WO2013030786A1

WO2013030786A1 - Method for diagnosing or predicting hepatocellular carcinoma outcome

Info

Publication number: WO2013030786A1
Application number: PCT/IB2012/054470
Authority: WO
Inventors: Pierre-Yves Bochud; Darius Moradpour; Christian Lange; Zoltán KUTALIK
Original assignee: Centre Hospitalier Universitaire Vaudois
Priority date: 2011-08-31
Filing date: 2012-08-30
Publication date: 2013-03-07

Abstract

The present invention relates to in vitro method of determining a susceptibility to hepatocellular carcinoma development in a subject.

Description

METHOD FOR DIAGNOSING OR PREDICTING HEPATOCELLULAR

CARCINOMA OUTCOME FIELD OF THE INVENTION

The present invention relates to in vitro methods of determining a susceptibility to hepatocellular carcinoma development in a subject. BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC) is the most common type of primary tumour of the liver. Worldwide, it is the fifth most common tumour type and the third most common cause of death from cancer. In 30% to 70% of all cases, the tumour arises after chronic infection with the hepatitis C virus (HCV). Indeed transformation to HCC commonly occurs after chronic liver disease. HCC commonly arises after hepatic cirrhosis and chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), which are the most important causes of cirrhosis and HCC.

Although recent medical advances have made great progress in diagnosing the disease, a large number of patients with HCCs are still diagnosed at advanced stages. Most of the patients are not cured by surgical resection because of severe liver dysfunction, widespread and/or multiple tumours, or high incidence of recurrence. Indeed HCC is difficult to diagnose, in particular in early stages of the disease. Due to such poor prognosis, by the time a subject is diagnosed with HCC the disease has progresses to such an extent that current therapies are largely ineffective. Only a minority of patients with HCC are candidates for potentially curative treatments of resection, transplantation, or ablation.

As current therapies are ineffective for most HCC patients, prevention of hepatitis B virus (HBV) and hepatitis C virus (HCV) transmission and identification of high-risk populations have been proposed as alternative strategies. Strategies for identification of high- risk populations include alpha fetoprotein measurements and liver imaging. These techniques are costly and are hindered by suboptimal sensitivity and specificity. To this end,

identification of molecular markers associated with an increased risk of HCC would better define populations at highest risk for HCC and can additionally define important therapeutic targets for prevention and treatment.

Thus there is still a profound need to develop an effective predictive method of determining a susceptibility to develop hepatocellular carcinoma. To date, no efficient methods or strategies have been developed to overcome this problem.

SUMMARY OF THE INVENTION

The present invention provides a method of determining a susceptibility to

hepatocellular carcinoma development in a subject, comprising

(a) providing a biological sample from said subject,

(b) determining the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from said biological sample, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7, and

wherein the presence of the at least one polymorphic marker is an indication that said subject has a decreased or an increased susceptibility to hepatocellular carcinoma.

The present invention further provides a method for reducing the risk of developing a hepatocellular carcinoma in a subject, comprising

i) providing a biological sample from said subject,

ii) determining the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from said biological sample, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7,

iii) and treating said subject based upon whether the at least one polymorphic markers of the subject is associated with increased susceptibility to hepatocellular carcinoma.

The present invention also provides a kit for determining susceptibility to

hepatocellular carcinoma development in a subject according to the method of the present invention, said kit comprising i) reagents for selectively detecting the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from a biological sample obtained from said subject, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7 and ii) instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows risk of hepatocellular carcinoma in chronic hepatitis C patients according to single nucleotide polymorphisms (SNPs) in the CYP2R1 gene. (A) Uni- and multivariate Cox regression models were performed to assess the risk of HCC by CYP2R1 SNPs. Rsl993116, rs2060793 and rsl0741657 were in strong linkage disequilibrium (pairwise R2>=0.94).

Multivariate analyses were adjusted for age at infection, sex, diabetes, HIV co-infection, alcohol consumption (>40g/d > 5 years), and steatosis. The ranking of SNPs according to the level of significance is comparable to the strength of their association with vitamin D insufficiency in a genome-wide association study [2]. (B) The probability to develop HCC from the time of HCV infection by CYP2R1 SNPs was assessed by using cumulative incidence curves, with censoring of data at the date of last follow-up or death. The cumulative incidence curve for the top SNP (rsl993116) is shown, which was based on the dominant model of inheritance. P value is for the log rank test.

Figure 2 shows rs2244546 in HCP5 is associated with HCV-induced hepatocellular carcinoma. Cumulative incidence of hepatocellular carcinoma (HCC) according to rs2244546 genotype. The probability to develop HCC from the time of hepatitis C virus (HCV) infection by rs2244546 genotype was assessed by using cumulative incidence curves, with censoring of data at the date of last follow-up or death. Only patients with known duration of infection were included in this analysis. Statistics are shown for the multivariate model including age, sex, presence of diabetes and excessive alcohol consumption (> 40 g/d > 5 years) as co- variates (univariate =0.005, hazard ratio (HR)=2.39, 95% CI= 1.29-4.42).

DETAILED DESCRIPTION OF THE INVENTION Applicants discovered that polymorphic variants in a number of sequences, SEQ ID

NOs: l to 7 are associated with susceptibility of developing hepatocellular carcinoma in subjects. The present invention thus provides SNPs associated with hepatocellular carcinoma, nucleic acid molecules containing SNPs, in vitro methods and reagents for the detection of the SNPs disclosed herein, and assays or kits that utilize such reagents. The hepatocellular carcinoma associated SNPs disclosed herein are useful for diagnosing, screening for, and evaluating predisposition to hepatocellular carcinoma and related pathologies in humans.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Alternatively this term "comprise" also encompasses the term "include".

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term "at least one" means "one or more" and also encompasses the terms "at least two", "at least three", "at least four", "at least five", etc.

As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject suffering from chronic liver diseases. However, in other embodiments, the subject can be a normal subject, which is not suffering from chronic liver diseases. The terms "subject" or "patient" do not denote a particular age or sex. Thus, adult, infant and newborn subjects, whether male or female, are intended to be covered. As used herein the term "susceptibility" refers to the likelihood, for the subject, or a predisposition, or a risk to develop hepatocellular carcinoma in subjects.

An "allele", as used herein, refers to one specific form of a genetic sequence or a single nucleotide position within a genetic sequence (such as a gene) within a cell, an individual or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequence may or may not be within a gene. The sequences at these variant sites that differ between different alleles are termed "variances", "polymorphisms", or "mutations". At each autosomal specific chromosomal location or "locus", an individual possesses two alleles, one inherited from one parent and one from the other parent, for example one from the mother and one from the father.

"Polymorphism," as used herein, refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A "polymorphic marker" or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at a frequency of preferably greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphic locus may be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, copy number variations (CNV) and insertion elements such as Alu. The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wild-type form. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms. A polymorphism between two nucleic acids can occur naturally, or be caused by exposure to or contact with chemicals, enzymes, or other agents, or exposure to agents that cause damage to nucleic acids, for example, ultraviolet radiation, mutagens or carcinogens. A particular kind of polymorphism, called a single nucleotide polymorphism, or SNP, is a small genetic change or variation that can occur within a person's DNA sequence. The genetic code is specified by the four nucleotide "letters" A (adenine), C (cytosine), T (thymine), and G (guanine). SNP variation occurs when a single nucleotide, for example A (adenine), replaces one of the other three nucleotides, C (cytosine), T (thymine), and G (guanine). The term "Chronic liver diseases" encompasses diseases related to liver, such as chronic hepatitis C, hepatitis B, alcoholic liver disease or non-alcoholic steatohepatitis (NASH).

"CYP2R1 locus" generally refers, in humans, to a genomic DNA region located from base pair 14'899'554 to base pair 14'913'750 on the short arm of chromosome 11. CYP2R1 encodes the CYP2R1 protein, a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This enzyme is a microsomal vitamin D hydroxylase that is important in the conversion of vitamin D into the active ligand for the vitamin D receptor. A mutation in this gene has been associated with selective 25-hydroxyvitamin D deficiency.

"HCP5 locus" generally refers, in humans, to a genomic DNA region located from base pair 31 '368 '479 to base pair 31 '445 '283 on chromosome 6 and consists of 76'805 bases.

The present invention thus provides individual SNPs associated with hepatocellular carcinoma. The invention includes in vitro methods of detecting these polymorphisms in a nucleic acid sample isolated from a biological sample and in vitro methods of determining a susceptibility (i.e. the risk) of an individual of having or developing hepatocellular carcinoma.

When the presence in the genome of an individual of a particular base, e.g., adenine, at a particular location in the genome correlates with an increased probability of that individual contracting hepatocellular carcinoma vis-a-vis a population not having that base at that location in the genome, that individual is said to be at "increased risk" of contracting hepatocellular carcinoma, i.e., to have an increased susceptibility. In certain cases, this effect can be a "dominant" effect in which case such increased probability exists when the base is present in one or the other or both alleles of the individual. In certain cases, the effect can be said to be "recessive", in which case such increased probability exists only when the base is present in both alleles of the individual.

When the presence in the genome of an individual of a particular base, e.g., adenine, at a particular location in the genome decreases the probability of that individual contracting hepatocellular carcinoma vis-a-vis a population not having that base at that location in the genome, that individual is said to be at "decreased risk" of contracting hepatocellular carcinoma, i.e., to have a decreased susceptibility. Such an allele is sometimes referred to in the art as being "protective". As with increased risk, it is also possible for a decreased risk to be characterized as dominant or recessive.

Applicants performed a retrospective analysis on the impact of the SNPs on the risk of hepatocellular carcinoma (HCC) development in subjects enrolled in the Swiss Hepatitis C Cohort Study (SCCS) who underwent a GWAS on response to treatment [3]. Of these, 858 patients with proven chronic hepatitis C, of whom 32 developed HCC, met the inclusion criteria (availability of genomic DNA, written informed consent for genetic testing, known putative date of infection with the hepatitis C virus [HCV], Caucasian ancestry). Univariate and multivariate Cox regression models were used to calculate the relative hazards to develop HCC associated with the SNPs. Several SNPs in CYP2R1 were significant independent predictors of HCC development in Applicant's cohort (top SNP rsl993116; univariate p=0.02, hazard ratio 0.44, 95% confidence interval (CI) 0.22-0.89, details in Figure 1).

CYP2R1 encodes a hepatic microsomal enzyme, that seems to be involved in the 25- hydroxylation of vitamin D in the liver [2]. CYP2R1 was reported to be down-regulated in HCV-infected livers [4], and patients with chronic hepatitis C are at high risk of vitamin D insufficiency [5]. Both findings may provide a biological basis for the strong association of SNPs in CYP2R1 with HCC development [2, 4, 5]. These observations point to an important role of vitamin D insufficiency in the development of HCV-related HCC.

Indeed Applicants found that SNPs in the CYP2R1 gene (top SNP rs 1993116) were significant independent predictors of hepatocellular carcinoma (HCC) development in subjects suffering from chronic hepatitis C. Specifically, subjects with a CYP2R1 genotype predictive for vitamin D insufficiency had a higher risk to develop HCC. These data appear to be important, since subjects suffering from chronic hepatitis C are at high risk of severe vitamin D deficiency. Moreover, the incidence of HCC is expected to strongly increase in the next years. Thus SNPs in the CYP2R1 gene may help to identify those subjects who have a high risk of HCC development and who may benefit from vitamin D supplementation as a potential chemopreventive strategy. In a particular embodiment of the invention, said subjects are suffering from chronic liver diseases.

In addition, the Applicants found another SNP, namely rs2244546 in a group consisting of 938 SCCS patients with chronic hepatitis C, from whom genomic DNA and written informed consent for genetic testing was available, and in whom co-infection with hepatitis B virus has been excluded. In the combined analysis of the entire cohort, rs2244546 showed the strongest association for HCV-related HCC. Rs2244546 is located in HCP5, a gene located between MICA and HLA-DQA I HLA-DQB.

In the case of a method of determining a susceptibility to hepatocellular carcinoma development in a subject, the presence of the at least one polymorphic marker is an indication that said subject has a decreased or an increased susceptibility to develop hepatocellular carcinoma. In a particular embodiment of the invention, said subjects are suffering from chronic liver diseases.

A number of methods are available for analyzing the presence or absence of at least one single nucleotide polymorphism (SNP), which can be applied to the CYP2R1 locus and/or HCP5 locus in a nucleic acid sample isolated from a biological sample obtained from said subject. Assays for detection of polymorphisms or mutations fall into several categories, including but not limited to direct sequencing assays, fragment polymorphism assays, hybridization assays, and computer based data analysis. Protocols and commercially available kits or services for performing multiple variations of these assays are available. In some embodiments, assays are performed in combination or in hybrid (e.g., different reagents or technologies from several assays are combined to yield one assay). The following assays are useful in the present invention, and are described in relationship to detection of the various SNP found in the CYP2R1 locus and/or HCP5 locus.

In one aspect of the present invention, SNPs are detected using a direct sequencing technique. In these assays, DNA samples are first isolated from a subject using any suitable method. In some embodiments, the region of interest is cloned into a suitable vector and amplified by growth in a host cell (e.g., a bacterium). In other embodiments, DNA in the region of interest is amplified using the Polymerase Chain Reaction (PCR).

Following amplification, DNA in the region of interest (e.g., the region containing the SNP) is sequenced using any suitable method, including but not limited to manual sequencing using radioactive marker nucleotides, or automated sequencing. The results of the sequencing are displayed using any suitable method. The sequence is examined and the presence or absence of a given SNP is determined.

In one aspect of the present invention, SNPs are detected using a PCR-based assay. In some embodiments, the PCR assay comprises the use of oligonucleotide primers ("primers") to amplify a fragment containing the repeat polymorphism of interest. Amplification of a target polynucleotide sequence may be carried out by any method known to the skilled artisan. See, for instance, [6] and [7]. Amplification methods include, but are not limited to, PCR including real time PCR (RT-PCR), strand displacement amplification [8]; [9], strand displacement amplification using Phi29 DNA polymerase (US Patent No. 5,001,050), transcription-based amplification [10], self-sustained sequence replication ("3SR") [11]; [12], the Q.beta. replicase system ([13]; [14]), nucleic acid sequence-based amplification

("NASBA") ([15]), the repair chain reaction ("RCR") ([15], supra), and boomerang DNA amplification (or "BDA") ([15]). PCR is the preferred method of amplifying the target polynucleotide sequence.

PCR may be carried out in accordance with techniques known by the skilled artisan. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with a pair of amplification primers. One primer of the pair hybridizes to one strand of a target polynucleotide sequence. The second primer of the pair hybridizes to the other, complementary strand of the target polynucleotide sequence. The primers are hybridized to their target polynucleotide sequence strands under conditions such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand. The extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer. After primer extension, the sample is treated to denaturing conditions to separate the primer extension products from their templates. These steps are cyclically repeated until the desired degree of amplification is obtained.

The amplified target polynucleotide may be used in one of the detection assays described elsewhere herein to identify the GT-repeat polymorphism present in the amplified target polynucleotide sequence.

In one aspect of the present invention, SNPs are detected using a fragment length polymorphism assay. In a fragment length polymorphism assay, a unique DNA banding pattern based on cleaving the DNA at a series of positions is generated using an enzyme (e.g., a restriction endonuclease). DNA fragments from a sample containing a polymorphism will have a different banding pattern than wild type. In one aspect of the present invention, fragment sizing analysis is carried out using the Beckman Coulter CEQ 8000 genetic analysis system, a method well-known in the art for microsatellite polymorphism determination.

In one aspect of the present invention, SNPs are detected using a restriction fragment length polymorphism assay (RPLP). The region of interest is first isolated using PCR. The PCR products are then cleaved with restriction enzymes known to give a unique length fragment for a given polymorphism. The restriction-enzyme digested PCR products are separated by agarose gel electrophoresis and visualized by ethidium bromide staining and compared to controls (wild-type). In one aspect, polymorphisms are detected using a CLEAVASE fragment length polymorphism assay (CFLP; Third Wave Technologies, Madison, Wis.; see e.g., US Patent No. 5,888,780). This assay is based on the observation that, when single strands of DNA fold on themselves, they assume higher order structures that are highly individual to the precise sequence of the DNA molecule. These secondary structures involve partially duplexed regions of DNA such that single stranded regions are juxtaposed with double stranded DNA hairpins. The CLEAVASE I enzyme, is a structure-specific, thermostable nuclease that recognizes and cleaves the junctions between these single-stranded and double-stranded regions.

The region of interest is first isolated, for example, using PCR. Then, DNA strands are separated by heating. Next, the reactions are cooled to allow intrastrand secondary structure to form. The PCR products are then treated with the CLEAVASE I enzyme to generate a series of fragments that are unique to a given polymorphism. The CLEAVASE enzyme treated PCR products are separated, detected (e.g., by agarose gel electrophoresis), visualized (e.g., by ethidium bromide staining) and compared to controls (wild-type). In other aspects of the present invention, SNPs are detected by hybridization assay. In a hybridization assay, the presence or absence of a given polymorphism or mutation is determined based on the ability of the DNA from the sample to hybridize to a complementary DNA molecule (e.g., an oligonucleotide probe). A variety of hybridization assays using a variety of technologies for hybridization and detection are available. A description of a selection of assays is provided below. In a preferred aspect, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels may be incorporated by any of a number of means well known to those of skill in the art. In one embodiment, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids. Thus, for example, polymerase chain reaction (PCR) with labelled primers or labelled nucleotides will provide a labelled amplification product. In another embodiment, transcription amplification using a labelled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to the original nucleic acid sample (e.g., mR A, polyA mR A, cDNA, genomic DNA etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example, nick translation or end- labeling (e.g. with a labeled R A) by kinasing the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore). In another embodiment label is added to the end of fragments using terminal deoxytransferase (TdT).

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include, but are not limited to: biotin for staining with labeled streptavidin conjugate; anti-biotin antibodies; magnetic beads (e.g., Dynabeads™); fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, and the like); radiolabels (e.g., Ή, ¹ 1 , S, ¹⁴C, or P); phosphorescent labels; enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA); and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters;

fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label. The label may be added to the target nucleic acid(s) prior to, or after the hybridization. So-called "direct labels" are detectable labels that are directly attached to or incorporated into the target nucleic acid prior to hybridization. In contrast, so-called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids. See Tijssen, 1993, Laboratory

Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization with Nucleic Acid Probes, which is hereby incorporated by reference in its entirety for all purposes.

In one aspect, hybridization of a probe to the sequence of interest (e.g., polymorphism like SNP) is detected directly by visualizing a bound probe (e.g., a Northern or Southern assay; See e.g., Ausabel et al. (Eds.), 1991, Current Protocols in Molecular Biology, John Wiley & Sons, NY). In these assays, genomic DNA (Southern) or RNA (Northern) is isolated from a subject. The DNA or RNA is then cleaved with a series of restriction enzymes that cleave infrequently in the genome and not near any of the markers being assayed. The DNA or RNA is then separated (e.g., agarose gel electrophoresis) and transferred to a membrane. A labeled (e.g., by incorporating a radionucleotide) probe or probes specific for the mutation being detected is allowed to contact the membrane under a condition of low, medium, or high stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe.

In one aspect of the present invention, SNPs are detected using a DNA chip hybridization assay. In this assay, a series of oligonucleotide probes are affixed to a solid support. The oligonucleotide probes are designed to be unique to a given single nucleotide polymorphism. The DNA sample of interest is contacted with the DNA "chip" and

hybridization is detected.

In some embodiments, the DNA chip assay is a GeneChip (Affymetrix, Santa Clara, Calif; see e.g., US Patent No. 6,045,996) assay. The GeneChip technology uses miniaturized, high-density arrays of oligonucleotide probes affixed to a "chip". Probe arrays are

manufactured by Affymetrix's light-directed chemical synthesis process, which combines solid-phase chemical synthesis with photolithographic fabrication techniques employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density arrays of oligonucleotides, with each probe in a predefined position in the array. Multiple probe arrays are synthesized simultaneously on a large glass wafer. The wafers are then diced, and individual probe arrays are packaged in injection-molded plastic cartridges, which protect them from the environment and serve as chambers for hybridization.

The nucleic acid to be analyzed is isolated from a biological sample obtained from the subject, amplified by PCR, and labelled with a fluorescent reporter group. The labelled DNA is then incubated with the array using a fluidics station. The array is then inserted into the scanner, where patterns of hybridization are detected. The hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the target, which is bound to the probe array. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, by complementarity, the identity of the target nucleic acid applied to the probe array can be determined.

In another aspect, a DNA microchip containing electronically captured probes

(Nanogen, San Diego, Calif.) is utilized (see e.g., US Patent No. 6,068,818). Through the use of microelectronics, Nanogen's technology enables the active movement and concentration of charged molecules to and from designated test sites on its semiconductor microchip. DNA capture probes unique to a given polymorphism or mutation are electronically placed at, or "addressed" to, specific sites on the microchip. Since DNA has a strong negative charge, it can be electronically moved to an area of positive charge.

First, a test site or a row of test sites on the microchip is electronically activated with a positive charge. Next, a solution containing the DNA probes is introduced onto the microchip. The negatively charged probes rapidly move to the positively charged sites, where they concentrate and are chemically bound to a site on the microchip. The microchip is then washed and another solution of distinct DNA probes is added until the array of specifically bound DNA probes is complete.

A test sample is then analyzed for the presence of target DNA molecules by determining which of the DNA capture probes hybridize, with complementary DNA in the test sample (e.g., a PCR amplified gene of interest). An electronic charge is also used to move and concentrate target molecules to one or more test sites on the microchip. The electronic concentration of sample DNA at each test site promotes rapid hybridization of sample DNA with complementary capture probes (hybridization may occur in minutes). To remove any unbound or nonspecifically bound DNA from each site, the polarity or charge of the site is reversed to negative, thereby forcing any unbound or nonspecifically bound DNA back into solution away from the capture probes. A laser-based fluorescence scanner is used to detect binding.

In still another aspect, an array technology based upon the segregation of fluids on a flat surface (chip) by differences in surface tension (ProtoGene, Palo Alto, Calif.) is utilized (see e.g., US Patent No. 6,001,311). Protogene's technology is based on the fact that fluids can be segregated on a flat surface by differences in surface tension that have been imparted by chemical coatings. Once so segregated, oligonucleotide probes are synthesized directly on the chip by ink-jet printing of reagents. The array with its reaction sites defined by surface tension is mounted on an X/Y translation stage under a set of four piezoelectric nozzles, one for each of the four standard DNA bases. The translation stage moves along each of the rows of the array, and the appropriate reagent is delivered to each of the reaction site. For example, the A amidite is delivered only to the sites where amidite A is to be coupled during that synthesis step and so on. Common reagents and washes are delivered by flooding the entire surface followed by removal by spinning. DNA probes unique for the polymorphism of interest are affixed to the chip using

Protogene's technology. The chip is then contacted with the PCR-amplified genes of interest. Following hybridization, unbound DNA is removed and hybridization is detected using any suitable method (e.g., by fluorescence de-quenching of an incorporated fluorescent group).

In yet other aspects, a "bead array" is used for the detection of SNPs (Illumina, San Diego, Calif; see e.g., PCT Publications W099/67641 and WO00/39587, each of which is herein incorporated by reference). Illumina uses a bead array technology that combines fiber optic bundles and beads that self-assemble into an array. Each fiber optic bundle contains thousands to millions of individual fibers depending on the diameter of the bundle. The beads are coated with an oligonucleotide specific for the detection of a given polymorphism or mutation. Batches of beads are combined to form a pool specific to the array. To perform an assay, the bead array is contacted with a prepared subject sample (e.g., DNA). Hybridization is detected using any suitable method like Enzymatic Detection of Hybridization In some aspects of the present invention, genomic profiles are generated using an assay that detects hybridization by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; see e.g., US Patent No. 6,001,567). The INVADER assay detects specific DNA and RNA sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes. Elevated temperature and an excess of one of the probes enable multiple probes to be cleaved for each target sequence present without temperature cycling. These cleaved probes then direct cleavage of a second labelled probe. The secondary probe oligonucleotide can be 5'-end labelled with fluorescein that is quenched by an internal dye. Upon cleavage, the dequenched fluorescein labelled product may be detected using a standard fluorescence plate reader.

The INVADER assay detects specific mutations and polymorphisms in unamplified genomic DNA. The isolated DNA sample is contacted with the first probe specific either for a polymorphism/mutation or wild type sequence and allowed to hybridize. Then a secondary probe, specific to the first probe, and containing the fluorescein label, is hybridized and the enzyme is added. Binding is detected using a fluorescent plate reader and comparing the signal of the test sample to known positive and negative controls.

In some aspects, hybridization of a bound probe is detected using a TaqMan assay (PE Biosystems, Foster City, Calif; see e.g., US Patent No. 5,962,233). The assay is performed during a PCR reaction. The TaqMan assay exploits the 5 '-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe, specific for a given allele or mutation, is included in the PCR reaction. The probe consists of an oligonucleotide with a 5 '-reporter dye (e.g., a fluorescent dye) and a 3 '-quencher dye. During PCR, if the probe is bound to its target, the 5 '-3' nucleo lytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.

In some aspects, a MassARRAY system (Sequenom, San Diego, Calif.) is used to detect polymorphisms (see e.g., US Patent No. 6,043,031). DNA is isolated from blood samples using standard procedures. Next, specific DNA regions containing the polymorphism of interest are amplified by PCR. The amplified fragments are then attached by one strand to a solid surface and the non- immobilized strands are removed by standard denaturation and washing. The remaining immobilized single strand then serves as a template for automated enzymatic reactions that produce genotype specific diagnostic products.

Very small quantities of the enzymatic products, typically five to ten nanoliters, are then transferred to a SpectroCHIP array for subsequent automated analysis with the

SpectroREADER mass spectrometer. Each spot is preloaded with light absorbing crystals that form a matrix with the dispensed diagnostic product. The MassARRAY system uses MALDI- TOF (Matrix Assisted Laser Desorption Ionization- Time of Flight) mass spectrometry. In a process known as desorption, the matrix is hit with a pulse from a laser beam. Energy from the laser beam is transferred to the matrix and it is vaporized resulting in a small amount of the diagnostic product being expelled into a flight tube. As the diagnostic product is charged when an electrical field pulse is subsequently applied to the tube they are launched down the flight tube towards a detector. The time between application of the electrical field pulse and collision of the diagnostic product with the detector is referred to as the time of flight. This is a very precise measure of the product's molecular weight, as a molecule's mass correlates directly with time of flight with smaller molecules flying faster than larger molecules. The entire assay is completed in less than 0.0001 second, enabling samples to be analyzed in a total of 3-5 second including repetitive data collection. The SpectroTYPER software then calculates, records, compares and reports, the genotypes at the rate of three seconds per sample. Usually, the "nucleic acid sample" of the invention is isolated from a biological sample obtained from the subject, such as whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, and biopsies of muscle, liver, brain tissue, nerve tissue and hair. The nucleic acid sample may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, and RNA (including mRNA, mi RNA and rRNA). Preferably the biological sample can be the whole blood or liver biopsy. The nucleic acid sample can be isolated from a biological sample using standard techniques. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis. Genomic DNA samples are usually amplified before being brought into contact with a probe. Genomic DNA can be obtained from any biological sample. Amplification of genomic DNA containing a SNP generates a single species of nucleic acid if the individual from whom the sample was obtained is homozygous at the polymorphic site, or two species of nucleic acid if the individual is heterozygous.

RNA samples also are often subject to amplification. In this case, amplification is typically preceded by reverse transcription. Amplification of all expressed m NA can be performed as described in, for example, in [16] and [17] which are hereby incorporated by reference in their entirety. Amplification of an RNA sample from a diploid sample can generate two species of target molecules if the individual providing the sample is

heterozygous at a polymorphic site occurring within the expressed RNA, or possibly more if the species of the RNA is subjected to alternative splicing. Amplification generally can be performed using the polymerase chain reaction (PCR) methods known in the art. Nucleic acids in a target sample can be labeled in the course of amplification by inclusion of one or more labeled nucleotides in the amplification mixture. Labels also can be attached to amplification products after amplification (e.g., by end-labeling). The amplification product can be RNA or DNA, depending on the enzyme and substrates used in the amplification reaction.

The genotype of an individual polymorphism comprises the sum of at least two alleles and may be homozygous (i.e. comprising identical alleles) or heterozygous (i.e. comprising different alleles).

In some aspect of the invention, the isolated nucleic acid sample of the present invention can be produced or synthesized using conventional nucleic acid synthesis or by recombinant nucleic acid methods known in the art (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (2001, Current Protocols in Molecular Biology, Green & Wiley, New York).

Surprisingly the Inventors of the present invention have shown that the presence of the at least one polymorphic marker in the CYP2R1 locus and/or HCP5 locus in a nucleic acid sample isolated from a biological sample obtained from a subject is an indication that said subject has a decreased or an increased susceptibility to develop hepatocellular carcinoma. In a particular embodiment of the invention, said subject is suffering from chronic liver diseases.

Thus the present invention relates to a method of determining a susceptibility to hepatocellular carcinoma development in a subject, comprising (a) providing a biological sample from said subject,

(b) determining the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from said biological sample, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7 (see Table 1) and ,

The at least one polymorphic marker of the invention is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of rsl993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546.

Although detecting the at least one SNP of the invention is sufficient to provide a diagnostic value, detecting two or more SNPs of the invention can improve the accuracy and precision of the method. Specifically the at least one polymorphic marker, which presence is an indication that said subject has a decreased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of G/A for rsl993116, G/A for rs2060793, G/A for rsl0741657, C/T for rs7116978.

Specifically the at least one polymorphic marker, which presence is an indication that said subject has an increased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of T/G for rsl0500804, G/A for rsl2794714 G/A and C/G for rs2244546.

In a particular embodiment of the invention, said subject is suffering from chronic liver diseases.

Preferably the biological sample used in the method of the present invention is whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin or biopsies of muscle, liver, brain tissue, nerve tissue and hair.

Table 1 (Note that the order of the alleles is not indicative of which one is the major allele and which one is the minor allele)

Preferably the at least one SNP of the invention is located on human chromosome 11 within a region comprising about 14Ί96 b and/or on human chromosome 6 within a region comprising 76 '805 bases. For example, according to one embodiment of the present invention, Figures 1 and 2 shows a strong genetic association between the SNPs and a susceptibility to develop hepatocellular carcinoma in a subject suffering from chronic liver diseases.

In an embodiment of the present invention, the at least one polymorphic marker of the invention is a polymorphic site being in complete or strong linkage disequilibrium with at least one SNP selected from the group consisting of rsl993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546.

In a further embodiment of the present invention, the at least one polymorphic marker is a combination of at least two SNPs selected from the group consisting of rs 1993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546.

In another embodiment of the present invention, the at least one SNP selected from the group consisting of rsl993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546 is in complete or strong linkage disequilibrium with any other genetic or epigenetic polymorphic marker.

The present invention also contemplates determining the presence or absence of at least one, i.e. one or more as defined supra, i.e. a combination of, single nucleotide polymorphism (SNP) in the CYP2R1 locus and/or HCP5 locus in a nucleic acid sample isolated from a biological sample obtained from said subject. Preferably, the polymorphic marker is a polymorphic site associated with at least one SNP selected from the group consisting of rsl993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714 and rs2244546.

According to an embodiment of the present invention, the at least one polymorphic marker, which presence is an indication that said subject has a decreased susceptibility to hepatocellular carcinoma, is selected from the group comprising G/A for rs 1993116, G/A for rs2060793, G/A for rs 10741657, C/T for rs7116978.

According to another embodiment of the present invention, the at least one

polymorphic marker, which presence is an indication that said subject has an increased susceptibility to hepatocellular carcinoma, is selected from the group consisting of T/G for rsl0500804, G/A for rsl2794714 and C/G for rs2244546. SNP Common Rare MAF Comment

allele allele

CYP2R1 rsl9931 16 G A 0.363 Carriage of the rare allele is associated with a decreased risk of HCC

CYP2R1 rs 10741657 G A 0.358 Carriage of the rare allele is associated with a decreased risk of HCC

CYP2R1 rs2060793 G A 0.358 Carriage of the rare allele is associated with a decreased risk of HCC

CYP2R1 rs71 16978 C T 0.329 Carriage of the rare allele is associated with a decreased risk of HCC

CYP2R1 rs 10500804 T G 0.486 Carriage of the rare allele is associated with an increased risk of HCC

CYP2R1 rs 12794714 G A 0.482 Carriage of the rare allele is associated with an increased risk of HCC

HCP5 rs2244546 C G 0.093 Carriage of the rare allele is associated with an increased risk of HCC

Table 2 - * Minor allele frequency in studied our cohort

In one aspect, the nucleic acid sample useful for the determination of the

polymorphism, as described herein, are isolated from the biological sample obtained from the subject. The biological sample is then prepared on one hand for the isolation of the nucleic acid sample useful for determining the presence or absence of the at least one polymorphic marker of the invention.

"Linkage disequilibrium" (LD) describes a situation in which some combinations of alleles or genetic markers occur more or less frequently in a population than would be expected from a random formation of haplotypes from alleles based on their frequencies. When a particular allele at one locus is found together on the same chromosome with a specific allele at a second locus-more often than expected if the loci were segregating independently in a population-the loci are in disequilibrium. This concept of LD is formalized by one of the earliest measures of disequilibrium to be proposed (symbolized by D). D, in common with most other measures of LD, quantifies disequilibrium as the difference between the observed frequency of a two-locus haplotype and the frequency it would be expected to show if the alleles are segregating at random. Adopting the standard notation for two adjacent loci-A and B, with two alleles (A, a and B, b) at each locus-the observed frequency of the haplotype that consists of alleles A and B is represented by PAB. Assuming the independent assortment of alleles at the two loci, the expected halotype frequency is calculated as the product of the allele frequency of each of the two alleles, or PAxPB, where PA is the frequency of allele A at the first locus and PB is the frequency of allele B at the second locus. So, one of the simplest measures of disequilibrium is D=PAB-PAxPB. LD is created when a new mutation occurs on a chromosome that carries a particular allele at a nearby locus, and is gradually eroded by recombination. Recurrent mutations can also lessen the association between alleles at adjacent loci. The importance of recombination in shaping patterns of LD is acknowledged by the moniker of "linkage". The extent of LD in populations is expected to decrease with both time (t) and recombinational distance (r, or the recombination fraction) between markers. Theoretically, LD decays with time and distance according to the following formula, where DO is the extent of disequilibrium at some starting point and Dt, is the extent of disequilibrium t generation later: Dt=(l-r)tD0

A wide variety of statistics have been proposed to measure the amount of LD, and these have different strengths, depending on the context. Although the measure D has the intuitive concepts of LD, its numerical value is of little use for measuring the strength of and comparing levels of LD. This is due to the dependence of D on allele frequencies. The two most common measures are the absolute value of D' and r2.

The absolute value of D' is determined by dividing D by its maximum possible value, given the allele frequencies at the two loci. The case of D'=l is known as complete LD. Values of D'<1 indicate that the complete ancestral LD has been disrupted. The magnitude of values of D'<1 has no clear interpretation. Estimates of D' are strongly inflated in small samples. Therefore, statistically significant values of D' that are near one provide a useful indication of minimal historical recombination, but intermediate values should not be used for comparisons of the strength of LD between studies, or to measure the extent of LD.

The measure r2 is in some ways complementary to D'. r2 is equal to D2 divided by the product of the allele frequencies at the two loci. Hill and Roberson deduced that E [r2]=l/l+4Nc where c is the recombination rate in morgans between the two markers and N is the effective population size. This equation illustrates two important properties of LD. First, expected levels of LD are a function of recombination. The more recombination between two sites, the more they are shuffled with respect to one another, decreasing LD. Second, LD is a function of N, emphasizing that LD is a property of populations. In the present application, strong linkage disequilibrium presents a correlation termed r2 of at least 0.6 and/or a D' of 0.5 with said SNPs in the HapMap European dataset and/or in the population experimentally analyzed by the Inventors. Vitamin D insufficiency is believed to be associated with the risk of cancer

development. However, vitamin D serum levels vary during season or aging, and are influenced by concomitant diseases and behavior. In contrast, genetic polymorphisms within the CYP2R1 gene are associated with vitamin D insufficiency in the long-term, independently from short-term variations of vitamin D serum levels. Thus, CYP2R1 SNPs may be suitable markers for the vitamin D insufficiency-related risk of HCC development.

These genetic tests are useful for prognosing and/or diagnosing hepatocellular carcinoma and often are useful for determining whether an individual is at an increased or decreased risk of developing or having hepatocellular carcinoma. Consequent optimization of the vitamin D status of these "risk" population may reduce the risk of HCC development. Thus, the invention includes a method for identifying a subject at risk of hepatocellular carcinoma, which includes detecting in a nucleic acid sample from the subject the presence or absence of a SNP associated with hepatocellular carcinoma at a polymorphic site in a nucleotide sequence identified as SEQ ID NOs: l to 7.

Preferably the method of the invention includes detecting one SNP associated with hepatocellular carcinoma at a polymorphic site in a nucleotide sequence identified as SEQ ID NOs: l to 7. More preferably the method of invention includes detecting two, three, four, five, six or seven SNPs associated with hepatocellular carcinoma at a polymorphic site in a nucleotide sequence identified as SEQ ID NOs: l to 7. Detecting two or more SNPs can improve the accuracy and precision of the method. Results from prognostic tests may be combined with other test results to diagnose

HCC. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to hepatocellular carcinoma, the patient sample analyzed, and the results of the analysis may be utilized to diagnose hepatocellular carcinoma. Also hepatocellular carcinoma diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into

subpopulations having different progressions of hepatocellular carcinoma. In another embodiment, prognostic results may be gathered; a patient's risk factors for developing hepatocellular carcinoma analyzed (e.g., age, family history); and a patient sample may be ordered based on a determined predisposition to hepatocellular carcinoma. In an alternative embodiment, the results from predisposition analyses may be combined with other test results indicative of hepatocellular carcinoma, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of hepatocellular carcinoma, and the combination can be utilized as a hepatocellular carcinoma diagnostic.

Risk of hepatocellular carcinoma sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of one or more of the SNP variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating risk based upon patient data are well known (Agresti, 2001). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for hepatocellular carcinoma are useful in a variety of applications. In one embodiment, hepatocellular carcinoma risk determinations are used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these.

In a further embodiment, the present invention also provides a method for reducing the risk of developing a hepatocellular carcinoma in a subject, comprising

i) providing a biological sample from said subject,

ii) determining the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from said biological sample, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No

1 to SEQ ID No 7,

The at least one polymorphic marker is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of rs 1993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546. Specifically the at least one polymorphic marker, which presence is an indication that said subject has a decreased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of G/A for rsl993116, G/A for rs2060793, G/A for rsl0741657, C/T for rs7116978.

Specifically the at least one polymorphic marker, which presence is an indication that said subject has an increased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of T/G for rsl0500804, G/A for 12794714 G/A and C/G for rs2244546.

In a particular embodiment of the present invention, said subject is suffering from chronic liver diseases.

Preferably the biological sample is whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin or biopsies of muscle, liver, brain tissue, nerve tissue and hair. Preferably the treatment comprises a vitamin D supplementation treatment or any other suitable treatment.

Further encompassed in the present invention is a kit for determining susceptibility to hepatocellular carcinoma development in a subject according to the method of the present invention, said kit comprising i) reagents for selectively detecting the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from a biological sample obtained from said subject, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7 and ii) instructions for use. Alternatively, the reagents used in the kits comprise an isolated nucleic acid, preferably a primer, a set of primers, or an array of primers, as described elsewhere herein. The primers may be fixed to a solid substrate. The kits may further comprise a control target nucleic acid and primers. One skilled in the art will, without undue experiments, be able to select the primers in accordance with the usual requirements. The isolated nucleic acids of the kit may also comprise a molecular label or tag.

Usually, the primer, set of primers, or array of primers, are directed to detect the presence or absence of at least one single nucleotide polymorphism (SNP) in the CYP2R1 locus and/or HCP5 locus.

In addition to the primers, set of primers, or array of primers, directed to detect the presence or absence of at least one single nucleotide polymorphism (SNP) in the CYP2R1 locus and/or HCP5 locuse from a nucleic acid sample isolated from a biological sample obtained from a subject, the reagents of the kit may comprise, for example, an other primer, set of primers, or array of primers, directed to separately detect the viral genotype isolated from a biological sample obtained from a subject. These set of primers, or array of primers used are generally known in the art or may be readily generated knowing the usual

requirements.

In additional embodiments, the kits of the present invention comprise various reagents, such as buffers, necessary to practice the methods of the invention, as known in the art.

These reagents or buffers may for example be useful to extract and/or purify the nucleic from the biological sample obtained from the subject.

The kit may also comprise all the necessary material such as microcentrifuge tubes necessary to practice the methods of the invention.

A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit can include one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of interest, where the fragment includes a polymorphic site. The kit sometimes comprises a polymerizing agent, for example, a thermo-stable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664. Also, the kit often comprises an elongation oligonucleotide that hybridizes to the nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it can also include chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermo-stable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP. The kit can include one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this specification, each of which is

incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Patients were included from the Swiss Hepatitis C Cohort Study (SCCS). The SCCS is a multicenter study of >3600 HCV-infected patients enrolled at 8 major Swiss hospitals and their local affiliated centers since 2001. The characteristics of SCCS patient selection and data collection have been described elsewhere (18). Patients provided written consent for genetic analyses were included in the genetic study. Single nucleotide polymorphisms were extracted from a genome-wide association (GWA) study-generated dataset (19) or genotyped by using KASP SNP genotyping system, a competitive allele-specific PCR incorporating a FRET quencher cassette

with the primers presented in Table 3. Genotyping for the GWA was performed by the Genomics Platform of the National Center of Competence in Research "Frontiers in Genetics" at the University of Geneva in Geneva, Switzerland, by using Illumina HumanlM-Duo BeadChips (Illumina, San Diego, CA).

Table 3. Primers used for SNP genotyping

The association between polymorphisms and HCC was first performed with a case control design, by using a logistic regression model. Since this approach does not take into account the time at risk to develop HCC, Applicants calculated the cumulative incidence of HCC for the different polymorphisms, using the putative infection date (whenever available) as a starting point, with censoring at death or lost follow-up. Finally, the association between the polymorphisms and the risk to develop HCC was performed using a univariate and multivariate Cox regression model, accounting for all relevant covariates. REFERENCES

1. Rosen CJ. Clinical practice. Vitamin D insufficiency. N Engl J Med

2011;364:248-54.

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3. Rauch A, Kutalik Z, Descombes P et al. Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. Gastroenterology 2010;138: 1338-45.

4. Petta S, Camma C, Scazzone C et al. Low vitamin D serum level is related to severe fibrosis and low responsiveness to interferon-based therapy in genotype 1 chronic hepatitis C. Hepatology 2010;51 :1158-67.

5. Lange CM, Bojunga J, Ramos-Lopez E et al. Vitamin D deficiency and a CYP27B 1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy. J Hepatol 2011, in press (published online ahead of print on December 10, 2010.

6. Kwoh et al. ,1990, Am. Biotechnol. Lab. 8: 14-25.

7. Hagen-Mann, et al, 1995 Exp. Clin. Endocrinol. Diabetes 103 : 150- 155.

8. Walker et al, 1992 PNAS, 89:392-396.

9. Walker et al, 1992 Nucleic Acids Res. 20: 1691-1696.

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11. Guatelli et al, 1990, PNAS 87: 1874-1878.

12. Mueller et al, 1997, Histochem. Cell Biol. 108:431-437.

13. Lizardi et al, 1988, BioTechnology 6: 1 197-1202.

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Claims

1. A method of determining a susceptibility to hepatocellular carcinoma development in a subject, comprising

(a) providing a biological sample from said subject,

2. The method of claim 1 , wherein the at least one polymorphic marker is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of rsl9931 16, rs2060793, rsl0741657, rs71 16978, rsl0500804, rsl2794714, rs2244546.

3. The method of any one of claims 1 to 2, wherein the at least one polymorphic marker, which presence is an indication that said subject has a decreased susceptibility to

hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of G/A for rs 19931 16, G/A for rs2060793, G/A for rs 10741657, C/T for rs71 16978.

4. The method of any one of claims 1 to 2, wherein the at least one polymorphic marker, which presence is an indication that said subject has an increased susceptibility to

hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of T/G for rsl0500804, G/A for 12794714 G/A and C/G for rs2244546.

5. The method of any one of claims 1 to 4, wherein said subject is suffering from chronic liver diseases.

6. The method of any one of claims 1 to 5, wherein said biological sample is whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin or biopsies of muscle, liver, brain tissue, nerve tissue and hair.

7. The method of any one of claims 1 to 6, wherein the at least one polymorphic marker is a polymorphic site being in complete or strong linkage disequilibrium with at least one SNP selected from the group according to claim 2.

8. The method of any one of claims 1 to 7, wherein the at least one polymorphic marker is a combination of at least two SNPs selected from the group according to claim 2.

9. The method of any one of claims 1 to 8, wherein the at least one SNP selected from the group according to claim 2 is in complete or strong linkage disequilibrium with any other genetic or epigenetic polymorphic marker.

10. A method for reducing the risk of developing a hepatocellular carcinoma in a subject, comprising

i) providing a biological sample from said subject,

11. The method of claim 10, wherein the at least one polymorphic marker is a

polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of rsl993116, rs2060793, rsl0741657, rs7116978, rsl0500804, rsl2794714, rs2244546.

12. The method of any one of claims 10 to 11, wherein the at least one polymorphic marker, which presence is an indication that said subject has a decreased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of G/A for rs 1993116, G/A for rs2060793, G/A for rs 10741657, C/T for rs7116978.

13. The method of any one of claims 10 to 11, wherein the at least one polymorphic marker, which presence is an indication that said subject has an increased susceptibility to hepatocellular carcinoma, is a polymorphic site associated with at least one single nucleotide polymorphism (SNP) selected from the group consisting of T/G for rsl0500804, G/A for 12794714 G/A and C/G for rs2244546.

14. The method of any one of claims 10 to 13, wherein said subject is suffering from chronic liver diseases.

15. The method of any one of claims 10 to 14, wherein said biological sample is whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin or biopsies of muscle, liver, brain tissue, nerve tissue and hair.

16. The method of any one of claims 10 or 15, wherein the treatment comprises a vitamin D supplementation treatment.

17. A kit for determining susceptibility to hepatocellular carcinoma development in a subject according to the method of any of claims 1 to 9, said kit comprising i) reagents for selectively detecting the presence or absence of at least one polymorphic marker in a nucleic acid sample isolated from a biological sample obtained from said subject, wherein the at least one polymorphic marker is located in a nucleic acid segment selected from the group consisting of SEQ ID No 1 to SEQ ID No 7 and ii) instructions for use.