WO2013113761A1

WO2013113761A1 - Methods and kits for predicting the risk of having a basal cell carcinoma in a subject

Info

Publication number: WO2013113761A1
Application number: PCT/EP2013/051809
Authority: WO
Inventors: Nadem Soufir
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2012-01-31
Filing date: 2013-01-30
Publication date: 2013-08-08

Abstract

The present invention relates to a method of determining whether a subject is at risk of having or developing a basal cell carcinoma, comprising testing for said subject the presence of at least one genetic alteration in the Damaged-DNA binding protein 2(DDB2) gene, wherein the presence of said genetic alteration indicates an increased risk of having or developing basal cell carcinoma.

Description

METHODS AND KITS FOR PREDICTING THE RISK OF HAVING A BASAL CELL

CARCINOMA IN A SUBJECT

FIELD OF THE INVENTION:

The present invention relates to in vitro methods and kits for predicting the risk of having basal cell carcinoma in a subject.

BACKGROUND OF THE INVENTION:

Basal cell carcinoma (BCC) is the most common form of skin cancer and the most common form of cancer of any type in Caucasians. It develops in the basal germinative cell layer of the epidermis, often on sun-exposed areas of the skin. Although BCC rarely spreads (i.e., metastasizes) to other parts of the body, it can be very destructive and disfiguring. BCC may cause local tissue destruction that may lead to disfigurement or functional impairment of surrounding non-cancerous tissue. Disfigurement may be a particular concern of BCC patients because many BCC tumors occur on the sun-exposed— and, therefore, also typically otherwise exposed— skin of the head and neck. Larger tumors, tumors that have been present for long periods of time, and tumors that have recurred after initial therapy may be biologically more aggressive and especially difficult to cure. While the mortality rate of BCC is relatively low, its increasing incidence and prolonged morbidity means that the disease can be very costly to treat. In 1996, mutations in the tumor suppressor gene PATCHED (PTCH) have been found to be associated to the nevoid basal cell carcinoma (Hahn et al, 1996, J. Biol. Chem. 271, 12125; Johnson et al, 1996, Science 272, 1668; W097/43414). Nonetheless, no single biomarker is sufficiently specific to provide adequate clinical utility for the predisposition for basal cell carcinoma in an individual subject. Therefore, there is a need for identifying other factors that provide a more accurate prediction of basal cell carcinoma. Thus, the invention aims to provide a novel method for determining whether a subject is at risk to basal cell carcinoma using new genetic biomarkers

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SUMMARY OF THE INVENTION:

DETAILED DESCRIPTION OF THE INVENTION:

A "subject" in the context of the present invention can be a male or female. A subject can also be one who has not been previously diagnosed as having basal cell carcinoma. In one embodiment of the invention, the subject having or being at risk of having or developing a basal cell carcinoma may be a substantially healthy subject, which means that the subject has not been previously diagnosed or identified as having or suffering from basal cell carcinoma. In another embodiment, said subject may also be one that is asymptomatic for basal cell carcinoma. As used herein, an "asymptomatic" subject refers to a subject that does not exhibit the traditional symptoms of basal cell carcinoma. In another embodiment of the invention, said subject may be one that is at risk of having or developing a basal cell carcinoma, as defined by clinical indicia such as ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, a family history of basal cell carcinoma...

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to a basal cell carcinoma, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to basal cell carcinoma conversion and therapeutic geographic atrophy form of basal cell carcinoma conversion risk reduction ratios.

"Determining whether a subject is at risk of having or developing a basal cell carcinoma" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that basal cell carcinoma may occur. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of basal cell carcinoma, such as excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, a family history of basal cell carcinoma... The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to basal cell carcinoma, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for basal cell carcinoma . In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for basal cell carcinoma.

According to the invention the presence of the genetic alteration is tested from a sample obtained from the subject.

A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. As used herein "blood" includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood. According to the invention, the sample comprises nucleic acids, wherein nucleic acids may be genomic DNA, heterogenous nuclear RNA (hnRNA, also referred as incompletely processed single strand of ribonucleic acid) and/or cDNA.

As used herein the term "DDB2 gene" has its general meaning in the art and refers to the gene encoding for damage-specific DNA binding protein 2 (Gene ID : 1643 ; NG 009365.1). Typically the mRNA sequence of this gene is deposited in the database Genbank under accession number NM 000107.2. The corresponding polypeptide sequence is deposited in databases under accession number NP 000098.1. This gene encodes a protein that is necessary for the repair of ultraviolet light-damaged DNA. This protein is the smaller subunit of a heterodimeric protein complex that participates in nucleotide excision repair, and this complex mediates the ubiquitylation of histones H3 and H4, which facilitates the cellular response to DNA damage. This subunit appears to be required for DNA binding. Mutations in this gene cause xeroderma pigmentosum complementation group E, a recessive disease that is characterized by an increased sensitivity to UV light and a high predisposition for skin cancer development, in some cases accompanied by neurological abnormalities.

A "genetic alteration" has its general meaning in the art and refers to a genomic polymorphic site. Each genetic alteration has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, a genetic alteration implies that there is association to at least one specific allele of that particular genetic alteration. The alteration can comprise any allele of any variant type found in the genome, including splicing mutations, non sense mutation, missense mutations, insertions, deletions, or duplications. Genetic alterations can be of any measurable frequency in the population.

In the context of the instant application, mutations identified in DDB2 gene or protein are designated pursuant to the nomenclature of Dunnen and Antonarakis (Dunnen et Antonarakis (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutation. 15 :7-12; Erratum in: Hum Mutat 2002 ;20(5):403). As defined by Dunnen and Antonarakis at the nucleic acid level, substitutions are designated by ">". Deletions are designated by "del" after the deleted interval (followed by the deleted nucleotides). Insertions are designated by "ins," followed by the inserted nucleotides. Intron mutations are designated by the intron number (preceded by ' VS") or cDNA position; positive numbers starting from the G of the GT splice donor site, whereas negative numbers starting from the G of the AG splice acceptor site. When the full-length genomic sequence is known, the mutation is best designated by the nucleotide number of the genomic references.

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

Typically, the genetic alteration of the invention compromises the function of the gene product. Thus, a genetic alteration of the invention typical confers a loss of function. For example the genetic alteration is a mutation that truncates at least 10%, 15%, 20%, 25%, 30%, 40%), 50%), 60%), or more of the DDB2 gene product. Other loss function genetic alterations include region mutations in one or more essential functional domains or essential conserved structures of the DDB2 gene product.

The term "polymorphism" or "allelic variant" means a mutation in the normal sequence of a gene, Allelic variants can be found in the exons, introns, or the coding region of the gene, or in the sequences that control expression of the gene. In particular, genetic alterations that are associated to a high risk of having or developing a basal cell carcinoma are depicted in table 1.

In the methods described herein, subject at risk for basal cell carcinoma is one in whom a particular genetic alteration is present in the DDB2 gene. In other words, carriers of the genetic alteration are at a different risk for basal cell carcinoma than non-carriers. In certain embodiments, significance associated with risk of a genetic alteration is measured by a relative risk (RR). In another embodiment, significance associated with a genetic alteration is measured by an odds ratio (OR). In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.2, including but not limited to: at least 1.5, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, and at least 15.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 2.0 is significant. In another particular embodiment, a risk of at least 3.0 is significant. In yet another embodiment, a risk of at least 4.0 is significant. In a further embodiment, a relative risk of at least 5.0 is significant. In another further embodiment, a significant increase in risk is at least 10.0 is significant. However, other values for significant risk are also contemplated, e.g., at least 2.5, 3.5, 4.5, 5.5, or any suitable other numerical values, and such values are also within scope of the present invention. In other embodiments, a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, and 1500%. In one particular embodiment, a significant increase in risk is at least 100%. In other embodiments, a significant increase in risk is at least 200%, at least 300%, at least 400%, at least 500%, at least 700%, at least 800%, at least 900% and at least 1000%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention. In certain embodiments, a significant increase in risk is characterized by a p-value, such as a p-value of less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001 , less than 0.0000001 , less than 0.00000001, or less than 0.000000001.

In a particular embodiment, the method of the invention comprises testing for said subject the presence of at least one genetic alteration in linkage disequilibrium with at least one genetic alteration of the invention, wherein the presence of said genetic alteration indicates an increased risk of having or developing basal cell carcinoma.

The term "linkage disequilibrium" (LD) refers to a population association among alleles at two or more loci. It is a measure of co-segregation of alleles in a population. Linkage disequilibrium or allelic association is the preferential association of a particular allele or any other genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. Accordingly, two particular alleles at different loci on the same chromosome are said to be in LD if the presence of one of the alleles at one locus tends to predict the presence of the other allele at the other locus.

Linked variants are readily identified by determining the degree of linkage disequilibrium (LD) between the allele genotyped for one SNP and a candidate linked allele at a polymorphic site located in the chromosomal region where said SNP is located or elsewhere on the chromosome. The candidate linked variant may be an allele of a polymorphism that is currently known. Other candidate linked variants may be readily identified by the skilled artisan using any technique well-known in the art for discovering polymorphisms. One of the most frequently used measures of linkage disequilibrium is r, which is calculated using the formula described by Devlin et al. (Genomics, 29(2):311-22 (1995)). "r" is the measure of how well an allele X at a first locus predicts the occurrence of an allele Y at a second locus on the same chromosome. The measure only reaches 1.0 when the prediction is perfect (e.g. X if and only if Y).

Detecting the specific genetic alterations according to the invention can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of genetic alterations can be used, such as fluorescence-based techniques (e.g., Chen, X. et al, Genome Res. 9(5): 492-98 (1999); Kutyavin et al, Nucleic Acid Res. 34:el28 (2006))., utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, realtime PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology (e.g., Affymetrix GeneChip; Perlegen), BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave). Thus, by use of these or other methods available to the person skilled in the art, one or more alleles at genetic alterations can be identified.

Typically, the determination of the said genetic alteration may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridization, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said polymorphism may be detected in a

RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to genotype the polymorphism (or identify the allele).

Actually numerous strategies for genotype analysis are available (Antonarakis et al, 1989; Cooper et al, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.

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

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides.

Their length may be shorter than 400, 300, 200 or 100 nucleotides.

In a particular embodiment, the genetic variation is detected at the protein level. A variety of methods can be used for detecting genetic variations at protein level, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. A sample from a subject is assessed for the presence of an alteration in the polypeptide encoded by the nucleic acid having the genetic alteration according to the invention. Such alteration can, for example, be an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced). An alteration in the composition of a polypeptide can be an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide). Both such alterations (quantitative and qualitative) can also be present. An alteration in the expression or composition of the polypeptide can be the result of a particular genetic alteration (insertions, duplications...). In one embodiment, an antibody (e.g., an antibody with a detectable label) that is capable of binding to a particular target polypeptide (e.g., a polypeptide encoded by a nucleic acid associated with a genetic alteration as described herein) can be used. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end- labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. In one embodiment, the level or amount of polypeptide in the sample obtained from the subject is compared with the level or amount of the polypeptide in a control sample. Typically, when a level or amount of the polypeptide in the sample is lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of a genetic alteration in the in the DDB2 gene.

In some embodiments, the method of the invention is performed by a laboratory that will generate a test report. The test report will thus indicates whether the genetic alteration is present or absent, and preferably indicates whether the patient is heterozygous or homozygous for said genetic alteration. Accordingly, if the patient is homozygous for the risk allele, then the test report further indicates that the patient is positive for a genetic alteration associated with a high risk of having or developing basal cell carcinoma. If the patient is heterozygous for the risk allele, then the test report further indicates that the patient is positive for a genetic alteration associated with a risk of having or developing basal cell carcinoma. In some embodiments, the test result will include a probability score, which is derived from running a model that include the risk factor determined for the genetic alteration of the invention that are tested. For calculating the score, the risk factor determined for a genetic alteration of the invention may be pondered by a coefficient depending on what is the contribution of said genetic alteration in the determination of the risk in comparison with another genetic alteration. Typically, the method for calculating the score is based on statistical studies performed on various cohorts of patients. The score may also include other various patient parameters (e.g., age, gender, weight, race, test results for other genetic risk factors or other typical risk factors such as excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, family history of basal cell carcinoma... The weight given to each parameter is based on its contribution relative to the other parameters in explaining the inter- individual variability of having basal cell carcinoma in the relevant disease population. In some embodiments, the test report may be thus generated by a computer program for establishing such a score.

This probability score may be used as a guide in selecting a therapy or treatment regimen for the subject. Accordingly; when the subject is considered at risk according to the method of the invention, one or more basal cell carcinoma treatments or prophylactic regimens may be prescribed to said subject. Subjects genotyped as having one or more of the alleles described herein that are associated with increased risk of basal cell carcinoma often are prescribed a prophylactic regimen designed to minimize the occurrence of basal cell carcinoma. An example of a prophylactic regimen often prescribed is directed towards minimizing ultraviolet (UV) light exposure. Such a regimen may include, for example, prescription of a lotion applied to the skin that minimizes UV penetration and/or counseling individuals of other practices for reducing UV exposure, such as by wearing protective clothing and minimizing sun exposure. In certain embodiments, a treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing basal cell carcinoma assessed by the method of the invention. The treatment sometimes is preventative (e.g., is prescribed or administered to reduce the probability that a basal cell carcinoma arises or progresses), sometimes is therapeutic, and sometimes delays, alleviates or halts the progression of a basal cell carcinoma. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of a basal cell carcinoma can be prescribed and/or administered.

Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes the genetic alteration detection, restriction enzymes (e.g., for RFLP analysis), nucleic acid probes, optionally labelled with suitable labels (e.g., fluorescent labels), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of the nucleic acids as described herein, means for analyzing the nucleic acid sequence as described herein, means for analyzing the amino acid sequence of a polypeptide encoded, etc. The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids, and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays for basal cell carcinoma.

In one preferred embodiment, the kit comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease. As explained in the above, the detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)). The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the polymorphism to be detected. Preferably, the polymorphism is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.

The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.

In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.

In one embodiment, the DNA template is amplified by means of Whole Genome Amplification (WGA) methods, prior to assessment for the presence of specific genetic alterations as described herein. Standard methods well known to the skilled person for performing WGA may be utilized, and are within scope of the invention. In one such embodiment, reagents for performing WGA are included in the reagent kit.

Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.

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

EXAMPLE 1: DDB2 IS A MAJOR GENE INVOLVED IN PREDISPOSITION TO MULTIPLE BASAL CELL CARCINOMA OR FAMILIAL BASAL CELL CARCINOMA

The protein DDB2 (Damaged-DNA binding protein 2) is one of the elements of the

NER (nucleotide excision repair) involved in the recognition of UV-induced mutations, forming a heterodimeric complex with the protein DDB1. In addition, it possesses an E3 ubiquitin ligase, and active cell apoptosis. Mutations in DDB2 are responsible for a subgroup rare of xeroderma pigmentosum (XP-E). We investigated the DDB2 gene as a candidate gene in the predisposition to basal cell carcinoma (BCC), the most common cancer in Caucasians.

One hundred patients with CBC multiple (at least 2 BCCs) and / or familial (at least 2 affected related by family) were included in the study. Constitutional DNA was extracted from blood of patients after having signed an informed consent. All exons and adjacent exon junctions of DDB2 were amplified by PCR and sequenced (Applied Biosystems 3130). Computer analysis of sequences was performed by SeqScape version 2 and the putative deleterious effects of missense and intronic variants has been studied by bioinformatic prediction sites (Sift, Polyphen, SNP3D, SSF). The allele frequency of functional variants was then compared to that of 3010 Caucasian individuals from a reference control population project exome sequencing (http://snp.gs.washington.edu/EVS/).

Eight deleterious variants of the gene DDB2/XPE were identified in 6 patients (allelic frequency= 0.04) (Table 1). Two heterozygous intronic variants were characterized in two patients, and were predicted to have an effect on splicing; one homozygous nonsense mutation was detected in one patient; one patient was a compound heterozygous for a frameshift and a missense variant; finally, two patients carried heterozygous missense variants. All missense variants were predicted to be deleterious by all three prediction sites: Sift, polyphen, Snp3D. By comparison, the allelic frequency of deleterious variants in the exome database (exome variant server) was 15 deleterious alleles out of 7020 alleles (allelic frequency, 0.0013; P <0.0001, odds ratio = Odd Ratio= 19.46 [7.5- 49.5], relative risk 13 [6.35-21.09]).

While mutations of the XPE/DDB2 gene is responsible for one of the rarest subgroups of xeroderma pigmentosum, our study shows that DDB2 mutations are also clearly involved in genetic predisposition to some forms of basal cell carcinomas i.e., multiple basal cell carcinomas patients and/or familial basal cell carcinomas patients. Interestingly, four patients were heterozygous for DDB2 mutations, although xeroderma pigmentosum is an autosomal recessive disease. However, heterozygous DDB2 + / - mice, when irradiated by UV light, develop skin cancers, strongly suggesting that inactivation of one DDB2 allele may be pathogenic. Segregation analysis of mutations is in progress in some families, as well as functional studies (notably DNA repair studies in skin fibroblasts from mutated patients) to confirm their deleterious effect. These results may have important implications for genetic counselling. Table 1: mutations identified in DDB2 gene and associated with basal cell carcinomas patient mutation

s DDB2 mutation localisation mutation effect hetero/homozygous c.127+5 T>G

O 305 hetero IVS1 splicing heterozygous

c.702+5G>A

0 415 hetero IVS5 splicing heterozygous

C.640 OT

0 711 p.Arg214Ter exon 5 Stop homozygous

c.994 OT

0 685 p.Arg332Cys exon 7 missense heterozygous

c.1126 G>A

B544 p.Asp376Asn exon 8 missense heterozygous

C.620 T>C

p.Leu207Pro;

C.970 971dupC

p.Leu324ProfsX3 missense, heterozygous

P581 6 exon 5, exon 7 splicing composite REFERENCES

l .Stoyanova T, Roy N, Kopanja D, Raychaudhuri P, Bagchi S. DDB2 (damaged-DNA binding protein 2) in nucleotide excision repair and DNA damage response. Cell Cycle. 2009 Dec 15;8(24):4067-71.

2. Itoh T, Cado D, Kamide R, Linn S. DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not achemical carcinogen. Proc Natl Acad Sci USA. 2004 Feb 17;101(7):2052-7.

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

A method of determining whether a subject is at risk of having or developing a basal cell carcinoma, comprising testing for said subject the presence of at least one genetic alteration in the Damaged-DNA binding protein 2(DDB2) gene, wherein the presence of said genetic alteration indicates an increased risk of having or developing basal cell carcinoma.

The method according to claim 1 wherein said genetic alteration is selected from the group consisting of Table 1.