WO2003064687A2 - Procedes - Google Patents

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WO2003064687A2
WO2003064687A2 PCT/GB2003/000337 GB0300337W WO03064687A2 WO 2003064687 A2 WO2003064687 A2 WO 2003064687A2 GB 0300337 W GB0300337 W GB 0300337W WO 03064687 A2 WO03064687 A2 WO 03064687A2
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patient
factor
risk
hcv
liver
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WO2003064687A3 (fr
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Howard Thomas
Adrian Hill
Mark Wright
Mark Thursz
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Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Publication of WO2003064687A3 publication Critical patent/WO2003064687A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention relates to prognostic and treatment methods relating to f ⁇ brotic liver disease, including fibrotic liver disease arising from Hepatitis C virus (HCV) infection.
  • HCV Hepatitis C virus
  • HCV infects 170 million people worldwide, and the majority of the morbidity and mortality associated with infection is due to the development of cirrhosis and its complications.
  • the rate of progression to cirrhosis through the deposition of fibrous tissue varies among individuals. However, only a small proportion of the variability in the rate of fibrosis can be explained by the known demographic and environmental factors (18% in one large study(l)).
  • the coagulation pathways may be of importance in liver injury as elsewhere in the body with supporting evidence from carbon tetrachloride induced fibrosis in the rat demonstrating increased fibrin and fibrinogen deposition in the injured liver(7).
  • Thrombin receptors on stellate cells are up regulated during liver damage(8) and Thrombin is also considered to be a stellate cell mitogen(9).
  • a number of coagulation factor polymorphisms have been shown to have functional effects on the clotting cascade and subsequent risk of thrombosis.
  • an Adenine for Guanine substitution at position 20210 in the 3 'untranslated region of the thrombin gene is associated with excess thrombin generation(l ⁇ ) and an increased risk of both arterial and venous thrombosis.
  • Factor V Lieden results from an amino acid substitution at position 506 of Gin for Arg and follows a single nucleotide substitution (Adenine for Guanine at position 1691).
  • the polymorphism confers resistance to activated protein C, part of a negative feed back loop which inhibits activated factor V. Together with activated factor X, factor V converts prothrombin to thrombin.
  • the polymorphism may lead to increased conversion of prothrombin to thrombin.
  • the factor V Leiden mutation has been shown to increase the risk of venous thromboembolism(l l).
  • the Coagulation factor VII Arg353Gln polymorphism has been shown to affect plasma levels, with the highest in Gln/Gln homozygotes (12).
  • liver disease especially liver disease arising from infection with a hepatitis virus, particularly HCV, and for aiding the clinician in the management of liver disease or viral hepatitis or viral infection, for example HCV infection.
  • an object of the invention is to provide a method of assessing the likely rate of liver fibrosis. Further objects of the invention include the provision of methods of treatment of liver disease (including any chronic inflammatory disease of the liver which may develop cirrhosis) and infection with a hepatitis virus..
  • a first aspect of the invention provides a method for assessing a patient's risk of development or progression of liver cirrhosis, comprising the step of determining the patient's genotype or phenotype for a coagulation factor.
  • the patient's genotype is a genotype associated with a procoagulant tendency (in particular Factor V Leiden), or the patient's phenotype is a procoagulant tendency or indicative of a procoagulant tendency, then the patient is considered to be at a higher risk of developing liver cirrhosis or fibrosis and/or of rapid progression of liver cirrhosis or fibrosis.
  • a procoagulant tendency in particular Factor V Leiden
  • determining whether the patient has a procoagulant tendency may in itself be predictive of development of clinically significant liver disease, or it may be used by the clinician as an aid in reaching a diagnosis or prognosis.
  • the method may be used as an adjunct to known assessment or prognostic methods such as histopathological examination of biopsy tissue, or imaging or serum marker assays (Imbert-Bismut F et al (2001) Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 357(9262): 1069-1075; Rosenberg W et al (2001) Serum Markers Predict Liver Fibrosis. British Association for the study of the Liver Meeting 2001, Book of Abstracts, 23).
  • the method may also be used in conjunction with consideration of other risk factors, for example sex (males may be at higher risk of rapid fibrosis) and age at infection (higher age at infection may be associated with a higher risk of rapid fibrosis).
  • Fibrosis and rate of fibrosis may be assessed as described in Example 1.
  • the degree of fibrosis may be scored using the modified histological activity index (HAI-Ishak) scoring system (Ishak et al (1995) Hepatol 22(6), 696-699), which assigns a score of 0-6 for the degree of fibrosis and a score of 0-18 for necro-inflammatory activity.
  • HAI-Ishak modified histological activity index
  • the rate of fibrosis in a patient may be estimated by assuming that the fibrosis stage at the time of infection is zero and may be expressed in terms of units per year.
  • a rate of fibrosis of more than about 0.3, 0.4 or 0.5 units, still more 0.6, 0.7, 0.8, 0.9, 1 or 2 units per year may be considered to be fast, whilst a rate of fibrosis of less than about 0.25 or (more preferably) 0.2, for example about 0.18, 0.15, 0.13, 0.11, 0.1, 0.09, 0.08, 0.07, 0.06 or 0.05 units per year may be considered to be slow.
  • a rate between about 0.2 or 0.25 and 0.3 units per year may be classified as intermediate rate.
  • Cirrhosis corresponds to a score of 6 on the HAI-Ishak scoring system and to a score of 4 using the METAVIR System (Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
  • a procoagulant tendency in particular presence of Factor V Leiden indicates that the patient is likely to have a rate of fibrosis of > 0.3 following liver damage, for example caused by HCV infection.
  • factors other than presence or absence of a procoagulant tendency for example age and sex may also influence the likely rate of fibrosis in a particular patient.
  • the presence of a procoagulant tendency may increase the likely rate of fibrosis (in the absence of the procoagulant tendency) by a factor of between about 1.2 and 4, for example between about 1.3, 1.4 or 1.5 and 2.0, 2.5 and 3.
  • determination of the coagulation factor genotype or phenotype will be useful to the clinician in determining how to manage the patient's disease (for those with liver disease or infected with a hepatitis virus) or in determining what activities an individual may undertake or should not undertake (for example to reduce the risk of exposure to a hepatitis virus for an individual at risk of rapid fibrosis in view of a procoagulant tendency).
  • a procoagulant tendency in particular the presence of Factor V Leiden
  • the clinician may use the information concerning the coagulation factor genotype or phenotype to facilitate decision making regarding treatment of the patient.
  • the genotype or phenotype is indicative of the absence of a procoagulant tendency (in particular if Factor V Leiden is absent)
  • early antiviral intervention and/or anticoagulant treatment may be the preferred treatment.
  • the determination of the coagulation factor genotype or phenotype may be exploited diagnostically to predict whether a given individual is likely to develop clinically significant liver fibrosis or cirrhosis, since the presence of a procoagulant tendency (in particular Factor V Leiden) is believed to correspond to possible future cirrhosis.
  • the coagulation factor may preferably be a coagulation factor involved in the regulation of thrombin activity, for example involved in (ie upstream of (in the intrinsic or extrinsic clotting pathway) or directly involved in) the cleavage of prothrombin to thrombin; or the inhibition or inactivation of thrombin; or thrombin itself.
  • the coagulation factor is Factor V. It is still further preferred that the method comprises the step of determining whether the patient has a Factor V genotype or phenotype which confers resistance to activated protein C, for example the Factor V Leiden genotype.
  • Factor V Leiden results from an amino acid substitution at position 506 of Gin for Arg and follows a nucleotide substitution (Adenine for Guanine at position 1691).
  • Factor V Leiden may be detected by genotypic analysis, for example using a PCR-based method (for example as described in Example 1).
  • Activated Protein C resistance (most frequently arising from Factor V Leiden) may be determined by known tests, for example the Activated Protein C (APC) sensitivity ratio, which is a partial thromboplastin time based clotting test in which results are compared in the presence and absence of APC.
  • APC Activated Protein C
  • the Coatest APC Resistance test supplied by Chromogenix, Sweden may be used in assessing APC resistance.
  • Other suitable assays may be described in Thrombosis and Haemostasis (1994) 72, 880-886; Vogel et al (1996) Blood 88(10), Suppll, part 1-2, 175a. A ratio of less than 2 may be indicative of APC resistance, though this value may vary slightly depending on the details of the test system used.
  • the coagulation factor is thrombin (including prothrombin). It is further preferred that the method comprises the step of determining whether the patient has an Adenine for Guanine substitution at position 20210 in the 3 'untranslated region of the thrombin gene (ie a mutation associated with excess thrombin generation(l ⁇ ) and an increased risk of both arterial and venous thrombosis).
  • the coagulation factor may be Factor X, Factor VIII, Factor IX, Factor XI or Factor VII.
  • the coagulation factor may be Tissue Factor, HMW Kininogen or Prekallikrein. These factors may be involved in the activation of thrombin (including the modulation of such activation).
  • the coagulation factor may be an antithrombin (for example antithrombin III), Protein C, Protein S, Protein C inhibitor, thrombomodulin or ⁇ 2 -Macroglobulin. These factors may be involved in the inactivation or inhibition of thrombin (including the modulation of such inactivation or inhibition).
  • Deficiency states of antithrombin 111, protein C and S are known and have a genetic basis (Bertina RM.Genetic approach to thrombophilia. Thromb Haemost 2001 Jul;86(l):92-103).
  • Protein C and Protein S function are well known in the art and include tests described in WO 96/42018 and references cited therein; US 5,169,786 and references cited therein; WO 91/01382 and references cited therein.
  • Protein S is measured using an ELISA kit.
  • Protein C and antithrombin III are measured as activity levels using a chromogenic assays. These are conducted according to standard protocols in clinical haematology labs by those skilled in the art.
  • the method may comprise testing the genotype or phenotype of the patient in relation to more than one coagulation factor.
  • the method may comprise testing the patient's genotype in relation to Factor V and thrombin (and optionally also Factor VII).
  • the method may comprise a multi-target PCR amplification step, for example as described in Example 1.
  • the targets may be restricted to coagulation factors for convenience (ie need not include all the targets in the test kit used in Example 1).
  • the patient genotype in relation to one or more coagulation factors is determined.
  • Methods of genotypic analysis will be well known to those skilled in the art.
  • the genotype may preferably be determined by testing a sample from the patient.
  • the sample contains nucleic acid, such as genomic DNA or mRNA (preferably genomic DNA), and the genotype is determined by a method which involves contacting said nucleic acid (or nucleic acid derived from it, for example cDNA when the target nucleic acid is an RNA, for example a mRNA) with a nucleic acid which hybridises selectively to said coagulation factor nucleic acid.
  • genotyping may be performed by any one of a large number of assays, for example Sequencing, RFLP, ARMS PCR, PCR and sequence- specific oligonucleotide hybridisation, Snapshot PCR, Ligase detection reaction, PCR and Maldi-TOF, Pyrosequencing.
  • assays for example Sequencing, RFLP, ARMS PCR, PCR and sequence- specific oligonucleotide hybridisation, Snapshot PCR, Ligase detection reaction, PCR and Maldi-TOF, Pyrosequencing.
  • the sample may conveniently be whole blood or genomic DNA extracted from whole blood, for example a 5 ml sample collected into EDTA and extracted using a commercial kit (for example as supplied by Nucleon II, Scotlabs, UK).
  • a commercial kit for example as supplied by Nucleon II, Scotlabs, UK.
  • nucleic acid has sufficient nucleotide sequence similarity with the said (human) nucleic acid that it can hybridise under moderately or highly stringent conditions.
  • stringency of nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence.
  • any nucleic acid which is capable of selectively hybridising as said is useful in the practice of the invention.
  • Nucleic acids which can selectively hybridise to the said (human) nucleic acid include nucleic acids which have >95% sequence identity, preferably those with >98%, more preferably those with >99% sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid.
  • human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene would not match perfectly along its entire length with the said human genomic DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA.
  • the invention specifically includes nucleic acids which selectively hybridise to said coagulation factor mRNA or cDNA but may not hybridise to a said coagulation factor gene (and vice versa).
  • nucleic acids which span the intron-exon boundaries of the said coagulation factor gene may not be able to selectively hybridise to the said coagulation factor mRNA or cDNA.
  • Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, incorporated herein by reference.
  • the hybridisation is performed at 68°C.
  • the nylon membrane, with the nucleic acid immobilised may be washed at 68°C in 1 x SSC or, for high stringency, 0.1 x SSC.
  • 20 x SSC may be prepared in the following way. Dissolve 175.3 g of NaCl and 88.2 g of Na + citrate in 800 ml of H 2 0. Adjust the pH to 7.0 with a few drops of a IO N solution of NaOH. Adjust the volume to 1 litre with H 2 0. Dispense into aliquots. Sterilize by autoclaving.
  • An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:
  • the optimal temperature for hybridization is usually chosen to be 5 C below the Tj for the given chain length.
  • Tj is the irreversible melting temperature of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637 discusses the determination of Tjs.
  • the recommended hybridization temperature for 17-mers in 3 M TMAC1 is 48-50 > C; for 19-mers, it is 55-57 * 0; and for 20-mers, it is 58- 66 * C.
  • nucleic acid which selectively hybridises is also included nucleic acids which will amplify DNA from the said coagulation factor genomic DNA or mRNA by any of the well known amplification systems such as those described in more detail below, in particular the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Suitable conditions for PCR amplification include amplification in a suitable 1 x amplification buffer:
  • 10 x amplification buffer is 500 mM KC1; 100 mM Tris.Cl (pH 8.3 at room temperature); 15 mM MgCl 2 ; 0.1% gelatin.
  • a suitable denaturing agent or procedure (such as heating to 95°C) is used in order to separate the strands of double-stranded DNA.
  • the annealing part of the amplification is between 37 * 0 and 60 * 0, preferably 50 > C.
  • nucleic acid which is useful in the methods of the invention may be RNA or DNA
  • DNA is preferred.
  • nucleic acid which is useful in the methods of the invention may be double-stranded or single- stranded, single-stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.
  • the nucleic acid which is useful in the methods of the invention may be any suitable size. However, for certain diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than 1000, more preferably still from 10 to 100, and in further preference from 15 to 30 base pairs (if the nucleic acid is double- stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.
  • the nucleic acid for use in the methods of the invention is a nucleic acid capable of hybridising to the said coagulation factor genomic DNA or mRNAs. Fragments of the said coagulation factor genes and cDNAs derivable from the mRNA encoded by the said coagulation factor genes are also preferred nucleic acids for use in the methods of the invention.
  • the nucleic acid for use in the methods of the invention is an oligonucleotide primer which can be used to amplify a portion of the said coagulation factor (for example Factor V or Factor V Leiden) nucleic acid, particularly said coagulation factor genomic DNA (for example Factor V or Factor V Leiden genomic DNA).
  • said coagulation factor for example Factor V or Factor V Leiden
  • Nucleic acids for use in the invention may hybridise to more than one allele of a coagulation factor or may hybridise only to one allele. Single-allele- specific and multi-allele-specific nucleic acids may be used in combination, for example in an amplification reaction.
  • Preferred nucleic acids for use in the invention are those that selectively hybridise to a particular allele (ie a particular polymorphism) and do not hybridise to other alleles (polymorphisms) of the coagulation factor.
  • Such selectively hybridising nucleic acids can be readily obtained, for example, by reference to whether or not they hybridise to the said coagulation factor allele (preferably genomic) nucleic acid and not to other allele (preferably genomic) nucleic acid.
  • Example 1 Details of suitable primers are given in references in Example 1, for example Cheng et al (1999) Genome Res 9, 936-949. Methods and nucleic acids as described, for example, in Example 1, may be used.
  • polymorphisms for example associated with Protein C and Protein S
  • Suitable methods and nucleic acids for assessing such polymorphisms may be designed or selected by methods well known to those skilled in the art, for example as indicated above and discussed further below.
  • the methods are suitable in respect of any fibrosing liver disease (which term excludes thrombotic conditions such as Budd Chiari syndrome and Portal vein thrombosis) but it is preferred if the patient has or is at risk of hepatitis virus infection, particularly HCV infection.
  • the patient is a patient with or at risk of viral hepatitis, for example with or at risk of infection with Hepatitis A virus (HAV), Hepatitis B virus (HBV) or Hepatitis C virus (HCV).
  • HCV Hepatitis A virus
  • HBV Hepatitis B virus
  • HCV Hepatitis C virus
  • the patient is infected with, or at risk of infection by, HCV. Infection may be judged by the presence of antibodies to the relevant virus and preferably also by the presence of RNA from the relevant virus. This may be determined by methods well known to those skilled in the art, and as noted in Example 1.
  • the patient may have acute HCV infection or may have or be at risk of chronic HCV infection.
  • a patient may be at risk of viral hepatitis due to occupational exposure or an intravenous drug habit.
  • the method may be useful in identifying persons whose exposure to hepatitis virus should be minimised due to a higher risk of rapid disease progression should infection occur.
  • the aforementioned methods may be used for presymptomatic screening of a patient who is in a risk group for liver disease, particularly viral hepatitis, for example HCV infection.
  • a risk group for liver disease particularly viral hepatitis, for example HCV infection.
  • healthcare workers may be at a greater risk of HCV infection (for example as a consequence of needle stick injuries) than other occupational groups.
  • Intravenous drug users may also be at greater risk.
  • the sample tested is blood or is derived from blood from the patient.
  • any sample containing nucleic acid derived from the patient is useful in the methods of the invention when determining genotype, it is preferred if the sample is readily obtainable from the patient, for example blood, urine, semen or skin cells (for example a sample of cells from the buccal cavity). Liver tissue taken at biopsy may be used. Most conveniently the sample is blood. Although it is preferred that the sample containing nucleic acid from the patient is, or is derived directly from, a cell of the patient, a sample indirectly derived from a patient, such as a cell grown in culture, is also included within the invention. Equally, although the nucleic acid derived from the patient may have been physically within the patient, it may alternatively have been copied from nucleic acid which was physically within the patient.
  • the nucleic acid capable of selectively hybridising to the said human nucleic acid such as genomic DNA and which is used in the methods of the invention may further comprise a detectable label.
  • detecttable label is included any convenient radioactive label such as
  • the term "detectable label” also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphatase can convert colourless o- nitrophenylphosphate into coloured o-nitrophenol).
  • the nucleic acid probe may occupy a certain position in a fixed array and whether the nucleic acid hybridises to the said coagulation factor nucleic acid can be determined by reference to the position of hybridisation in the fixed array.
  • PCR polymerase chain reaction
  • PCR primers do not contain any complementary structures with each other longer than 2 bases, especially at their 3' ends, as this feature may promote the formation of an artifactual product called "primer dimer”.
  • primer dimer When the 3' ends of the two primers hybridize, they form a “primed template” complex, and primer extension results in a short duplex product called “primer dimer”.
  • Optimum annealing temperatures may be determined empirically and may be higher than predicted.
  • Taq DNA polymerase does have activity in the 37-55 > C region, so primer extension will occur during the annealing step and the hybrid will be stabilized.
  • concentrations of the primers are equal in conventional (symmetric) PCR and, typically, within 0.1- to l ⁇ M range.
  • nucleic acid amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and ligase chain reaction.
  • NASBA nucleic acid sequence based amplification
  • 3SR can be used as described in Compton
  • a pair of suitable nucleic acids of the invention When a pair of suitable nucleic acids of the invention are used in a PCR it is possible to detect the product by gel electrophoresis and ethidium bromide staining.
  • a labelled oligonucleotide capable of hybridising to the amplified DNA as a probe.
  • the oligonucleotide probe hybridises to the interprimer sequence as defined by the two primers.
  • the oligonucleotide probe is preferably between 10 and 50 nucleotides long, more preferably between 15 and 30 nucleotides long.
  • the probe may be labelled with a radionuclide such as P, P and S using standard techniques, or may be labelled with a fluorescent dye.
  • a radionuclide such as P, P and S
  • the amplified DNA product may be detected in solution (see for example Balaguer et al (1991) "Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescence adsorbent" Anal. Biochem. 195, 105-110 and Dilesare et al (1993) "A high-sensitivity electrochemiluminescence-based detection system for automated PCR product quantitation" BioTechniques 15, 152-157.
  • PCR products can also be detected using a probe which may have a fluorophore-quencher pair or may be attached to a solid support or may have a biotin tag or they may be detected using a combination of a capture probe and a detector probe.
  • Fluorophore-quencher pairs are particularly suited to quantitative measurements of PCR reactions (eg RT-PCR). Fluorescence polarisation using a suitable probe may also be used to detect PCR products.
  • the level or activity of a coagulation factor protein is measured.
  • the level of said protein is measured by contacting the protein with a molecule which selectively binds to said coagulation factor, or other known technique for quantifying the said coagulation factor.
  • Manuals relating to assays for coagulation factors are well known to those skilled in the art, and include "Dacie and Lewis's Practical Haematology", 9 th Edition (2001), Churchill Livingstone, Dacie, Lewis Eds.
  • the sample containing protein derived from the patient is conveniently blood.
  • the molecule which selectively binds to the coagulation factor is an antibody.
  • Antibodies which can selectively bind to a particular coagulation factor can be made using techniques well known to those skilled in the art, or may be obtained from, for example, Sigma (Sigma-Aldrich Company Limited, Fancy Road, Poole, Dorset, BH12 4QH, UK; see "Antibodies and Reagents for Coagulation Research section of catalogue).
  • the antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference.
  • the level of a coagulation factor which is indicative of a procoagulant tendency may be defined as the increased level (for procoagulant factors) or decreased level (for inhibitors of coagulation) present in known procoagulant patients over individuals with normal coagulation.
  • the level may be, for example, at least 1.5 Standard deviations [SD] higher (or lower, for inhibitors of coagulation) in patients with a procoagulant tendency, or it may be at least 2 SD or 3 SD higher (or lower, as appropriate) when compared with a population of individuals with normal coagulation.
  • the relative amount of said coagulation factor protein is meant the amount of said coagulation factor protein per unit mass (or volume) of sample tissue or per unit number of sample cells compared to the amount of said coagulation factor protein per unit mass of known normal tissue or per unit number of normal cells.
  • the relative amount may be determined using any suitable protein quantitation method.
  • antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.
  • synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs)
  • dAbs domain antibodies
  • a further aspect of the invention provides the use of an agent which is capable of use in determining the genotype or phenotype of a patient for a coagulation factor (for example for characterising the patient's nucleic acid or coagulation process) in the manufacture of a reagent for assessing the patient's risk of development or progression of liver cirrhosis, for example following HCV infection.
  • a coagulation factor for example for characterising the patient's nucleic acid or coagulation process
  • the agent may suitably be a nucleic acid which selectively hybridises to a coagulation factor nucleic acid (preferably Factor V nucleic acid, for example Factor V Leiden nucleic acid) or the agent may be a molecule which selectively binds to a coagulation factor protein or the agent may be an agent useful in selectively assaying the activity of the coagulation factor or all or part of the patient's coagulation process.
  • reagents useful in measuring the APC sensitivity ratio test include platelet poor plasma, kaolin 5g/l in barbitone buffered saline pH 7.4, phospholipid, CaC12 and APC.
  • the agents as defined are therefore useful in a method of prognosing liver fibrosis or cirrhosis, particularly arising from HCV (or other hepatitis virus) infection.
  • a further aspect of the invention comprises a kit of parts useful for prognosis of liver fibrosis or cirrhosis, especially arising from HCV infection, comprising means for use in determining the genotype or phenotype of a patient for a coagulation factor (for example for characterising the patient's nucleic acid or coagulation process).
  • the means may be or comprise an agent which may suitably be a nucleic acid which selectively hybridises to a coagulation factor nucleic acid, for example genomic DNA (preferably Factor V nucleic acid, for example Factor V Leiden nucleic acid).
  • the agent may be a molecule which selectively binds to a coagulation factor protein or the agent may be an agent useful in selectively assaying the activity of the coagulation factor or all or part of the patient's coagulation process, for example as noted above.
  • the kit further comprises a control sample containing coagulation factor nucleic acid or protein wherein the control sample may be a negative control (which contains a coagulation factor nucleic acid or protein which is not associated with a procoagulant tendency) or it may be a positive control (which contains a coagulation factor nucleic acid or protein which is associated with a procoagulant tendency).
  • the kit may contain both negative and positive controls.
  • the kit may usefully contain controls of coagulation factor protein or nucleic acid which correspond to different amounts such that a calibration curve may be made.
  • the kit further comprises means suitable for use in determining whether the patient has been exposed to or infected with a hepatitis virus (preferably HCV) (for example by checking for antibodies to the virus) and/or whether the patient has continuing infection (for example by checking for viral RNA).
  • a hepatitis virus preferably HCV
  • the kit may preferably comprise (as an example) nucleic acids suitable for use as PCR primers for amplifying a hepatitis virus nucleic acid (for example following reverse transcription of viral RNA), preferably HCV nucleic acid.
  • a further aspect of the invention provides a method for treating a patient with or at risk of liver fibrosis or cirrhosis comprising the step of administering to the patient an anticoagulant agent.
  • a further aspect of the invention provides an anticoagulant agent for use in the treatment of a patient with or at risk of liver fibrosis or cirrhosis.
  • a still further aspect of the invention provides an anticoagulant agent for use in the treatment of a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV).
  • a further aspect of the invention provides the use of an anticoagulant in the manufacture of a medicament for the treatment of a patient with or at risk of liver fibrosis or cirrhosis, or with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV).
  • a further aspect of the invention provides a method for treating a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV) comprising the step of administering to the patient an anticoagulant agent.
  • a further aspect of the invention provides an anticoagulant agent for use in the treatment of a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV).
  • a still further aspect of the invention provides a method for treating a patient with or at risk of liver fibrosis or ci ⁇ hosis, comprising the steps of (1) assessing the patient's risk of development or progression of liver ci ⁇ hosis, using a method comprising the step of determining the patient's genotype or phenotype for a coagulation factor; (2) if the patient's risk of development or progression of liver ci ⁇ hosis is high (ie the patient is at risk of rapid fibrosis), administering to the patient an anticoagulant agent and/or other therapeutic agent suitable for treating liver fibrosis.
  • the therapeutic agent may be, for example, a steroid in autoimmune hepatitis or ursodeoxycholic acid in primary biliary ci ⁇ hosis.
  • a further aspect of the invention provides an anticoagulant agent and/or other therapeutic agent suitable for treating liver fibrosis for treatment of a patient with or at risk of liver fibrosis or ci ⁇ hosis, wherein the patient is one whose risk of development or progression of liver ci ⁇ hosis has been assessed using a method comprising the step of determining the patient's genotype or phenotype for a coagulation factor; and the patient's determined risk of development or progression of liver ci ⁇ hosis is high (ie whose predicted rate of fibrosis is rapid).
  • a still further aspect of the invention provides a method for treating a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), comprising the steps of (1) assessing the patient's risk of development or progression of liver ci ⁇ hosis, comprising the step of determining the patient's genotype or phenotype for a coagulation factor; (2) if the patient's risk of development or progression of liver ci ⁇ hosis is high (ie the patient is at risk of rapid fibrosis), administering to the patient an anticoagulant agent and/or other therapeutic agent suitable for treating viral hepatitis, for example an antiviral agent, for example -interferon.
  • hepatitis virus infection for example HBV, HAV or HCV, preferably HCV
  • a further aspect of the invention provides an anticoagulant agent and/or other therapeutic agent suitable for treating viral hepatitis, for example an antiviral agent, for example ⁇ -interferon, for treatment of a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), wherein the patient is one whose risk of development or progression of liver ci ⁇ hosis has been assessed, using a method comprising the step of determining the patient's genotype or phenotype for a coagulation factor; and the patient's determined risk of development or progression of liver ci ⁇ hosis is high (ie whose predicted rate of fibrosis is rapid).
  • an antiviral agent for example ⁇ -interferon
  • the invention provides a method for treating a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), comprising the steps of (1) determining the patient's genotype or phenotype for Factor V; (2) if the patient has Factor V Leiden, administering to the patient an anticoagulant agent and/or other therapeutic agent suitable for treating viral hepatitis, for example an antiviral agent, for example ⁇ -interferon.
  • hepatitis virus infection for example HBV, HAV or HCV, preferably HCV
  • a further aspect of the invention provides an anticoagulant agent and/or other therapeutic agent suitable for treating viral hepatitis, for example an antiviral agent, for example ⁇ -interferon, for treatment of a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), wherein the patient has Factor V Leiden.
  • an anticoagulant agent and/or other therapeutic agent suitable for treating viral hepatitis for example an antiviral agent, for example ⁇ -interferon, for treatment of a patient with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), wherein the patient has Factor V Leiden.
  • anticoagulant will be well known to those skilled in the art and includes agents (or their prodrugs or precursors) which have the effect or reducing clotting tendency or extending the time taken for clotting to occur in response to an initiating event.
  • Particularly useful anticoagulants may be those useful in the treatment of patients with Factor V Leiden (homozygous or heterozygous) and thrombosis. These include (often after initial treatment with heparins) oral vitamin K antagonists (for example Warfarin, phenindione and acenocoumarol/nicoumalone) .
  • oral vitamin K antagonists for example Warfarin, phenindione and acenocoumarol/nicoumalone
  • prefened anticoagulants are considered to include direct thrombin inhibitors (or their precursors), for example Hirudin, Bivalirudin, melagatran and napsagatran.
  • Activated protein C which has been administered therapeutically in meningococcal sepsis, or modified forms with longer half-lives and greater activity may also be useful.
  • prefened agents suitable for treating viral hepatitis may include agents discussed in the following patent applications (all incorporated herein by reference): PCT/GBO 1/03901 (relating to agents useful in inhibiting HCV replication in the CNS); PCT/GBO 1/04636 (relating to HCV prophylactic and therapeutic vaccines) and PCT/GB01/05722 (relating to recombinant bone ma ⁇ ow derived stem cells or cells derived therefrom capable of expressing therapeutic products).
  • a patient with a procoagulant allele or polymorphism may be treated using recombinant bone ma ⁇ ow derived stem cells or cells derived therefrom (as described in PCT/GBO 1/05722) capable of expressing (for example when differentiated into liver cells) a "normal" (non-procoagulant) allele.
  • a patient with Factor V Leiden may be treated using recombinant cells capable of expressing (for example when differentiated into liver cells) a normal Factor V (ie not Factor V Leiden).
  • a further aspect of the invention provides a bone-ma ⁇ ow derived stem cell comprising a recombinant nucleic acid capable of expressing, in the bone-ma ⁇ ow derived stem cell or cell derived therefrom, normal Factor V.
  • a still further aspect of the invention provides a recombinant polynucleotide suitable for expressing in a bone-ma ⁇ ow derived stem cell or cell derived therefrom normal Factor V.
  • the recombinant polynucleotide is suitable for expressing the normal Factor V under control of a liver-specific promoter and/or an inducible promoter.
  • a further aspect of the invention provides a recombinant polynucleotide construct (chimaeroplasty construct) comprising RNA and DNA elements suitable for co ⁇ ecting a Factor V Leiden genotype to a normal Factor V genotype in a bone-ma ⁇ ow derived stem cell or cell derived therefrom.
  • Chimaeric RNA/DNA polynucleotide constructs and their use in gene therapy are described in, for example, Lai & Lien (2001) Expert Opin Biol Ther 1(1), 41-47 and Lai & Lien (1999) Exp Nephrol 7(1), 11-14.
  • Chimaeric constructs have been shown to be useful in co ⁇ ecting point mutations in several genetic disease models, without using viral vectors.
  • Chimaeric constructs may be used to co ⁇ ect the Factor V genotype of bone- ma ⁇ ow derived stem cells (or their progeny) obtained from the patient, and the co ⁇ ected cells (or their progeny) returned
  • the construct may comprise a Moloney Leukaemia Virus (MLV) based retroviral vector or a lentiviral vector or adeno-associated vector (AAV), or a baculovirus based vector.
  • MMV Moloney Leukaemia Virus
  • AAV adeno-associated vector
  • Pieroni et al 2001) Hum Gene Ther 12(8), 871-881 and Pieroni & La Monica (2001) Curr Opin Mol Ther 3(5), 464-467 discuss the use of baculovirus vectors as gene therapy vectors.
  • Baculovirus vectors may have the advantages that they do not replicate in mammalian cells, do not cause a cytopathic effect upon infection and are able to cany large DNA inserts. Although baculovirus vectors may not be suited to in vivo gene delivery by systemic administration, due to rapid removal by the complement system, they may be used in ex-vivo systems, in which cells are removed from the patient, treated with the virus and then returned to the patient (or progeny of the patient-derived cells treated or returned to the patient).
  • a further aspect of the invention provides a bone-ma ⁇ ow derived stem cell, recombinant polynucleotide or gene therapy construct according to the invention for use in medicine.
  • a further aspect of the invention provides a bone-manow derived stem cell, recombinant polynucleotide or gene therapy construct according to the invention for use in treating a patient with or at risk of liver fibrosis and/or with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV).
  • the patient may be selected on the basis of coagulation factor genotype or phenotype; preferably the patient has Factor V Leiden.
  • a further aspect of the invention provides a pharmaceutical preparation comprising a bone-manow derived stem cell, recombinant polynucleotide or gene therapy construct of the invention and a pharmaceutically acceptable canier.
  • the ca ⁇ ier(s) must be "acceptable" in the sense of being compatible with the bone-ma ⁇ ow derived stem cell, recombinant polynucleotide or gene therapy construct and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • a further aspect of the invention provides a method for treating a patient with or at risk of liver fibrosis and/or with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV) comprising the steps of (1) providing a bone-ma ⁇ ow derived stem cell according to the invention, or a recombinant nucleic acid of the invention (2) introducing the stem cell or recombinant nucleic acid into the patient.
  • hepatitis virus infection for example HBV, HAV or HCV, preferably HCV
  • the bone-ma ⁇ ow derived stem cell may be provided by a method comprising the steps of (1) obtaining a bone-manow derived stem cell from the patient, (2) introducing a recombinant nucleic acid encoding normal Factor V (ie not Factor V Leiden) into the bone-manow derived stem cell. It is strongly prefened that the bone-ma ⁇ ow derived stem cells are derived from the patient being treated, but this not essential.
  • the recombinant nucleic acid may comprise an inducible promoter controlling expression of the Factor V and the method may comprise the step of administering to the patient a molecule that regulates expression from the inducible promoter.
  • the recombinant nucleic acid may further comprise an inducible promoter controlling expression of a cytotoxic product and the method may comprise the step of administering to the patient a molecule (cytotoxic inducer) that regulates expression from the inducible promoter controlling expression of the cytotoxic product.
  • a molecule cytotoxic inducer
  • the mobilising composition may comprise G- CSF (Granulocyte-colony stimulating factor).
  • the colonisation composition may comprise T3, HGF and/or KGF.
  • a further aspect of the invention provides a kit of parts or composition comprising (1) (a) a bone-ma ⁇ ow derived stem cell of the invention or (b) a recombinant polynucleotide or gene therapy vector of the invention and an agent useful in promoting transfection of bone-ma ⁇ ow derived stem cells, and (2) a mobilising composition or colonising composition as defined above and/or means for testing whether the patient has Factor V Leiden.
  • a further aspect of the invention provides a method for identifying a compound for treating a patient with or at risk of liver fibrosis and/or hepatitis virus infection, the method comprising contacting a test compound with Factor V or Factor V Leiden (which term includes activated (thrombin- cleaved) Factor V or Factor V Leiden); determining whether said compound is capable of modulating the activity of Factor V or Factor V Leiden; and selecting a compound which has an activity which has an anticoagulant effect.
  • the compound may inhibit activated Factor V Leiden, or may render activated Factor V Leiden susceptible to inhibition by activated Protein C.
  • the method may comprise a step of contacting a test compound with Factor V or Factor V Leiden and determining whether the compound is capable of binding to Factor V or Factor V Leiden; compounds which are capable of binding may then be tested for modulation of the activity of Factor V or Factor V Leiden.
  • a further aspect of the invention provides a compound identified by the screening method of the invention for use in medicine.
  • a further aspect of the invention provides such a compound for use in treating a patient with or at risk of liver fibrosis and/or with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV).
  • a further aspect of the invention provides a method for treating a patient with or at risk of liver fibrosis and/or with or at risk of hepatitis virus infection (for example HBV, HAV or HCV, preferably HCV), comprising the step of administering to the patient such a compound.
  • the patient may be selected for treatment on the basis of coagulation factor genotype or phenotype as described above; preferably the patient has Factor V Leiden.
  • Figure 4 Potential mechanisms by which Factor V Leiden may lead to more rapid fibrosis
  • Example 1 Factor V Leiden polymorphism and the rate of fibrosis development in chronic hepatitis C virus infection.
  • HCV Hepatitis C Virus
  • Date of infection was documented; in persons infected by blood products the date of transfusion was recorded. When the exact date was not known, but the year was, the estimated date of infection was recorded as the middle of that year. In persons infected by intravenous drug use the date of infection was estimated as the middle of the year of 1 st drug use as elsewhere (1).
  • a second cohort of patients collected as part of the HENCORE collaboration was also used in this study and has been described else where(5).
  • Biopsies from all patients in both groups were stained with both haematoxylin & eosin and reticulin. These were scored by a single pathologist (RG) using the modified histological activity index (HAI-Ishak) scoring system(13). This scoring system assigns a score of 0-6 for the degree of fibrosis and a score of 0-18 for necro-inflammatory activity. Fibrosis stage was assumed to be zero at the time of infection. DNA collection
  • Genomic DNA was extracted from a 5ml sample of whole blood collected into EDTA. Extraction was performed using a commercial kit (Nucleon II, Scotlabs, UK) according to the manufacturers instructions.
  • Genotyping was performed by a reverse line blot hybridisation linear a ⁇ ay assay (Roche Molecular Systems, Alameda USA). Extracted genomic DNA was subjected to multi target PCR amplifications using a blend of primers. PCR products were then genotyped by hybridising to sequence specific oligonucleotides impregnated onto nylon strips and detected in a colorimetric reaction as described in(14). Strips were then examined for the presence of coloured bands indicating the presence of an allele.
  • Rate of fibrosis was calculated as the ratio of the fibrosis score to duration of infection at biopsy. Demographic differences between fast (predicted ci ⁇ hosis in less than 20 years) and slow (not predicted to reach ci ⁇ hosis for more than 30 years) groups were assessed using independent samples t tests, analysis of variance (ANOVA) and Chi squared tests as appropriate.
  • Rate of fibrosis was logarithmically transformed to give a normal distribution. Comparison of rates between groups of individuals with different genotypes was performed using analysis of variance. Genotype and allele frequency were compared between subsets of patients with fast or slow fibrosis using the Chi Squared or Fishers Exact test as appropriate.
  • Variations in rates of fibrosis with respect to genotype were sought in the St Mary's cohort. Reproducibility of the observed variations was then sought in the Hencore cohort. The two cohorts were then pooled to allow subgroup analysis. In order to account for demographic differences between the phenotypic groups, subgroup analysis was performed by gender and analysis of covariance (ANCOVA) to examine the effect of age at infection and gender.
  • ANCOVA analysis of covariance
  • factor V Leiden leads to an increased rate of fibrosis in HCV infection.
  • the functional significance of factor V Leiden is well described in that this mutation confers resistance to activated protein C which normally degrades factor V.
  • Increased activity of factor V leads to increased thrombin activity and hence fibrin production.
  • those with the polymorphism have a procoagulant state in response to the liver inflammation resulting from HCV which gives rise to increased thrombin generation and increased fibrin deposition.
  • Thrombin is a stellate cell mitogen(9).
  • Increased thrombin levels affect stellate cell activation and hence may enhance fibrosis deposition. We have observed this association in 2 populations.
  • the weaker association in the HENCORE patients from mainland Europe may reflect the greater genetic heterogeneity in this group(15).
  • the procoagulant factor V Leiden is associated with rapid development of liver fibrosis and its effect appears to be predominant in males with only a trend observed in females. As has been previously reported by Poynard et al (1) age at infection and male gender were found to be associated with a rapid fibrosis rate in our study. Lack of association between alcohol and rate of fibrosis may relate to different drinking patterns between our pan European and Poynards(l) predominantly French patients. Genetic risk factors need to be examined in the context of environmental ones. Factor V Leiden may influence the rate of fibrosis in hepatitis of other aetiologies. Establishing the influence of this genetic factor on rate of fibrosis opens the possibility of therapeutic intervention in the form of anticoagulation, for example for patients who do not respond to anti viral therapy.

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Abstract

L'invention porte sur un procédé visant à évaluer le risque chez un patient à développer une cirrhose du foie. Ce procédé consiste à déterminer le génotype ou le phénotype du patient pour un facteur de coagulation. Si le génotype du patient est un génotype associé à une tendance procoagulante ou indiquant une tendance procoagulante, le patient est alors considéré être un patient à très haut risque dans le développement et/ou l'évolution rapide de la cirrhose du foie ou de la fibrose. Le facteur de coagulation peut être le Facteur V. Les patients présentant le Facteur V Leiden peuvent être des patients à très haut risque dans le développement et/ou l'évolution rapide de la cirrhose du foie ou de la fibrose.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010106140A1 (fr) 2009-03-19 2010-09-23 Universite D'angers Méthode non invasive pour évaluer la progression d'une fibrose hépatique
WO2012139970A1 (fr) * 2011-04-15 2012-10-18 Johann Wolfgang Goethe-Universität, Frankfurt Am Main Bivalirudine et autres analogues de l'hirudine pour la conservation de la fonctionnalité cellulaire dans la thérapie cellulaire clinique
WO2026039728A1 (fr) * 2024-08-16 2026-02-19 Regeneron Pharmaceuticals, Inc. Stratégies thérapeutiques pour réduire le risque thrombotique dans des supports du facteur v leiden (fvl)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BERTINA R M ET AL: "MUTATION IN BLOOD COAGULATION VACTOR V ASSOCIATED WITH RESISTANCE TO ACTIVATED PROTEIN C" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 369, no. 6475, 5 May 1994 (1994-05-05), pages 64-67, XP000563812 ISSN: 0028-0836 cited in the application *
CARMASSI ET AL.: "Modulation of hemostatic balance with antithrombin III replacement therapy in a case of liver cirrhosis associated with recurrent venous thrombosis" JOURNAL OF MOLECULAR MEDICINE, vol. 73, no. 2, 1995, pages 89-93, XP008021759 *
CORNBERG ET AL.: "Hepatitis C: therapeutic perspectives" FORUM (GENOVA), vol. 11, no. 2, 2001, pages 154-162, XP008021753 *
MALLAT ET AL.: "Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 42, 1998, pages 27300-27305, XP002253845 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010106140A1 (fr) 2009-03-19 2010-09-23 Universite D'angers Méthode non invasive pour évaluer la progression d'une fibrose hépatique
US10861582B2 (en) 2009-03-19 2020-12-08 Centre Hospitalier Universitaire D'angers Non-invasive method for assessing liver fibrosis progression
WO2012139970A1 (fr) * 2011-04-15 2012-10-18 Johann Wolfgang Goethe-Universität, Frankfurt Am Main Bivalirudine et autres analogues de l'hirudine pour la conservation de la fonctionnalité cellulaire dans la thérapie cellulaire clinique
WO2026039728A1 (fr) * 2024-08-16 2026-02-19 Regeneron Pharmaceuticals, Inc. Stratégies thérapeutiques pour réduire le risque thrombotique dans des supports du facteur v leiden (fvl)

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