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EP1114180A2 - Marqueur au niveau du gene recepteur de la vitamine d, utilise pour la determination de la sensibilite au cancer du sein - Google Patents

Marqueur au niveau du gene recepteur de la vitamine d, utilise pour la determination de la sensibilite au cancer du sein

Info

Publication number
EP1114180A2
EP1114180A2 EP99944189A EP99944189A EP1114180A2 EP 1114180 A2 EP1114180 A2 EP 1114180A2 EP 99944189 A EP99944189 A EP 99944189A EP 99944189 A EP99944189 A EP 99944189A EP 1114180 A2 EP1114180 A2 EP 1114180A2
Authority
EP
European Patent Office
Prior art keywords
vitamin
breast cancer
receptor
gene
genotype
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99944189A
Other languages
German (de)
English (en)
Inventor
François Rousseau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Laval
Original Assignee
Signalgene Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Signalgene Inc filed Critical Signalgene Inc
Publication of EP1114180A2 publication Critical patent/EP1114180A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to breast cancer.
  • the invention further relates to a marker at the vitamin D receptor gene or equivalents thereof to prognose, diagnose or treat breast cancer.
  • the invention relates to a method for determining breast cancer susceptibility, prognosis and response to therapy based on a determination of a genotype at the vitamin D receptor locus or at a marker in linkage disequilibrium therewith.
  • the invention further relates to screening assays to identify and select agents which can be used in the treatment of breast cancer.
  • BRCA1 and BRCA2 have been identified up to now as being associated with some familial forms of breast cancer. Together, these two genes account for 5% to 10% of all cases of breast cancer and possibly up to 70% of familial breast cancer cases.
  • a breast examination by a patient or a physician begins with a visual inspection for asymmetric breast size, nipple inversion, bulging, or dimpling.
  • An underlying cancer is sometimes detected by having the patient press both hands against the hips or the palms together in front of the forehead. This contracts the pectoral muscles, and a subtle dimpling of the skin may appear if a Cooper's ligament has been entrapped by a growing tumor.
  • Breast cancers have the same clinical characteristics in older as in younger women Cancer is usually suspected when changes are noted on mammography or when a breast lesion is seen or felt Lesions usually can be felt as firm nodules within the breast Ulcerations may occur, and lesions within or near the nipple may produce discharge Sometimes breast cancer is discovered only after metastatic lesions cause bone fractures, neurologic changes, hypercalcemia, liver failure, or ascites
  • VDR gene polymorphisms studied i e Bsml or Fokl
  • Bsml or Fokl VDR gene polymorphisms studied
  • Fokl a gene polymorphism that has been associated with osteoporosis as well as prostate cancer
  • Ruggiero et al study did not find any effect of VDR in general susceptibility to breast cancer but only an association of VDR genotype with the presence of metastases of breast cancer
  • a prediction of the general susceptibility of women to develop breast cancer in their lifetime, or of an association between the VDR gene and breast cancer independently of metastases of breast cancer has yet to be reported
  • One aim of the present invention is to provide a genetic assay for determining the predisposition to breast cancer and/or response to breast cancer treatment
  • the determination of a predisposition to breast cancer is assessed by a determination of the genotype of the VDR gene, at at least one locus thereof, or at a locus in linkage disequilibrium therewith
  • a determination of the VDR genotype comprises a determination of the Bsml polymorphism, wherein certain combinations of VDR gene btallelic Bsml polymorphism (bb genotype, presence of the restriction site on both alleles) have a significant but slightly increased risk of developing breast cancer as compared to the category with the smallest risk (BB genotype, absence of the restriction site on both alleles)
  • the assessment of a predisposition and/or protection to breast cancer comprises a determination of the VDR genotype, comprising a determination of the Fokl btallelic polymorphism, (wherein a ff genotype is designated as the presence of the restriction site on both alleles and a FF genotype designates the absence of the restriction site on both alleles) or a marker in linkage disequelibnum therewith, and a determination of the genotype of another marker, wherein the combination of alleles at the Fokl polymorphism and at the other marker, shows a significant association with breast cancer
  • Another aim of the present invention is to use a combination of polymorphisms of the vitamin D receptor (VDR) gene or an equivalent thereof, and a second polymorphism of the VDR or an equivalent thereof, as markers for breast cancer susceptibility and/or response to breast cancer preventive or curative therapy
  • VDR vitamin D receptor
  • One combination of a polymorphism of the vitamin D receptor (VDR) gene, or any polymorphism in linkage disequilibrium therewith, when combined with another polymorphism of the VDR gene, or any polymorphism in linkage disequilibrium therewith, can be used as a test for breast cancer susceptibility or protection, for responsiveness to treatment of breast cancer, for breast cancer prognosis or severity, or as a means to classify patients in clinical trial for breast cancer (screening, diagnosis, prognosis or treatment)
  • the VDR genotype is determined at two loci, the Bsml polymorphism (or at a locus in linkage disequilibrium therewith) and at an other locus of VDR In one especially preferred embodiment of the present invention this additional locus of VDR is the Fokl polymorphism
  • the determination of the predisposition and/or protection and/or prognosis of breast cancer is carried out by analyzing the combination of alleles at the Bsml and Fokl polymorphisms of VDR, or combinations of alleles at loci in linkage disequilibrium with the Bsml and Fokl polymorphisms.
  • such a determination of a combination of alleles at the Bsml and Fokl polymorphisms enables the identification of a striking association with breast cancer.
  • the identification of the combination of allele BBff is strongly associated with breast cancer in a very signifcant fraction of breast cancer patients, while the BBFf and BBFF are shown to have a protecting effect against breast cancer.
  • the present invention indeed provides, in accordance with a preferred embodiment thereof, the strongest susceptibility marker for breast cancer ever identified, as it explains ten times more cases of the disease than the BRCA1 and BRCA2 genes combined. Indeed, when the number of total cases of breast cancer attributable to these two VDR polymorphisms was computed, it was shown that 45% of all cases of breast cancer were attributable to VDR gene polymorphism, as detected by the combined testing of the Bsml and the Fokl genotypes.
  • the present invention also relates to vectors, including expression vectors harboring a VDR gene (or fragment or fusion thereof) having a genotype in accordance with the present invention (i.e. a predisposing genotype, Bbff, or alternatively, a protecting genotype, BBFF; or other genotypes isolated from patients or genetically engineered), cells harboring such vectors, and non-human animals harboring such vectors or cells.
  • a VDR gene or fragment or fusion thereof
  • BBFF protecting genotype
  • the present invention aims at providing a method of determining at least two polymorphisms in the vitamin D receptor gene, wherein this determination can be correlated with a predisposition or a protection to breast cancer This determination can be based on a variety of genotyping methods at the DNA, RNA or protein level
  • Another aim of the present invention is to provide a method of prognosing and/or forecasting the development of breast cancer in a patient, which comprises determining at least two polymorphisms of the VDR gene of the present invention, associated with breast cancer, or any polymorphism in linkage disequilibrium therewith, in a biological sample of the patient, wherein a combination of the polymorphisms at the VDR loci shows a significant association with breast cancer
  • Another aim of the present invention is to provide means of identifying young women that will be at risk of developing breast cancer and to categorize those that are likely to respond significantly to preventive therapy
  • An aim of the present invention is thus to provide means of identification of target sub-groups of women for breast cancer prevention measures/programs
  • Another aim of the present invention is to provide means to determine which sub-group of women will most benefit from breast cancer treatment(s) and eventually predict their response to therapy or choose the optimal preventive pharmacotherapy
  • Another aim of the present invention is to identify means of predicting and managing interventions for breast cancer as well as identifying and/or characterizing biological parameters which could enable the establishment of population-based breast cancer prevention and intervention programs
  • an aim of the present invention to provide a method of selecting alleles of the VDR gene or in linkage disequilibrium therewith, which are suitable for designing an assay to screen compounds which can modulate the activity of a vitamin D receptor
  • Another aim of the present invention is to provide an assay to screen for drugs for the treatment and/or prevention of breast cancer. Having identified alleles which predispose to breast cancer (and those which predispose to a "resistance" to breast cancer), assays can be set-up to screen agents and select drugs which could be used in the treatment or prevention of breast cancer. Since some alleles of the VDR have been shown to affect the functionality of the vitamin D receptor (Tut et al. 1997, J. Clin.
  • assays could be designed based on chosen genotypes of the VDR gene.
  • a non-limiting example of a type of assay which could be designed includes, cis-trans assays similar to those described in USP 4,981 ,784.
  • a cis-trans assay could be set-up, based on the use of a chosen alleles of VDR, shown here to predispose to breast cancer (i.e. the combination of allele BBff of the VDR gene) as compared to a alleles of VDR, shown here to be associated with lower risk and/or protection of breast cancer (i.e.
  • BBFF allele BBFF or BBFf of the VDR gene
  • a non-limiting example of such an assay could be based on 2 cell lines (one expressing a predisposing genotype of VDR and one expressing a non-predisposing and/or protecting genotype of VDR) which could be used in parallel to screen for VDR-f unction modulating compounds.
  • the cell line expressing the non-predisposing genotype of VDR (BBFF, for example) can be used as a positive control for the functionality of the vitamin D receptor.
  • such assays can be designed using cells from patients having a known genotype at the loci of the present invention, these cells harboring recombinant vectors could enable an assessment of the functionality of the VDR and dissect the structure-function relationship of the vitamin D receptor and its role in breast cancer.
  • the polymorphisms of the VDR and/or the determination of allelic variations in the VDR gene can be combined to the determination of allelic variations in other genes/markers linked to the predisposition to breast cancer and/or responsiveness to therapy therefor. This combination of genotype analyses could lead to better diagnoses programs and/or treatment of breast cancer.
  • Non-limiting examples of such markers include BRCA1 and BRCA2
  • breast cancer is significantly more preponderant in women, it can also be a deadly disease in men.
  • the present invention is meant to also cover men.
  • a method of determining an individual's predisposition to breast cancer, protection to breast cancer, development of breast cancer and/or responsiveness to therapy for breast cancer which comprises determining a genotype of the vitamin D receptor gene (directly or indirectly by linkage disequilibrium) in a biological sample of the individual and analyzing allelic variation in the vitamin D receptor of the individual, thereby determining an individual's predisposition to breast cancer, protection to breast cancer, development of breast cancer and/or responsiveness to therapy therefor.
  • a method for determining susceptibility to breast cancer, and/or response to therapy therefor comprises the step of determining the vitamin D receptor genotype of the individual, thereby determining an individual's susceptibility to breast cancer and/or response to therapy therefor.
  • genotype determination include a restriction endonuclease digestion, a hybridization with allele specific oligonucleotides, a sequencing of the polymorphism, and an amplification of a segment of the vitamin D receptor (i.e.
  • a method of determining an individual's predisposition to breast cancer, protection to breast cancer, development of breast cancer and/or responsiveness to therapy therefor comprises determining vitamin D receptor polymorphism at at least one of the Bsml and Fokl polymorphic loci (directly or indirectly using a marker in linkage disequilibrium with these polymorphic loci) in a biological sample of the individual and analyzing allelic variation in the vitamin D receptor gene of the individual, thereby determining an individual's predisposition to breast cancer, protection to breast cancer, development of breast cancer and/or responsiveness to therapy therefor.
  • the model comprises two vitamin D receptor gene polymorphisms at the Bsml and Fokl loci thereof, that allow to identify a subset of women that are at significantly increased risk of breast cancer as compared to those bearing other variants of this gene
  • a single gene, the vitamin D receptor gene has been identified as such a target to assess this predisposition.
  • the vitamin D receptor polymorphism is selected from the Bsml polymorphism located in the last intron of the VDR gene and the Fokl polymorphism located in the first exon of the s gene, or any DNA variant or mutation which shows some degree of linkage disequelibrium with one of these polymorphisms.
  • at least one pair of primers is designed to specifically amplify a segment of the vitamin D receptor.
  • the region of the VDR gene which is amplified is in exon 1 and/or in in the last intron thereof
  • This at least one pair of primers is preferably derived from a nucleic acid sequence of the vitamin D receptor gene or flanking portion thereof, to amplify a segment of the vitamin D receptor gene, as commonly known.
  • other primer pairs can be designed, based on the known sequence of the VDR gene Method to design primer pairs form known sequences are commonly known in the art.
  • primers used for amplifying a segment of the vitamin D receptor are defined as:
  • the method of the present invention includes detecting the vitamin D receptor polymorphisms by analyzing the restriction fragment length polymorphisms using an endonuclease digestion.
  • the method can further include a step prior to the vitamin D receptor gene digestion, wherein at least a fragment of the vitamin D receptor is amplified, for example, by polymerase chain reaction.
  • the step of determining the vitamin D receptor genotype could also comprise hybridizing with allele specific oligonucleotides.
  • Suitable endonucleases for genotyping the vitamin D receptor gene, in accordance with a preferred embodiment of the present invention are known in the art. Non-limiting examples thereof include, Bsml, Apal, Taql, Fokl and their isoschizomers.
  • the vitamin D receptor genotype can be determined using a polymorphic variant site in linkage disequilibrium with at least one allelic variant as detected with Bsml, Apal, Taql, Fokl, and isoschizomers thereof, in the restriction endonuclease digestion, or otherwise.
  • the polymorphism of the vitamin D receptor gene can be detected using at least one oligonucleofide specific to the normal or variant vitamin D receptor gene allele. Methods to design specific probes from a known nucleic acid sequence are commonly known in the art.
  • the present invention also provides a kit for determining predisposition to low, intermediate or high risk of breast cancer or to a protection to breast cancer of a patient, which includes at least a probe specific for the vitamin D receptor; a polymorphism selected from: a) a Bsml polymorphism; b) a Fokl polymorphism; c) a polymorphism of VDR showing a significant association with breast cancer; and d) a polymorphism in linkage disequilibrium with the polymorphisms of a)-c).
  • a polymorphism selected from: a) a Bsml polymorphism; b) a Fokl polymorphism; c) a polymorphism of VDR showing a significant association with breast cancer; and d) a polymorphism in linkage disequilibrium with the polymorphisms of a)-c).
  • the present invention provides a specific detection of a VDR polymorphism of the VDR gene using a nucleic acid for the specific detection of this VDR polymorphism in a sample comprising at a nucleic acid sequence which binds under stringent conditions to the VDR polymorphic nucleic acid sequence.
  • the present invention relates to nucleic acid probes which are complementary to a VDR polymorphic sequence, consisting of at least 10 consecutive nucleotides (preferably, 15, 20, 25, or 30) and which specifically hybridize to the VDR polymorphic sequence under high stringency condition.
  • a non-limiting example of a polymorphic specific probe according to the invention includes a probe which would bind to the Fokl polymorphic sequence (specific to the C to T transition, creating a new initiator AUG giving rise to a protein having a three amino acids difference).
  • a nucleic acid probe is immobilized on a solid support
  • solid supports include plastics (i.e. polycarbonate), acrylic resins (i.e. polyacrylamide and latex beads), and carbohydrates (i e agarose and sepharose) Techniques for coupling nucleic acid probes to solid supports are well known in the art
  • the antibodies (i e an antibody specific to a polymorphism of VDR) of the present invention can be immobilized on a solid support
  • similar supports as those used for probe immobilization can be used for antibody immobilization on a solid support
  • the techniques for coupling antibodies to such solid supports The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromatography according to known methods
  • test samples suitable for carrying the methods of the present invention include, cells or nucleic acid extracts of cells, or biological fluids
  • test samples suitable for carrying the methods of the present invention
  • the type of test sample used can vary according to the assay format, the method of detection, and the particular needs of the clinical practioner which will readily adapt the methods of preparation of the sample and the method of detection so that they are compatible, in accordance with the knowledge in the art
  • the allelic variation in the vitamin D receptor gene can be analyzed indirectly using a nucleic acid variant, or equivalent in linkage disequilibrium with one of the polymorphic sites of VDR
  • the allelic variation in the vitamin D receptor gene can also be analyzed directly by determining the genotype within the vitamin D receptor gene
  • the polymorphism of the vitamin D receptor gene can be used as a marker for breast cancer susceptibility
  • the polymorphism in linkage disequilibrium with the markers used can also be used as a test for breast cancer susceptibility, breast cancer protection, or for responsiveness to treatment for breast cancer, for breast cancer prognosis or severity, or as a means to classify patients in clinical trials for breast cancer (screening, diagnosis, prognosis or treatment)
  • RFLP restriction fragment length polymorphism
  • polymorphism refers to any sequence in the human genome which exists in more than one version or variant in the population.
  • linkage disequilibrium refers to any degree of non-random genetic association between one or more allele(s) of two different polymorphic DNA sequences, that is due to the physical proximity of the two loci.
  • Linkage disequilibrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain unseparated for several generations with the consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby.
  • testing of one of a marker in linkage desiquilibrium with the polymorphisms of the present invention at the VDR gene indirect testing
  • will give almost the same information as testing for the herein-identified polymorphisms of the VDR gene directly This situation is encountered throughout all the human genome when two DNA polymorphisms that are very close to each other are studied.
  • vitamin D receptor polymorphism or “genetic marker” are intended to include, without limitation, Bsml, Taql, Apal or Fokl and any other allelic variant of the vitamin D receptor gene that shows some degree of linkage disequilibrium in any population sub-group with at least one of the above-mentioned vitamin D receptor polymorphisms
  • the vitamin D receptor gene polymorphism sites in accordance with the present invention can be located within the vitamin D receptor gene, or on each side thereof, provided that is on the same chromosome and in linkage disequilibrium with the VDR polymorphism of the present invention Distances between markers in linkage disequilibrium can vary widely (below 50 kb to more than 1 mega base) depending on the genetic structure of the population and is ascertainable by a statistically significant association between the markers
  • the present invention should not be limited to the identification of polymorphisms at the DNA level (whether on genomic DNA, amplified DNA, cDNA or the like) Indeed, the herein-identified polymorphisms could be detected at the mRNA or protein level Such detections of polymorphism identification on mRNA or protein are known in the art Non-iimiting examples include detection based on oligos designed to hybridize to mRNA or ligands such as antibodies which are specific to the encoded polymorphism (I e specific to the 3 extra ammo acids encoded by the Fokl polymorphism for example)
  • one of the advantages of the present invention is to enable a determination of the polymorphisms in the VDR gene, in easily obtainable cells which express these genes
  • a non-limiting example thereof is lymphocytes, thereby enabling a genotyping from a simple blood sample
  • Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission
  • nucleic acid molecule refers to a polymer of nucleotides Non-limiting examples thereof include DNA (i e genomic DNA, cDNA) and RNA molecules (i e mRNA)
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense])
  • recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments This is often referred to as genetic engineering
  • DNA segment is used herein, to refer to a
  • DNA molecule comprising a linear stretch or sequence of nucleotides
  • This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of ammo acids which can be referred to as a polypeptide, protein, protein fragment and the like
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • the nucleic acid i.e. DNA or RNA
  • the nucleic acid for practicing the present invention may be obtained according to well known methods.
  • Oligonucleofide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
  • the oligonucleofide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucleofide probes and primers can be designed by taking into consideration the melting point of hydrizidation thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
  • oligonucleofide or "DNA” molecule or sequence refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a "regulatory element” according to the present invention, as the term is defined herein.
  • oligonucleofide or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction.
  • Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al , 1989, supra) and are commonly known in the art.
  • a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ⁇ g/ml denatured carrier DNA (i.e. salmon sperm DNA).
  • the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0 1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency).
  • the selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • RNA-DNA hybrids can also be formed and detected.
  • the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra).
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and -nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988,
  • Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • probes can be used include Southern blots (DNA detection), dot orslot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. More recently, PNAs have been described (Nielsen et al 1999, Current Opin. Biotechnol. 10:71-75). PNAs could also be used to detect the polymorphisms of the present invention Other detection methods include kits containing probes on a dipstick setup and the like
  • Probes can be labeled according to numerous well known methods (Sambrook et al , 1989, supra) Non-limiting examples of labels include 3 H, 14 C, 32 P, and 3!
  • Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies
  • Other detectable markers for use with probes include biotin and radionucleotides It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe
  • radioactive nucleotides can be incorporated into probes of the invention by several methods Non-limiting examples thereof include kinasmg the 5' ends of the probes using gamma 32 P ATP and poiynucleotide kinase, using the Klenow fragment of Pol I of E coli in the presence of radioactive dNTP (i e uniformly labeled DNA probe using random oligonucleofide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxynbonucleotides) The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill
  • An oligonucleofide can be synthesized chemically or derived by cloning according to well known methods
  • a ' primer' defines an oligonucleofide which is capable of annealing to a target sequence thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ rep case system and NASBA (Kwoh et al , 1989, Proc Natl Acad Sci USA 86, 1173-1177, ⁇ zardi et al , 1988, BioTechnology 6 1197- 1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra) Preferably, amplification will be carried out using PCR
  • PCR Polymerase chain reaction
  • a nucleic acid sample e g , in the presence of a heat stable DNA polymerase
  • An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith
  • the extension product synthesized from each p ⁇ mer can also serve as a template for further synthesis of extension products using the same primers Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess whether the sequence or sequences to be detected are present Detection of the amplified sequence may be carried
  • Ligase chain reaction is carried out in accordance with known techniques (Weiss, 1991 , Science 254 1292) Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al , 1992, Proc Natl Acad Sci USA 89 392-396, and ibid , 1992, Nucleic Acids Res 20 1691-1696)
  • SDA Strand displacement amplification
  • the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific ammo acid sequence thereby giving rise the a specific polypeptide or protein
  • a "heterotogous" i e a heterologous gene) region of a
  • DNA molecule is a subsegment segment of DNA within a larger segment that is not found in association therewith in nature
  • heterologous can be similarly used to define two polypeptidic segments not joined together in nature
  • Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned
  • vectors Numerous types of vectors exist and are well known in the art
  • expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more
  • expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host
  • the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often refered to as being operably linked to control elements or sequences.
  • Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript.
  • two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • a promoter and a reporter sequence are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest.
  • This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (i.e. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography).
  • the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies The purified protein can be used for therapeutic applications.
  • the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boses and “CCAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • an expression vector can be constructed to assess the functionality of specific alleles of the VDR gene and of the interaction of such alleles.
  • expression vectors include a vector comprising the vitamin D responsive element (the cis sequences [i.e. DNA sequence to which a factor binds] enabling vitamin D-dependent modulating effects of promoter activity are known in the art) operably linked to a chosen promoter and modulating the activity thereof, the promoter driving the expression of a reporter gene.
  • the modulating effect of the promoter activity can be assessed by determining the level of expression of the reporter gene.
  • the vector is transfected into a cell of a patient having the genotype of VDR shown herein to be associated with a low risk and/or protection of breast cancer, or in a cell from a patient having the genotype of VDR shown herein to be associated with a moderate or high risk of breast cancer.
  • VDR gene expressed by these cells can be modified at will (i e by in vitro mutagenesis or the like)
  • numerous combinations of genotypes can be tested in such assays to dissect the functional relationship between the VDR genotype and its function in vitamin D-dependent function and/or its function in breast cancer
  • indicator cells expressing VDR could also be engineered by choosing a cell line and transfecting thereinto, chosen genotypes of VDR and one expression vector as described above
  • Non-human transgenic animals expressing chosen alleles of VDR could also be prepared and used to screen compounds that affect vitamin D receptor function and possibly overcome a predisposition to breast cancer, perhaps to the level observed with the BBFF genotype
  • the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether an nucleic acid or am o acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence
  • This functional derivative or equivalent may be a natural derivative or may be prepared synthetically
  • Such derivatives include ammo acid sequences having substitutions, deletions, or additions of one or more am o acids, provided that the biological activity of the protein is conserved
  • derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained
  • the substituting ammo acid as chemico-physical properties which are similar to that of the substituted ammo acid
  • the similar chemico-physical properties include, similarities in charge, bulkmess, hydrophobicity, hydrophylicity and the like
  • the term “functional derivatives" is intended to include “fragments", "se
  • the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, all these methods are well known in the art.
  • chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are examplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
  • allele defines an alternative form of a gene which occupies a given locus on a chromosome.
  • a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell.
  • a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
  • Spontaneous mutations and experimentally induced mutations exist.
  • the result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule.
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
  • purified refers to a molecule having been separated from a cellular component.
  • a purified protein has been purified to a level not found in nature.
  • a “substantially pure” molecule is a molecule that is lacking in all other cellular components.
  • agent are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds.
  • the term “molecule” therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like
  • Non limiting examples of molecules include nucleic acid molecules, peptides, ligands, including antibodies, carbohydrates and pharmaceutical agents
  • the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling
  • the terms “rationally selected” or “rationally designed” are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention
  • macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule”
  • peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above
  • the polypeptides of the present invention are modified to enhance their stability It should
  • An indicator cell in accordance with the present invention can be used to identify antagonists
  • the test molecule or molecules are incubated with the host cell in conjunction with one or more agonists held at a fixed concentration
  • An indication and relative strength of the antagonistic properties of the molecule(s) can be provided by comparing the level of gene expression in the indicator cell in the presence of the agonist, in the absence of test molecules vs in the presence thereof
  • the antagonistic effect of a molecule can also be determined in the absence of agonist, simply by comparing the level of expression of the reporter gene product in the presence and absence of the test molecule(s)
  • the "in vivo" experimental model can also be used to carry out an "in vitro” assay
  • cellular extracts from the indicator cells can be prepared and used in an "in vitro” test
  • a non-limiting example thereof include binding assays
  • the recitation “indicator cells” refers to cells that express a given genotype of VDR according to the present invention
  • the indicator cells can be used in the screening assays of the present invention
  • the indicator cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of a genotype of the present invention
  • the cells can be yeast cells or higher eukaryotic cells such as mammalian cells
  • the indicator cell would be a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (Ausubel et al , 1994, supra) and can be used to test a compound or a library thereof
  • the cis-trans assay as described in USP 4,981 ,784 can be adapted and used in accordance with the present invention
  • Such an indicator cell could be used to rapidly screen at high-throughput a vast array of test molecules
  • the reporter gene is luciferase or ⁇
  • fusion proteins include a hemaglutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions.
  • GST Gluthione-S-transferase
  • MBP Maltose binding protein
  • protease cleavage sites between two heterologously fused polypeptides are well known in the art.
  • the protein of the present invention it might also be beneficial to fuse the protein of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell
  • Signal peptides from diverse organisms are well known in the art.
  • Bacterial OmpA and yeast Suc2 are two non limiting examples of proteins containing signal sequences.
  • Such fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like.
  • sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like.
  • VDR sequence of the present invention should encode a functional (albeit defective) VDR. It will be clear to the person of ordinary skill that whether the VDR sequence of the present invention, variant, derivative, or fragment thereof retains its function, can be determined by using the teachings and assays of the present invention and the general teachings of the art
  • VDR protein of the present invention can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds.
  • some derivative or analogs having lost their biological function may still find utility, for example for raising antibodies
  • These antibodies could be used for detection or purification purposes.
  • these antibodies could also act as competitive or non-competitive inhibitor and be found to be modulators of the activity of the VDR protein of the present invention.
  • a host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e.g a DNA construct) when such DNA has been introduced inside the cell.
  • the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transfecting DNA may be maintained on a episomal element such as a plasmid.
  • a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents
  • the DNA segments or proteins according to the present invention could be introduced into individuals in a number of ways
  • cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and re troduced to the afflicted individual in a number of ways
  • the DNA construct can be administered directly to the afflicted individual
  • the DNA construct can also be delivered through a vehicle such as a posome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes
  • a vitamin D receptor gene having the genotype associated with low risk of breast cancer could be introduced in cells or in an individual displaying the VDR polymorphism associated with high risk of breast cancer
  • the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (i e DNA construct, protein, cells), the response and condition of the patient as well as the seventy of the disease
  • composition within the scope of the present invention should contain the active agent (i e molecule, hormone) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects
  • the nucleic acids in accordance with the present invention can be administered to mammals (i e humans) in doses ranging from 0 005 to 1 mg per kg of body weight per day of the mammal which is treated
  • Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed , Mack Ed.).
  • the amount administered should be chosen so as to avoid adverse side effects.
  • the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient Typically, 0 001 to 50 mg/kg/day will be administered to the mammal.
  • kits for assessing a predisposition to breast cancer comprising a determination of the genotype at the VDR locus (or a locus in linkage desiquilibrium therewith) using a nucleic acid fragment, a protein or a ligand, or a restriction enzyme in accordance with the present invention
  • a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers
  • Such containers include small glass containers, plastic containers or strips of plastic or paper
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another
  • Such containers will include in one particular embodiment a container which will accept the test sample (DNA protein or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products.
  • nucleic acid sequences, probes, primers, antibodies and the like of the present invention enabling a detection of a VDR polymorphism of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
  • VDR vitamin D receptor
  • VDR vitamin D receptor
  • Genomic DNA was isolated from peripheral blood leukocytes by a mini-method necessitating only 200 ⁇ l of whole blood where all steps are processed in a single 1 5 ml tube (Rousseau et al Hum Mut , 4 51-54, 1994) Isolated DNA (5-7 ⁇ g) was resuspended into 100 ⁇ l TE 20 5 buffer (20mM T ⁇ s, 5mM EDTA), heated at 65°C for 4 hours and stored at 4°C until PCR was performed VDR genotype analysis
  • VDR genotype was first assessed for Bsml, Apal and Taql polymorphisms as described by Mo ⁇ sson et al (Morrison et al , Nature, 367 284-287, 1994 and USP 5,593,033) after amplification of a single 2 3 kb fragment by polymerase chain reaction (PCR) spanning 3' end of exon 8 to 5' end of non-translated exon 9, using forward primer (P1) 5'-CAACCAAGAC TACAAGTACC GCGTCAGTGA-3' (SEQ ID NO 1)(Morr ⁇ son et al , Nature, 367 284-287, 1994) and reverse primer (P2) ⁇ '-TATCGTGAGT AAGGCAGGAG AGGGAGACC-3' (SEQ ID NO 2) PCR was carried out in a Perkm-Elmer 480 DNA thermal cyclerTM (Perkm-Elmer Corporation, Norwalk, CT) Genomic DNA (200 ng) was amplified through 35 cycles in 50 ⁇ l containing 1 ⁇
  • a C-to-T transition polymorphism in the VDR resulting in an initiation codon (ATG) three codons proximal to a downstream start site and causing a three amino acids difference in the VDR sequence was previously described (Saijo et al., Am J. Hum Genet. 48:668-673, 1991). This polymorphism creates a Fokl site and was also tested for 543 subjects. PCR and digestion was conducted as described by Gross et al. (Gross et al., J. Bone Miner. Res. 11.1850-1855, 1996) except that digestion was achieved with 1 U of Fokl and was allowed overnight to minimize partial digests.
  • VDR Receptor Gene Polymorphism of the VDR Receptor Gene as a Marker for Breast Cancer Susceptibility
  • Table 1 shows that women with certain combination of VDR gene Bsml polymorphism (bb genotype) have a significant but slightly increased risk (OR of 1.6) of developing breast cancer as compared to the category with the smallest risk (BB genotype). However, these women represent about 35% of the general population.
  • the Fokl genotype was not associated with a significantly increased risk of breast cancer as the proportion of cases and control was roughly the same for each Fokl genotype
  • the combined VDR genotypes (Bsml and Fokl) were studied , a striking association with the disease was observed (Table 2).
  • the odds ratio for breast cancer became extremely strong and significant when compared to the genotype combination that had the least number of cases (Table 2)
  • VDR genotypes associated with an increased susceptibility to breast cancer are the most frequent ones.
  • VDR BBFF and VDR BBFf genotypes could be seen as "protective”. Together, these two latter genotypes represent about 18% of the genotypes found in the population.

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Abstract

L'invention concerne une méthode de détermination de la prédisposition d'un individu au cancer du sein, du développement du cancer du sein et/ou de la réactivité au traitement du cancer du sein. Ledit procédé consiste à déterminer le génotype du récepteur de la vitamine D de l'individu, la prédisposition de l'individu au cancer du sein, le développement du cancer du sein et/ou la réactivité au traitement du cancer du sein.
EP99944189A 1998-09-15 1999-09-15 Marqueur au niveau du gene recepteur de la vitamine d, utilise pour la determination de la sensibilite au cancer du sein Withdrawn EP1114180A2 (fr)

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