G) and homozygous for G has an increased risk to develop fast progression of liver fibrosis (homozygous and heterozygous = same risk)

rs9380516 • Homozygous for C (C/C) and heterozygous (C/T) has the baseline risk of developing liver fibrosis.

• As compared to C/C or C/T subjects, homozygous for T (T/T) has an increased risk to develop fast progression of liver fibrosis

Table B

The phrase "linkage disequilibrium" (LD) is used to describe the statistical correlation between two neighboring polymorphic genotypes. Typically, LD refers to the correlation between the alleles of a random gamete at the two loci, assuming Hardy- Weinberg equilibrium (statistical independence) between gametes. LD is quantified with either Lewontin's parameter of association (D') or with the so-called r² parameter which is based on Pearson correlation coefficient. Two loci with a LD value of 1 are said to be in complete LD. At the other extreme, two loci with a LD value of 0 are termed to be in linkage equilibrium. Linkage disequilibrium is calculated following the application of the expectation maximization algorithm (EM) for the estimation of haplotype frequencies. SNPs considered in LD with rsl6851720, rs4374383 or rs9380516 in the present invention are SNPs presenting a r2 value greater than 0,2, preferably greater than 0,4 and even more preferably greater than 0,5 with any of these three SNPs.

According to the invention the genotype of the single nucleotide polymorphism is tested from a sample obtained from the subject.

A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. As used herein "blood" includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood. According to the invention, the presence of the risk allele may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridization, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said polymorphism may be detected in a RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to genotype the polymorphism (or identify the allele). Actually numerous strategies for genotype analysis are available. Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.

Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the one of the allele of the polymorphism.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

In some embodiments, the method of the invention is performed by a laboratory that will generate a test report. The test report will thus indicate whether the risk allele is present or absent, and preferably indicates whether the patient is heterozygous or homozygous for the risk allele. In some embodiments, the test result will include a probability score for developing liver fibrosis, which is derived from running a model that include the risk factor determined for the one or the two single nucleotide polymorphisms of the invention that are tested. For calculating the score, the risk factor determined for a single nucleotide polymorphism of the invention may be pondered by a coefficient depending on what is the contribution of said single nucleotide polymorphism in the determination of the risk in comparison with the other one single nucleotide polymorphism. Typically, the method for calculating the score is based on statistical studies performed on various cohorts of patients. The score may also include other various patient parameters (e.g., age, gender, weight, alcohol consumption of the subject, HCV genotype, HIV infection). The weight given to each parameter is based on its contribution relative to the other parameters in explaining the inter- individual variability of developing liver fibrosis. In some embodiments, the test report may be thus generated by a computer program for establishing such a score.

It will, of course, be understood by practitioners skilled in the treatment or diagnosis of liver fibrosis that the present invention generally does not intend to provide an absolute identification of subjects who are at risk (or less at risk) of developing fast progression of liver fibrosis, and/or pathologies related to liver fibrosis (e.g. cirrhosis and hepatocarcinoma), but rather to indicate a certain increased (or decreased) degree or likelihood of developing fast progression of liver fibrosis based on statistically significant association results. However, this information is extremely valuable as it can be used to, for example, initiate preventive treatments or to allow an subject carrying one or more significant SNPs to foresee warning signs such as minor clinical symptoms, or to have regularly scheduled physical exams to monitor for appearance of a condition in order to identify and begin treatment of the condition at an early stage. Particularly with diseases that are extremely debilitating or fatal if not treated on time, the knowledge of a potential predisposition, even if this predisposition is not absolute, would likely contribute in a very significant manner to treatment efficacy.

In a different manner, the method of the invention is thus particularly suitable for determining if a subject is predisposed to cirrhosis or even more to hepatocarcinoma.

A further object of the invention is a kit for performing the methods of the invention, comprising at least one primer and/or at least one probe for amplification of a sequence comprising the polymorphisms of the invention and instructions for use. In some embodiments, the kit comprises primers and/or probes for amplification of at 1 or 2 sequences comprising the polymorphisms of the invention and instructions. In some embodiments, the kit comprise primers and/or probes for amplification of at least one region comprising a sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3. In one embodiment of the invention, the primer or probe may be labelled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. EXAMPLE:

Material & Methods

Patient subjects: The sample used for the primary screen combined data from two cohorts of adult patients of European descent from France and Switzerland with chronic HCV infection. We retained only patients who had liver biopsy before treatment. The French cohort (ANRS Genoscan study group) included patients from the hepatology units of several hospitals in Paris and Marseilles; the inclusion criteria applied, including no co-infection by HIV or HBV, have been described elsewhere.⁹ The Swiss Hepatitis C Cohort Study (SCCS) is a multicenter study of HCV-infected patients enrolled at eight major Swiss hospitals and the affiliated local centers. SCCS patient selection and data collection have also been described elsewhere,³'²¹ and patients with known HIV or active HBV co-infection were excluded for the present study. In total, 1,223 patients (490 from the French cohort and 733 from the SCCS) were eligible for genetic analyses. We studied three additional cohorts of European-descent adult patients with chronic HCV infection and not co-infected with HIV or HBV, with the aim of replicating the principal signals obtained in the primary cohorts. All 962 patients included in these cohorts had biopsy before treatment. A first cohort of 64 US patients was recruited at the Weill-Cornell Medical Center in New York, and an additional sample of 256 French patients was recruited from different hospitals in Marseilles. The third replication cohort included 642 patients recruited from centers in Australia, Germany, the United Kingdom and Italy, as described elsewhere.¹²'²² Finally, the seven signals showing evidence of true replication in the cohort combining primary and replication cohorts were also tested in an additional independent sample of Australian patients consisting of the primary cohort of a previous GWAS of response to hepatitis C treatment.¹² After excluding Australian patients with missing phenotype data, 219 individuals were kept for this analysis. When available, clinical risk factors, history of HCV acquisition and of alcohol consumption (assessed using time-line follow back interview) were recorded in the corresponding cohorts through face-to- face interviews conducted by physicians trained in addiction problems. The sampling of all the cohorts was approved by the appropriate institutional review boards, and written informed consent was obtained from all patients.

Determination of liver fibrosis phenotypes: The stage of liver fibrosis in patients with chronic HCV infection was determined by examination of a liver biopsy specimen obtained before treatment, with quantification according to the Metavir score, on a five-point scale from F0 to F4.²³ Fibrosis was assessed by experienced pathologists working in the local expert liver centers corresponding to the place of collection, and all biopsies were considered as interpretable by each local pathologist. Metavir scoring system is validated since many years and highly reproducible for pathologists specialized in liver disease.²⁴ A number of patients had several biopsies; for those who had developed fibrosis (>F2), we used the earliest biopsy specimen giving a F2 scorescore>F2, and for those who had not developed fibrosis (<F2), we used the most recent biopsy specimen obtained in the absence of treatment. We used three different approaches to define liver fibrosis phenotype on the basis of Metavir scores. The first of these approaches was a classic case/control approach in which FO-1 patients were considered as controls and F3-4 patients, as cases. Patients with an intermediate Metavir score (F2) were discarded for phenotypic analysis, which was thus a binary F0-1/F3- 4 phenotype analysis. Within this approach, we also used a more extreme definition of the phenotypes, in which we considered only F0 patients as controls and F4 patients as cases (binary F0/F4 phenotype analysis). In the second approach, we used survival analysis techniques to take into account the individual duration of infection in the analysis. Failure (F3-4 or only F4) and censored (FO-1 or only F0) events were defined as in the case/control study, and the duration of infection was estimated from the presumed year of HCV acquisition to the year in which the biopsy was carried out. The estimated date of HCV acquisition was obtained using the first reported major event at risk among blood transfusion, drug use (the first year of injecting drug use (IDU) was used as the starting point), accidental needle stick or other invasive medical procedures. In this analysis, the phenotype is denoted duration F0-1/F3-4 or duration F0/F4. In the third approach, liver fibrosis progression was considered as a quantitative phenotype, fibrosis progression rate (FPR), corresponding to the ratio of Metavir score to the estimated duration of infection in years (Metavir units per year). ' Given the distribution of FPR, this phenotype was log-transformed and then inverse normal quantile-transformed in each cohort before statistical analysis. The resulting phenotype, denoted as QTF phenotype, was further standardized such that the differences in mean QTF rates between genotypes for a given SNP could be expressed in standard deviation units, referred to as standardized Metavir units (SMUs). Also, we analyzed our GWAS primary data by modeling transition probabilities between Metavir stages using a Markov model. This approach was not further considered, because it provided no additional association signals with P values<5xl0^~6 in our primary cohort, with respect to analyses using the three other phenotype definitions.

Genotyping and quality controls: The French primary cohort was genotyped for -350,000 SNPs by the Post-Genomics Platform of the Pitie-Salpetriere Hospital, Paris, France, using the Illumina HumanCNV370-Duo beadchip (Illumina, San Diego, USA). The Swiss primary cohort was genotyped for -1,000,000 SNPs by the Genomics Platform of the National Center for Competence in Research "Frontiers in Genetics", University of Geneva, Switzerland, using the Illumina humanlM-Duo beadchip (Illumina). Genotype calling and quality control filters were applied in French and Swiss primary cohorts independently. Genotype calling was carried out with the default settings of the BeadStudio software (Illumina). For genetic markers, we discarded SNPs with (i) a genotype clustering score (Illumina GenTrain) < 0.2, (ii) a call rate < 90%, (iii) a MAF < 1% and (iv) a Hardy- Weinberg P-value < 10^"7. Quality-control filtering led to a total of 325,624 and 912,765 high quality SNPs in the French and the Swiss cohort, respectively.

For individuals, we discarded subjects with (i) a call rate < 95% (n=4 and 6 in French and Swiss cohorts, respectively), (ii) a sex mismatch, when comparing the sex reported in the clinical databases with the one determined from chromosome X heterozygosity rate and ratios of intensity of X and Y probes (n=0 and 24), (iii) evidence of cryptic relatedness, defined by relatedness coefficients > 0.125 (n=6 and 0) and (iv) a marked difference in genetic ancestry with the overall cohort (n=13 and 9). Calculation of relatedness coefficients and stratification in our entire sample were assessed using EIGENSOFT v3.0,²⁷ selecting only SNPs that were present in both beadchips and that were in linkage equilibrium (r²<0.1). Ancestry outliers were removed by Eigenstrat default removal procedure. After outlier removal, none of the 10 first principal components produced a significant difference in ancestry between cases and controls. Overall, a total of 1,161 filtered individuals (23 excluded from the French cohort and 39 from the Swiss cohort) were used for genome -wide statistical analyses. Table 1 presents the characteristics of these 1,161 subjects.

Replication cohorts were genotyped for 96 SNPs with Illumina GoldenGate genotyping on VeraCode technology (Illumina). In addition, the French primary cohort was genotyped for the 33 SNPs (out of these 96 SNPs) that were initially imputed in this cohort (see next section). Nine SNPs (including 4/33 imputed SNPs) were discarded based on the following quality-control filters: call rate < 90%, Hardy- Weinberg P-value<0.05, or a low- quality genotype clustering (evaluated by visual inspection). No individuals were excluded based on genotyping call rate.

Genotype Imputation: Almost all the SNPs (98.8%) of the French cohort were genotyped in the Swiss cohort, leading to a total of 321,744 common high-quality SNPs. In order to homogeneize the analysis of the two cohorts, genotype imputation was performed in the French primary cohort with MACH vl .O,²⁸ using the Swiss primary cohort as the template panel. SNPs with low imputation quality (r²-hat<0.3) and allele discrepancies were discarded, yielding 455,026 untyped markers confidently imputed in the French cohort. The analyses were therefore conducted on a total of 780,650 SNPs. In case of additive genetic model, allele dosages were used. We also considered dominant and recessive genetic models (when using the binary and duration phenotypes), for which the most likely genotype was inferred (by MACH). Whenever the posterior probability was below 80%> the genotype was assigned to be missing.

Among the 96 SNPs selected for replication, thirty-three SNPs were initially imputed in the French primary cohort. These 33 SNPs were thus genotyped in the French cohort for validation. For the 29 SNPs that passed quality controls, the overall concordance rate between imputed and genotyped SNPs was >94%>. Among the seven SNPs showing evidence for true replication, two SNPs, rs4374383 and rsl6851720, were not present in the filtered cohort of 219 Australian patients, genotyped by the Illumina Infinium HumanHap300 or the CNV370- Quad genotyping BeadChips (Illumina). We imputed these two SNPs using the same procedure as described above. For these two SNPs, the imputation accuracy observed in the French cohort was 96%, confirming the high efficiency of the imputation procedure for those SNPs.

Statistical analyses: For liver fibrosis considered as binary phenotypes, we used Fisher's exact test and logistic regression. For duration phenotypes, we used a Cox model considering estimated age at infection as the starting point and the first biopsy showing severe fibrosis (failure time) or the last biopsy showing an absence of severe fibrosis in the absence of treatment (censored time) as the endpoint. Logistic regression and Cox model analyses were used for stratified and multivariate adjusted analyses, with sex, HCV genotype, age at infection, alcohol consumption and/or mode of HCV acquisition as covariates. For the QTF phenotype, linear regression analysis was performed. All statistical analyses were carried out with procedures (LOGISTIC, PHREG) implemented in SAS software v.8.2 (SAS Institute, Cary, North Carolina, USA), R software (http://www.R-project.org ), Matlab (www.mathworks.com) and PLINK.²⁹ The power of this study was estimated for the binary F0- 1/3-4 phenotype and the duration F0- 1/3-4 phenotype, as described in. We carried out Gene Ontology overrepresentation analysis for the four corresponding genes using GeneTrail (http://genetrail.bioinf.uni-sb.de/index.php).³⁰

Results

Genome-wide analyses in the primary cohort: GWA analyses were conducted on a filtered primary cohort of 1,161 HCV-infected patients (Table 1), with a total of 780,650 SNPs. We first used the binary F0-1/F3-4 phenotype of liver fibrosis and assumed an additive genetic model. No significant deviations from expectations were observed on quantile- quantile plots before and after correction for population stratification, attesting to an absence of difference in ancestry between cases and controls (genomic inflation factor λ=1.013). In addition, none of the first 10 principal components of ancestry was associated with case- control status. We therefore carried out all subsequent analyses without adjustment for population stratification.

P-values<10^"6 were obtained for two SNPs on chromosomes 2 and 8. When restricting our cohort to the most extreme Metavir scores (binary F0/F4 phenotype), a single locus on chromosome 2, tagged by four SNPs in strong linkage disequilibrium (LD), achieved a P- value<10^"6. We then carried out GWA analyses taking into account the estimated duration of infection in 1,064 subjects for whom follow-up information was available. Two SNPs on chromosomes 11 and 18 provided P-values<10^"6 when considering the duration F0-1/F3-4 phenotype. When restricting the analysis to the duration F0/F4 phenotype, a P-value<10^"6 was obtained for one SNP on chromosome 6. Finally, the GWAS for the QTF phenotype identified a single SNP on chromosome 3 with a P-value<10^"6 (Table 2). For all phenotypes, 45 additional independent signals gave P-values<5xl0^"5. All the classical risk factors for fibrosis development available in our sample (i.e. sex, alcohol consumption, HCV genotype, HCV mode of acquisition and age at infection) were significantly and independently associated with fibrosis progression in our sample, in multivariate logistic regression analysis. Multivariate analyses of our binary and duration F0- 1/F3-4 pheno types adjusted for these factors did not substantially change our previous results. We also performed stratified analyses for these phenotypes. This involved conducting separate analyses on strata defined according to binary criteria such as sex (male/female), HCV genotype (1/others) or mode of acquisition (blood trans fusion/IDU). In analyses with the duration F0-1/F3-4 phenotype, six SNPs gave P-values<10^~6, including three with P- values<10^~7. For all phenotypes, 38 additional independent signals gave P-values<5xl0^~5.

Replication study: In the second phase, we genotyped a total of 96 independent SNPs presenting a P-value<5xl0^~5 in one of our previous GWA analyses in a replication cohort of 962 HCV-infected patients. Eighty-seven of these SNPs satisfying the quality-control filters were tested for association. Evidence for true replication (using the same model as in the primary analysis) at the 0.05 level was obtained for seven SNPs (Table 2). We further tested these seven SNPs in an additional filtered cohort of 219 Australian patients with liver biopsy data, who were previously genotyped for -310,000 SNPs by Illumina beadchips in a GWAS of response to chronic hepatitis C treatment.¹² Out of these seven SNPs, two (rs4374383 and rs 16851720) were not genotyped in the GWAS array, and were successfully imputed in this Australian cohort (Patients & Methods).

The combined analysis of these seven SNPs in the total sample of 2,342 patients identified two SNPs significant at the genome -wide level (Table 2). SNP rsl6851720 provided a combined P-value=8.9xl0^"9 in the whole sample, in the analysis of the QTF phenotype (additive for C, difference between genotypes of 0.23 SMU (0.15-0.31)). This intronic SNP is located in RNF7, encoding a redox-inducible antioxidant protein that protects against apoptosis.³¹ The second SNP was rs4374383, with P-value=2.1xl0^"9 in blood- transfused patients in analysis of the duration F0-1/F3-4 phenotype (recessive for minor allele A, hazard ratio of developing liver fibrosis (HR) for AA vs.AG/GG=0.18 (0.09-0.36)). We found that this association was independent of viral genotypes although blood-transfused patients are more often infected with HCV genotypes 1 and 2. SNP rs4374383 is intronic in the MEPvTK gene, a member of the three TAM receptor tyrosine kinases which are involved in the regulation of inflammatory responses.³² Of note, MERTK SNP rsl7174870 (r²=0.15 with rs4374383 in HapMap CEU) was identified as one of the 29 additional susceptibility loci recently involved in multiple sclerosis.³³

Out of the five remaining replicated SNPs, two were improved or unchanged by the addition of the Australian cohort (Table 2). One was rs2629751 in analysis of the binary F0/F4 phenotype (P=1.4xl0^"7; recessive for G, OR=7.10 (2.94-20.74)), which is intronic in the GLT8D2 gene. The other signal was identified using the duration F0-1/F3-4 phenotype in men at SNP rs9380516 (P=5.4xl0^~7; recessive for T, HR=4.53 (2.78-7.39)), which is located 21 kb downstream from the TULP1 gene. The three last signals (SNPs rs883924, rs7800244 and rs6485480) were clearly less significant when adding the Australian cohort (Table 2). Finally, we investigated the possible association of some previously published candidate genes with liver fibrosis progression in our primary cohorts, and we found that the previously reported risk allele G at the non-synonymous PNPLA3 variant rs738409^18"20 was strongly associated with liver fibrosis using the binary F0-1/F3-4 phenotype (additive for G, P=1.3xl0^" ⁴, OPv=1.21 (1.10-1.34)).

Discussion:

We report the first GWA study investigating liver fibrosis progression in a large sample of more than 2,300 HCV-infected patients of European descent. For all patients, liver biopsy data were obtained before treatment. We defined several fibrosis phenotypes on the basis of histological findings, because the Metavir system grading is not linear,²³ and because we wished to investigate various and complementary aspects of the fibrosis process. In this context, we also sought to use an additional, more sophisticated approach, involving a Markov model of the probability of transition between Metavir stages. This approach provided no additional signals with P values<5xl0^"6 for our primary cohort. Overall, our analyses covered a large panel of liver fibrosis phenotypes in a well characterized sample of HCV-infected patients, and identified four replicated signals including two that were significant at the genome-wide level in the combined cohort.

Gene ontology enrichment analysis of our four most interesting GWAS signals revealed significant enrichment for two categories, "photoreceptor outer segment" and "phagocytosis" ( =5xl0 ⁴ and =5xl0^"3, respectively, after Bonferroni correction). This result is explained by two SNPs: rs4374383, our most significant signal, and rs9380516, located within or close to MERTK and TULP1, respectively. Interestingly, rs4374383 is in strong LD (r^0.9 in CEU) with two MERTK non-synonymous SNPs, rs7604639 and rs381 1635 and rs9380516 is in strong LD (r²=0.9 in CEU) with an intronic SNP of TULP1, rs9296155. Although Mendelian defects of these two genes affect only the retina, ^" both genes have a broader spectrum of expression, with TULP1 expressed in the fetal liver³⁷ and MERTK expressed in macrophages.³² In addition, the MERTK and TULP1 proteins have been shown to interact during the phagocytosis of apoptotic cells.³⁸ Interestingly, mice lacking specifically the murine homolog of MERTK display a deficiency in the clearance of apoptotic thymocytes by macrophages.³⁹ Our results suggest a possible relationship between the clearance of apoptotic cells through phagocytosis and liver fibrosis, a pathogenic mechanism that has already been shown to be involved in fibrogenesis.⁴⁰'⁴¹

The second signal significant at the genome-wide level was that for SNP rs 16851720 in the whole sample. The effect of this SNP on fibrosis progression rate (FPR) was clearly additive in our combined cohort. If FPR means were calculated directly as a function of rs 16851720 genotypes, we found that each A risk allele increased the rate of liver fibrosis progression by 0.033 Metavir units/year, corresponding to ~1 Metavir unit over a 30-year period. This SNP is located in the first intron of RNF7, which is also known as SAG (sensitive to apoptosis gene). RNF7 is a cell-protecting molecule that acts as an antioxidant, inhibiting the apoptosis induced by metal ions and reactive oxygen species (ROS),³¹ thereby preventing DNA damage.⁴² In this context, it is interesting to note that HCV inhibits host DNA damage repair through ROS production.⁴³ The induction of these mechanisms in HCV-infected cells, leading to hepatocyte apoptosis, may also contribute to the development of liver fibrosis.⁴⁴ In conclusion, for the whole sample, we found a genome-wide significant effect of the

SNP rsl6851720 on liver fibrosis, and confirmed the role of the PNPLA3 non-synonymous I148M variant. We also found some interesting additional signals, including one genome- wide significant, in more refined subsamples, which require further investigation. Overall, our findings suggest that several variants in a number of genes with rather modest effects (in the whole sample) and/or more complex effects (e.g. limited to a specific subsample) may be involved in genetic susceptibility to liver fibrosis secondary to chronic HCV infection. They also point out the role of apoptosis potentially providing new insights into the mechanisms underlying liver fibrosis development, and opening new therapeutic avenues.⁴⁰'⁴¹

Covariate Category French cohort³ Swiss cohort³ Total

Sex

male 209 (44.8%) 433 (62.4%) 642 (55.3%) female 258 (55.2%) 261 (37.6%) 519 (44.7%)

Metavir score F0 41 (8.8%) 77 (11.1%) 118 (10.2%)

Fl 244 (52.2%) 217 (31.3%) 461 (39.7%)

F2 20 (4.3%) 192 (27.7%) 212 (18.3%)

F3 93 (19.9%) 86 (12.4%) 179 (15.4%)

F4 69 (14.8%) 122 (17.6%) 191 (16.5%)

Alcohol consumption

Low (<40g/day) 399 (85.4%) 542 (78.1%) 941 (81.1%)

High^b (>40g/day) 68 (14.6%) 132 (19.0%) 200 (17.2%)

Not available (NA) 0 (0.0%) 20 (2.9%) 20 (1.7%)

HCV mode of acquisition

IDU 157 (33.6%) 290 (41.8%) 447 (38.5%)

Blood transfusion 205 (43.9%) 133 (19.2%) 338 (29.1%)

OthersTNA 105 (22.5%) 271 (39.0%) 376 (32.4%)

HCV genotype

1 294 (63.0%) 362 (52.2%) 656 (56.5%)

2 41 (8.8%) 67 (9.7%) 108 (9.3%)

3 75 (16.1%) 193 (27.8%) 268 (23.1%)

4 10 (2.1%) 57 (8.2%) 67 (5.8%)

NA 47 (10.1%) 15 (2.2%) 62 (5.3%) yige at infection

Patients with available data 450 (96.4%) 614 (88.5%) 1064 (91.6%)

Age at infection < 20 years 139 (29.8%) 304 (43.8%) 443 (38.2%)

Age at infection > 20 years 311 (66.6%) 310 (44.7%) 621 (53.5%)

Mean (SD) age at infection (years) 28.00 (12.93) 21.20 (10.66) 24.08 (12.14)

Mean (SD) duration of infection (years) 20.17 (9.25) 22.39 (10.61) 21.45 (10.11)

Mean (SD) FPR (Metavir units / year) 0.11 (0.16) 0.11 (0.15) 0.11 (0.16)

Total 467 694 1,161

Table 1. Clinical and demographic characteristics of the two primary cohorts. aFrench patients were included if they were not co-infected with HIV or HBV and had no coexisting chronic liver disease. We also preferentially enrolled patients if (i) they had F0- 1 or F3-4 Metavir scores, (ii) their presumed date of HCV acquisition was known and (iii) they had low levels of alcohol consumption. Swiss patients with known HIV or active HBV co-infection were excluded for the purposes of this study. For both cohorts, relevant factors were retrieved from clinical databases, including sex, age at infection, mode of HCV acquisition and HCV genotype.

bThis category includes past heavy drinkers.

ther modes of HCV acquisition include situations at risk of exposure to blood (e.g. health workers) and invasive protocols (e.g. surgery, tattoo, piercing).

Table 2. Replicated SNPs from our GWA analyses of different fibrosis phenotypes in the final cohort of 2,342 patients with chronic HCV infection.

aThe first allele m refers to the minor allele, and M to the major allele, in the combined cohort. The genetic model is defined on the basis of the minor allele. ^bThe initial combined P- value was obtained in the cohort of 2,123 patients, including Swiss and French primary cohorts (Table 1), and our replication cohort (Cornell, Marseilles and International cohorts). The total combined P- value was obtained by combining this cohort of 2,123 patients with the sample of 219 Australian patients previously genotyped in another GWAS.¹²

The effect is computed from the total sample of 2,342 patients. Effects correspond to the standardized difference in QTF rates, relative to the first genotype (Patients & Methods), when using the quantitative QTF phenotype, hazard ratios when the duration phenotype is considered, and odds-ratios when the binary phenotype is considered.

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Claims

CLAIMS:

1. A method of determining if a subject is predisposed to fast progression of liver fibrosis, the method comprising testing for said subject the genotype of at least one single nucleotide polymorphism selected from the group consisting of rsl6851720, rs4374383 and rs9380516 or any single nucleotide polymorphism in linkage disequilibrium with rsl6851720, rs4374383 or rs9380516.

2. The method according to claim 1 the presence of allele A for rsl6851720 in heterozygous or homozygous form indicates that said subject has an increased risk to develop fast progression of liver fibrosis.

3. The method according to claim 2 wherein the presence of allele A for rsl6851720 in a homozygous form indicates a higher risk to to develop fast progression of liver fibrosis than the presence of allele A in a heterozygous form.

4. The method according to claim 1 the presence of allele G for rs4374383 in heterozygous or homozygous form indicates that said subject indicates that said subject has an increased risk to develop fast progression of liver fibrosis.

5. The method according to claim 1 the presence of allele T for rs9380516 in a homozygous form indicates that said subject indicates that said subject has an increased risk to develop fast progression of liver fibrosis.

6. The method according to claim 1 wherein the single nucleotide polymorphism in linkage disequilibrium has a r² value greater than 0.2 with rsl6851720, rs4374383 or rs9380516.

7. The method according to any of the preceding claims wherein said subject is tested for rsl6851720, rs4374383 and rs9380516.

8. The method according to any of the preceding claims wherein the genotype of the single nucleotide polymorphism is tested from a sample obtained from the subject.

9. The method according to any of the preceding claims, wherein said subject is suffering from a disease selected from the group of an hepatitis viral infection, an hepatotoxicity, a liver cancer, a non alcoholic fatty liver disease (NAFLD), an autoimmune disease, a metabolic liver disease, and a disease with secondary involvement of the liver.

10. The method according to claim 9 wherein said subject is a hepatitis C virus-infected subject.

11. A kit for performing the method according to any of the preceding claims, comprising primers and/or probes for amplification of at least one region comprising a sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3.