WO2000058509A2 - Gene de fibronectine humaine associe au cancer de la prostate et marqueurs bialleliques - Google Patents

Gene de fibronectine humaine associe au cancer de la prostate et marqueurs bialleliques Download PDF

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WO2000058509A2
WO2000058509A2 PCT/IB2000/000431 IB0000431W WO0058509A2 WO 2000058509 A2 WO2000058509 A2 WO 2000058509A2 IB 0000431 W IB0000431 W IB 0000431W WO 0058509 A2 WO0058509 A2 WO 0058509A2
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polynucleotide
sequence
biallelic marker
ofthe
nucleotide
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WO2000058509A3 (fr
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Marta Blumenfeld
Lydie Bougueleret
Ilya Chumakov
Annick Cohen-Akenine
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Genset
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    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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

  • Prostate cancer is a latent disease. Many men carry prostate cancer cells without overt signs of disease. Autopsies of individuals dying of other causes show prostate cancer cells in 30 % of men at age 50 and in 60 % of men at age 80. Furthermore, prostate cancer can take up to 10 years to kill a patient after the initial diagnosis.
  • PSA Prostate Specific Antigen
  • Fibronectin is an adhesive glycoprotein known to promote the anchorage of cells to substrata and contains binding domains for heparin, fibrin, collagens, DNA and cellular receptors. Fibronectin is known to be important as a structural protein, migratory substrate, source of signaling information for regulation of cell morphology, cytoskeletal rearrangement and signal transduction. Recent studies have concentrated on the role oi Fibronectin in malignant disease, particularly on its involvement in inhibition of tumor cell lodgment, in metastasis and in mechanisms of integrin- mediated signaling. A tripeptide sequence in the 10th Type III repeat i Fibronectin, Arg-Gly-Asp (RGD) as well as other possibly synergistically acting regions which are bound by several different integtins have been identified.
  • RGD Arg-Gly-Asp
  • Fibronectin has an average relative molecular mass of 250 kilodaltons (kDa).
  • the primary structure oi Fibronectin is variable, composed of several different internal repeats known as Types I, II and III of lengths of about 40, 60 and 90 amino acids residues, respectively.
  • the various different Fibronectin molecules result from alternative splicing of the primary mRNA transcript in at least three regions, including the production or removal of type III modules, ED-A and ED-B or portions of the IIICS region. Kornblihtt et al. (1985) The presence of extra domain (ED) sequence in Fibronectin. for example, differentiates the two main types of fibronectins. soluble plasma fibronectins (pE/V).
  • Fibronectin subunits are disulfide-linked to form various dimers or multimers derived from nonidentical polypeptides leading to the formation of a large isomorphic protein family. Kornblihtt and Gutman, (1988); Ruoslahti, (1988).
  • the present invention stems from the isolation and characterization of the genomic sequence of the Fibronectin gene. Oligonucleotide probes and primers hybridizing specifically with a genomic sequence oi Fibronectin are also part of the invention.
  • a further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the regulatory region of Fibronectin or a sequence encoding the Fibronectin enzyme, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors.
  • the invention also encompasses methods of screening of molecules which modulate or inhibit the expression of the Fibronectin gene.
  • the invention is also directed to biallelic markers that are located within the Fibronectin genomic sequence or that are in linkage disequilibrium with the Fibronectin gene, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles oi Fibronectin gene and diseases such as cancer, or prostate cancer. These association methods are within the scope of the invention.
  • the present invention stems from the identification of genetic associations between alleles of biallelic markers of the Fibronectin gene and cancer , or prostate cancer, as confirmed and characterized in a panel of human subjects.
  • Methods and products are provided for the molecular detection of a genetic susceptibility to cancer, or prostate cancer, the level of aggressiveness of cancer, or prostate cancer tumors, an early onset of cancer, or prostate cancer, a beneficial response to or side effects related to treatment against cancer, or prostate cancer. They can be used for diagnosis, staging, prognosis, and monitoring of such a disease, which processes can be further included within treatment approaches.
  • the invention also provides for the efficient design and evaluation of suitable therapeutic solutions including individualized strategies for optimizing drug usage, and screening of potential new medicament candidates.
  • Figure 1 is a table demonstrating the results of a haplotype association analysis between sporadic prostate and haplotypes which consist of biallelic markers of the invention. In this haplotype analysis, 294 sporadic cases and 313 controls were considered.
  • Figure 2 is a table demonstrating the results of a haplotype association analysis between familial prostate and haplotypes which consist of biallelic markers of the invention. In this haplotype analysis, 197 familial cases and 313 controls were considered.
  • Figure 7 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.
  • SEQ ID No 1 contains a genomic sequence of the EN gene comprising the 5 " regulatory region (upstream untranscribed region), the exons and introns, and the 3' regulatory region (downstream untranscribed region).
  • S ⁇ Q ID No 28 contains a primer containing the additional PU 5" sequence described further in Example 2.
  • SEQ ID No 29 contains a primer containing the additional RP 5' sequence described further in Example 2.
  • the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each of the alleles present at the polymo ⁇ hic base.
  • the code -'r" in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine, while the other allele is an adenine.
  • the code "y” in the sequences indicates that one allele of the polymo ⁇ hic base is a thvmine, while the other allele is a cytosine.
  • the code "m " ' in the sequences indicates that one allele of the polymo ⁇ hic base is an adenine, while the other allele is an cytosine.
  • the code “k” in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine, while the other allele is a thvmine.
  • the code “s' " in the sequences indicates that one allele of the polymo ⁇ hic base is a guanine. while the other allele is a cytosine.
  • the code “w” in the sequences indicates that one allele of the polymo ⁇ hic base is an adenine, while the other allele is an thymine.
  • the nucleotide code of the original allele for each biallelic marker is the following table:
  • the polymo ⁇ hic bases of the biallelic markers alter the identity of an amino acids in the encoded polypeptide. This is indicated in the accompanying Sequence Listing by use of the feature VARIANT, placement of an Xaa at the position of the polymo ⁇ hic amino acid, and definition of Xaa as the two alternative amino acids.
  • the codon CAC which encodes histidine
  • CAA which encodes glutamine
  • the Sequence Listing for the encoded polypeptide will contain an Xaa at the location of the polymo ⁇ hic amino acid. In this instance, Xaa would be defined as being histidine or glutamine.
  • the present invention provides the genomic sequence of the EN gene and further provides biallelic markers derived from the EN locus.
  • the EN-related biallelic markers of the present invention offer the possibility of rapid, high throughput genotyping of a large number of individuals.
  • the biallelic markers of the present invention can be used in any method of genetic analysis including linkage studies in families, linkage disequilibrium studies in populations and association studies of case-control populations.
  • An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. As part of the present invention an association between alleles of EN-related biallelic markers and prostate cancer was established.
  • the term '"EN gene when used herein, encompasses genomic, mR ⁇ A and cD ⁇ A sequences encoding the Fibronectin protein, including the untranslated regulatory regions of the genomic D ⁇ A.
  • F ⁇ means "'Fibronectin” throughout the specification.
  • heterologous protein when used herein, is intended to designate any protein or polypeptide other than the Fibronectin protein. More particularly, the heterologous protein is a compound which can be used as a marker in further experiments with a Fibronectin regulatory region.
  • isolated requires that the material be removed from its original environment (e. g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • purified does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1 % concentration to 10 % concentration is two orders of magnitude.
  • purified is used herein to describe a polynucleotide or polynucleotide vector of the invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides.
  • a polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently close).
  • a substantially pure polynucleotide typically comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%. and preferably is over about 99% pure.
  • Polynucleotide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain pu ⁇ oses higher resolution can be provided by using HPLC or other means well known in the art.
  • non-human animal refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single- stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides. meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a pu ⁇ ne or pyrimidine. a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • a sequence which is "operably linked" to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the nucleic acid of interest.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • twin and phenotype are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example.
  • phenotype are used herein to refer to symptoms of. or susceptibility to cancer or prostate cancer, the level of aggressiveness of cancer or prostate cancer tumors, an early onset of cancer or prostate cancer, a beneficial response to or side effects related to treatment against cancer or prostate cancer.
  • allele is used herein to refer to variants of a nucleotide sequence.
  • a biallelic polymo ⁇ hism has two forms. Typically the first identified allele is designated as the original allele whereas other alleles are designated as alternative alleles. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • heterozygosity rate is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2P a (l-P a ), where P a is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
  • genotyp refers the identity of the alleles present in an individual or a sample.
  • a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample.
  • genotyping a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.
  • haplotype refers to a combination of alleles present in an individual or a sample.
  • a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.
  • polymo ⁇ hism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals.
  • Polymo ⁇ hic refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a “polymo ⁇ hic site” is the locus at which the variation occurs.
  • a single nucleotide polymo ⁇ hism is the replacement of one nucleotide by another nucleotide at the polymo ⁇ hic site. Deletion of a single nucleotide or insertion of a single nucleotide also gives rise to single nucleotide polymo ⁇ hisms.
  • single nucleotide polymo ⁇ hism preferably refers to a single nucleotide substitution.
  • the polymo ⁇ hic site may be occupied by two different nucleotides.
  • biaselic polymo ⁇ hism and “biallelic marker” are used interchangeably herein to refer to a polymo ⁇ hism, usually a single nucleotide, having two alleles at a fairly high frequency in the population.
  • a “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site.
  • the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42).
  • a biallelic marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker".
  • the polymorphism, allele or biallelic marker is "at the center" of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymo ⁇ hism and the 3 " end of the polynucleotide. and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5' end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be "within 1 nucleotide of the center.” If the difference is 0 to 5.
  • the polymo ⁇ hism is considered to be "within 2 nucleotides of the center.” If the difference is 0 to 7, the polymo ⁇ hism is considered to be "within 3 nucleotides of the center.” and so on.
  • the terms "complementary” or “complement thereof are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. For the pu ⁇ ose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base.
  • the invention also relates to variants and fragments of the polynucleotides described herein, particularly of a E/Vgene containing one or more biallelic markers according to the invention.
  • Variants of polynucleotides are polynucleotides that differ from a reference polynucleotide.
  • a variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.
  • Variants of polynucleotides according to the invention include, without being limited to. 5 nucleotide sequences which are at least 95% identical to a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID No 1 or to any polynucleotide fragment of at least 12, 15, 18, 20, 25. 30. 50. 80, 100, 150.200.250, 300, 350, 400, 450. 500.
  • a polynucleotide fragment is a polynucleotide having a sequence that is entirely the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a EN gene, and variants thereof.
  • the fragment can be a portion of an intron of a EN gene. It can also be a portion of the regulatory regions of EN.
  • such fragments comprise at least one of the biallelic 30 markers A1-A78 or the complements thereto.
  • BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • the PAM or PAM250 matrices may also be used (see. e.g., Schwartz and Dayhoff. eds., 1978).
  • the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology.
  • a user-specified threshold of significance such as a user- specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g.. Karlin and Altschul. 1990).
  • the stringent hybridization conditions are the followings : the hybridization step is realized at 65°C in the presence of 6 x SSC buffer. 5 x Denhardt's solution. 0,5% SDS and lOO ⁇ g ml of salmon sperm DNA.
  • the hybridization step is followed by four washing steps : - two washings during 5 min, preferably at 65°C in a 2 x SSC and 0.1%SDS buffer;
  • hybridization conditions being suitable for a nucleic acid molecule of about 20 nucleotides in length.
  • hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art.
  • the suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985).
  • nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25. 30, 35, 40. 50, 60, 70. 80. 90, 100. 150, 200. 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous
  • the EN genomic nucleic acid comprises exons 1 to 47 of S ⁇ Q ID No 1 as listed below on Table A.
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of exons 1 to 47 of the EN gene, or a sequence complementary thereto.
  • the invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the EN gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5 " -end to the 3'-end of said nucleic
  • Intron 1 refers to the nucleotide sequence located between ⁇ xon 1 and ⁇ xon 2, and so on. The position of the introns is detailed in Table A.
  • the invention embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 46 introns of the EN gene, or a sequence complementary thereto.
  • the invention also concerns the polypeptide encoded by the nucleotide sequence of S ⁇ Q ID No 1 , or a fragment thereof or a complementary sequence thereto.
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of EN on either side or between two or more such genomic sequences.
  • the genomic sequence of the EN gene contains regulatory sequences both in the non-coding 5 '-flanking region and in the non-coding 3 '-flanking region that border the EN coding region containing the three exons of this gene.
  • the 5 '-regulatory sequence of the EN gene is localized between the nucleotide in position 1 and the nucleotide in position 2000 of the nucleotide sequence of S ⁇ Q ID No 1. This polynucleotide contains the promoter site.
  • the 3 '-regulatory sequence of the EN gene is localized between nucleotide position 76926 and nucleotide position 78925 of S ⁇ Q ID No 1.
  • the promoter activity of the 5' regulatory regions contained in EN can be assessed as described below.
  • each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, ⁇ galactosidase, or green fluorescent protein.
  • the sequences upstream the EN coding region are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell.
  • the resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity, such as described, for example, by Coles et al.( 1998), the disclosure of which is incorporated herein by reference in its entirety. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. This type of assay is well-known to those skilled in the art and is described in WO 97/17359, US Patent No.
  • the strength and the specificity of the promoter of the EN gene can be assessed through the expression levels of a detectable polynucleotide operably linked to the EN promoter in different types of cells and tissues.
  • the detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including a EN polypeptide or a fragment or a variant thereof.
  • the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof.
  • the invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, advantageously 99 % nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
  • Preferred fragments of the 5 " regulatory region have a length of about 1 00 or 1000 nucleotides, preferably of about 500 nucleotides, more preferably about 400 nucleotides. even more preferably 300 nucleotides and most preferably about 200 nucleotides.
  • Preferred fragments of the 3 ' regulatory region are at least 50, 100, 150, 200, 300 or 400 bases in length.
  • Bioly active polynucleotide derivatives of SEQ ID No 1 are polynucleotides comprising or alternatively consisting in a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. It could act either as an enhancer or as a repressor.
  • the regulatory polynucleotides of the invention may be prepared from the nucleotide sequence of SEQ ID No 1 by cleavage using suitable restriction enzymes, as described for example in the book of Sambrook et al.(1989).
  • the regulatory polynucleotides may also be prepared by digestion of SEQ ID No 1 by an exonuclease enzyme, such as Bal31 (Wabiko et al., 1986).
  • exonuclease enzyme such as Bal31 (Wabiko et al., 1986).
  • These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification.
  • the regulatory polynucleotides according to the invention may be part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
  • the recombinant expression vectors according to the invention are described elsewhere in the specification.
  • a further object of the invention consists of a purified or isolated nucleic acid comprising: a) a nucleic acid comprising a regulatory nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence comprising a polynucleotide of the 5 " regulatory region or a complementary sequence thereto;
  • nucleic acid comprising a 3'- regulatory polynucleotide. preferably a 3'- regulatory' polynucleotide of the EN gene.
  • the desired polypeptide encoded by the above-described nucleic acid may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin.
  • proteins of prokaryotic or eukaryotic origin include bacterial, fungal or viral antigens.
  • eukaryotic proteins such as intracellular proteins, like "house keeping” proteins, membrane-bound proteins, like receptors, and secreted proteins like endogenous mediators such as cytokines.
  • the desired polypeptide may be the F ⁇ protein, especially the protein of the amino acid sequence of S ⁇ Q ID No 3, or a fragment or a variant thereof.
  • the desired nucleic acids encoded by the above-described polynucleotide may be complementary to a desired coding polynucleotide, for example to the EN coding sequence, and thus useful as an antisense polynucleotide.
  • Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism.
  • Suitable recombinant vectors that contain a polynucleotide such as described herein are disclosed elsewhere in the specification.
  • the cDNA of SEQ ID No 2 includes a 3"-UTR region starting from the nucleotide at position 7347 and ending at the nucleotide at position 8039 of SEQ ID No 2.
  • the polyadenylation site starts from the nucleotide at position 5 8017 and ends at the nucleotide in position 8022 of SEQ ID No 2.
  • the invention concerns a purified, isolated, and recombinant nucleic acid comprising a nucleotide sequence of the 5'UTR or the 3' UTR of the ENcDNA, a sequence complementary thereto, or an allelic variant thereof.
  • nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of EN on either side or between two or more such genomic sequences. 5 Coding Regions
  • the EN open reading frame is contained in the corresponding mR ⁇ A of S ⁇ Q ID No 2. More precisely, the effective EN coding sequence (CDS) includes the region between nucleotide position 6 (first nucleotide of the ATG codon) and nucleotide position 7346 (end nucleotide of the TGA codon) of S ⁇ Q ID No 2.
  • CDS effective EN coding sequence
  • the present invention also embodies isolated, purified, and 0 recombinant polynucleotides which encode a polypeptide of S ⁇ Q ID No 3.
  • the invention concerns the polypeptide encoded by a nucleotide sequence selected from the group consisting of S ⁇ Q ID No 1 or 2, a complementary sequence thereof or a fragment thereto.
  • Polynucleotide constructs and “recombinant polynucleotide” are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as 5 contiguous nucleotide sequences in their initial natural environment.
  • DNA Construct That Enables Directing Temporal And Spatial Fibronectin Gene Expression In Recombinant Cell Hosts And In Transgenic Animals.
  • the invention also encompasses D ⁇ A constructs and recombinant vectors enabling a conditional expression of the EN genomic sequence or of a specific allele of the EN genomic sequence and also of a copy of this genomic sequence harboring substitutions, deletions, or additions of one or more bases as regards to the EN nucleotide sequences of S ⁇ Q ID ⁇ os 1 and 2, or a fragment thereof, these base substitutions, deletions or additions being located either in an exon, an intron or a regulatory sequence, but preferably in an exon of the EN genomic sequence.
  • the EN sequence comprises a biallelic marker of the present invention.
  • the EN sequence comprises one of the biallelic markers selected from the group consisting of Al to A19 and A31 to a64.
  • the present invention embodies recombinant vectors comprising any one of the polynucleotides described in the present invention. More particularly, the polynucleotide constructs according to the present invention can comprise any of the polynucleotides described in the "Genomic Sequences Of The Human Fibronectin Gene” section, the "ENcDNA Sequences” section, the “Coding Regions” section, and the "Oligonucleotide Probes And Primers" section.
  • a first preferred D ⁇ A construct is based on the tetracycline resistance operon tet from E. coli transposon Tn 10 for controlling the EN gene expression, such as described by Gossen et al.(1992.
  • Such a D ⁇ A construct contains seven tet operator sequences from TnlO (terop) that are fused to either a minimal promoter or a 5'-regulatory sequence of the EN gene, said minimal promoter or said EN regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide. including a EN nucleotide sequence, or for a polypeptide, including a EN polypeptide or a peptide fragment thereof.
  • This D ⁇ A construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of he ⁇ es simplex virus, placed under the control of a promoter, such as the HCMVI ⁇ 1 enhancer/promoter or the MMTV-LTR.
  • a preferred D ⁇ A construct of the invention comprise both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.
  • conditional expression D ⁇ A construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.
  • a second preferred DNA construct will comprise, from 5 " -end to 3 '-end: (a) a first nucleotide sequence that is comprised in the EN genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a 5 second nucleotide sequence that is comprised in the EN genomic sequence, and is located on the genome downstream the first EN nucleotide sequence (a).
  • this D ⁇ A construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c).
  • the negative selection marker consists of the thymidine kinase (tk) gene (Thomas et al.,
  • the positive selection marker is located within a EN exon sequence so as to interrupt the sequence encoding a EN protein.
  • the first and second nucleotide sequences (a) and (c) may be indifferently located within a
  • EN regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.
  • the PI phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs lox? site.
  • the lox? site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the Cre enzyme between two
  • the excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host.
  • the recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al.(1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al.(1993); (b) transfecting
  • the vector containing the sequence to be inserted in the EN gene by homologous recombination is constructed in such a way that selectable markers are flanked by lox? sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the EN sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-lox? system are described by Zou et al.(1994).
  • a third preferred D ⁇ A construct of the invention comprises, from 5 '-end to 3 '-end: (a) a first nucleotide sequence that is comprised in the EN genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a lox? site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the EN genomic sequence, and is located on the genome downstream of the first EN nucleotide sequence (a).
  • the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).
  • a promoter sequence preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).
  • DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a EN genomic sequence or a EN cD ⁇ A sequence, and most preferably an altered copy of a EN genomic or cD ⁇ A sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knockout homologous recombination) or to the replacement of a copy of the targeted gene by another copy
  • the D ⁇ A constructs described above may be used to introduce a EN genomic sequence or a EN cD ⁇ A sequence comprising at least one biallelic marker of the present invention, preferably at least one biallelic marker selected from the group consisting of Al to A19 and A31 to a64.
  • 20 recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50. 60. 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of S ⁇ Q ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1 , 2, 3, 5. or 10 of the nucleotide sequences of the following nucleotide positions of S ⁇ Q ID No 1 : 1-2000. 2154-2986. 31 16-4350. 4489-5847. 5980-6959. 7098-9473, 9633-12525, 12718-13485, 13666-14285, 14463-15574. 15728-17016,
  • probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60. 70, 80. 90, 100, 150, 200. 500, or 1000 nucleotides of S ⁇ Q ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide sequences of the following nucleotide positions of S ⁇ Q ID No 1 : 1-2000, 2154-2986,
  • the invention also relates to nucleic acid probes characterized in that they hybridize
  • Preferred probes and primers of the invention include isolated, purified, or recombinant
  • 20 polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25. 30, 35. 40, 50, 60, 70, 80, 90. 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises a nucleotide selected in the group consisting of a A at the position 49, a T at the position 383, a A at the position 2447, a C at the position 31 16. and a C at the position 3161 of SEQ ID No 2.
  • Tm melting temperature
  • Tm depends on the length of the primer or probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two.
  • the GC content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %,
  • a preferred probe or primer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of PI to P75 and the sequences complementary thereto.
  • a probe according to the invention consists of a nucleic acid comprising a biallelic marker selected from the group consisting of Al to A78 or the complements thereto, for which the respective locations in the sequence listing are provided in Table 2.
  • the invention also relates to a purified and/or isolated nucleotide sequence comprising a polymo ⁇ hic base of a EN-related biallelic marker, preferably of a biallelic marker selected from the group consisting of Al to A78, and the complements thereof.
  • the sequence has between 8 and 1000 nucleotides in length, and preferably comprises at least 8. 10. 12, 15. 18. 20, 25, 35. 40. 50, 60, 70, 80. 100, 250. 500 or 1000 contiguous nucleotides.
  • the invention encompasses isolated, purified, and recombinant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of S ⁇ Q ID ⁇ os 1, 2, or 4 to 27 and the complement thereof, wherein said span includes a EN-related biallelic marker in said sequence; optionally, wherein said EN-related biallelic marker is selected from the group consisting of Al to A78, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of Al to A19 and A31 to A64.
  • said EN-related biallelic marker is selected from the group consisting of A20 to A30 and A64 to A78: optionally, wherein said EN-related biallelic marker is selected from the group consisting of A3, A5, A4. A6, A21, A22, and A28, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A 19, A25, and A29, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.
  • nucleotide sequences comprise the polymo ⁇ hic base of either allele 1 or allele 2 of the considered biallelic marker.
  • said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide; optionally, wherein said contiguous span is 18 to 35 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein the 3' end of said contiguous span is present at the 3' end of said polynucleotide; and optionally, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said bialle
  • said polynucleotide may further comprise a label.
  • said polynucleotide can be attached to solid support.
  • the polynucleotides defined above can be used alone or in any combination.
  • said probes comprises, consists of, or consists essentially of a sequence selected from the following sequences: PI to P75 and the complementary sequences thereto.
  • the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos. 1 , 2, or 4 to 27 or the complements thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100.
  • EN-related biallelic marker is selected from the group consisting of Al to A78, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of Al to A19 and A31 to A64, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A20 to A30 and A64 to A78; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A3, A5, A4, A6, A21, A22, and A28, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A3, A5, A4, A6, A21, A22, and A28, and the complements thereof, or optionally the biallelic markers in link
  • polynucleotide A25, and A29, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said EN-related biallelic marker in said sequence; and optionally, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: Dl to D75 and ⁇ l to ⁇ 75.
  • said polynucleotide may further comprise a label.
  • said polynucleotide can be attached to solid support.
  • the polynucleotides defined above can be used alone or in any combination.
  • said E/V-related biallelic marker is selected from the group consisting of Al to A78. and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of Al to A19 and A31 to A64. and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A20 to A30 and A64 to A78; optionally, wherein said EN-related biallelic marker is selected from the group consisting of A3. A5. A4. A6, A21 , A22.
  • said polynucleotide may be attached to a solid support, array, or addressable array;
  • said polynucleotide may be labeled.
  • the primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of ⁇ arang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described in ⁇ P 0 707 592.
  • a method such as the phosphodiester method of ⁇ arang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described in ⁇ P 0 707 592.
  • the disclosures of all these documents are inco ⁇ orated herein by reference.
  • the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
  • the 3' hydroxyl group simply can be cleaved, replaced or modified,
  • U.S. Patent Application Serial No. 07/049.061 filed April 19. 1993 describes modifications, which can be used to render a probe non-extendable.
  • Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic. photochemical, biochemical, immunochemical. or chemical means.
  • useful labels include radioactive substances ( j2 P, 3:> S, ⁇ , 12:> I), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene. digoxigenin) or biotin.
  • radioactive substances j2 P, 3:> S, ⁇ , 12:> I
  • fluorescent dyes 5-bromodesoxyuridin, fluorescein, acetylaminofluorene. digoxigenin
  • biotin preferably, polynucleotides are labeled at their 3 " and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988).
  • the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent No. EP 0 225 807 (Chiron).
  • the invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID No 1 and a fragment or a variant thereof and a complementary sequence thereto in a sample, said kit comprising: a) a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID No 1 and a fragment or a variant thereof and a complementary sequence thereto; b) optionally, the reagents necessary for performing the hybridization reaction.
  • the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting of the nucleotide sequences of PI to P50 and the complementary sequence thereto, B l to B37, Cl to C37, Dl to D50, El to E50, or a biallelic marker selected from the group consisting of Al to A19 and A31 to A64 and the complements thereof.
  • an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in the EN gene and preferably in its regulatory region.
  • an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the EN gene.
  • 4L tiled array is implemented a set of four probes (A. C. G, T), preferably
  • the invention concerns an array of nucleic acid molecules comprising at least one polynucleotide described above as probes and primers.
  • the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers.
  • the invention also pertains to an array of nucleic acid sequences comprising either at least two of the sequences selected from the group consisting of PI to P75, Bl to B61, Cl to C61, Dl to D75, El to E75 or the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least two sequences comprising a biallelic marker selected from the group
  • biallelic markers of the present invention are disclosed in Table 2. They are described by location in the EN gene in Table 2 and in S ⁇ Q ID No 1 and as a single base polymorphism in the features of the related S ⁇ Q ID Nos 4 to 27.
  • the pairs of primers allowing the amplification of a nucleic acid containing the poiymo ⁇ hic base of one EN biallelic marker are listed in Table 1 of Example 2.
  • the present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention with a portion or all of the necessary reagents and instructions for 5 genotyping a test subject by determining the identity of a nucleotide at a EN-related biallelic marker.
  • the polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides.
  • the kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an 0 enzyme-based mismatch detection method.
  • test samples include biological samples, which can be tested by the methods ofthe present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions ofthe respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens.
  • the preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.
  • DNA samples can be pooled or unpooled for the amplification step.
  • DNA amplification techniques are well known to those skilled in the art.
  • Amplification techniques that can be used in the context ofthe present invention include, but are not limited to, the ligase chain reaction (LCR) described in EP-A- 320 308.
  • LCR ligase chain reaction
  • WO 9320227 and EP-A-439 182 the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli J.C., et al.(1990) and in Compton J.(1991), Q-beta amplification as described in European Patent Application No 4544610, strand displacement amplification as described in Walker et al.(1996) and EP A 684 315 and. target mediated amplification as described in PCT Publication WO 9322461.
  • NASBA nucleic acid sequence based amplification
  • NASBA nucleic acid sequence based amplification
  • NASBA nucleic acid sequence based amplification
  • NASBA nucleic acid sequence based
  • RT-PCR polymerase chain reaction
  • AGLCR is a modification of GLCR that allows the amplification of RNA.
  • the PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White ( 1997) and the publication entitled “PCR Methods and Applications” (1991. Cold Spring Harbor Laboratory Press).
  • PCR primers on either side ofthe nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase. Pfu polymerase, or Vent polymerase.
  • the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
  • the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
  • PCR has further been described in several patents including US Patents 4,683,195: 4,683, 202; and 4.965,188. Each of these publications is inco ⁇ orated herein by reference.
  • the PCR technology is the preferred amplification technique used to identify new biallelic markers.
  • a typical example of a PCR reaction suitable for the pu ⁇ oses of the present invention is provided in Example 2.
  • One of the aspects ofthe present invention is a method for the amplification of the human
  • EN gene particularly ofthe genomic sequences of S ⁇ Q ID No 1, or a fragment or a variant thereof in a test sample, preferably using the PCR technology.
  • This method comprises the steps of contacting a test sample suspected of containing the target EN encoding sequence or portion thereof with amplification reaction reagents comprising a pair of amplification primers, and eventually in some instances a detection probe that can hybridize with an internal region of amplicon sequences to confirm that the desired amplification reaction has taken place.
  • the present invention also relates to a method for the amplification of a human EN gene sequence, particularly of a portion of the genomic sequences of S ⁇ Q ID No 1 or a variant thereof in a test sample, said method comprising the steps of: a) contacting a test sample suspected of containing the targeted EN gene sequence comprised in a nucleotide sequence selected from a group consisting of S ⁇ Q ID No 1 and fragments or variants thereof with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side ofthe polynucleotide region to be amplified, and b) optionally, detecting the amplification products.
  • the amplification products generated as described above, are then sequenced using any method known and available to the skilled technician.
  • Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989).
  • Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al.(1996).
  • the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol.
  • the products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis.
  • the polymo ⁇ hism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymo ⁇ hic sequence, the polymo ⁇ hism has to be detected on both strands.
  • the above procedure permits those amplification products, which contain biallelic markers to be identified.
  • the detection limit for the frequency of biallelic polymo ⁇ hisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies.
  • more than 90% ofthe biallelic polymo ⁇ hisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele.
  • more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele thus a heterozygosity rate higher than 0.18, preferably higher than 0.32. more preferably higher than 0.42.
  • the determination ofthe least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative ofthe population as
  • the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals.
  • the larger the group the greater the accuracy ofthe frequency determination because of reduced sampling error.
  • determining the frequency of a biallelic marker allele in a population may be accomplished by determining the
  • determining the proportional representation may be accomplished by performing a genotyping method of the invention on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.
  • Methods well-known to those skilled in the art that can be used to detect biallelic polymo ⁇ hisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al.(1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.(1991 ), White et al.(1992), Grompe et al.(1989 and 1993).
  • Another method for determining the identity ofthe nucleotide present at a particular polymo ⁇ hic site employs a specialized exonuclease-resistant nucleotide derivative as described in US Patent 4,656,127.
  • a homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al.(1997).
  • amplified genomic DNA fragments containing polymo ⁇ hic sites are incubated with a 5'-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase.
  • the dye- labeled primer is extended one base by the dye-terminator specific for the allele present on the template.
  • the fluorescence intensities ofthe two dyes in the reaction mixture are analyzed directly without separation or purification.
  • reporter- detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993). or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712).
  • DNP dinitrophenyl
  • biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate WO 92/15712
  • Nyren et al.(1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).
  • ELIDA enzymatic luminometric inorganic pyrophosphate detection assay
  • One aspect ofthe present invention is a solid support which includes one or more microsequencing primers listed in Example 4, or fragments comprising at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides thereof, to the extent that such lengths are consistent with the primer described, and having a 3' terminus immediately upstream ofthe corresponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.
  • Allelic frequencies ofthe biallelic markers in a populations can be determined using one of the methods described above under the heading "Methods for genotyping an individual for biallelic markers", or any genotyping procedure suitable for this intended pu ⁇ ose.
  • Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population.
  • One way to reduce the number of genotypings required is to use pooled samples.
  • a major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools.
  • Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and: is the preferred method used in the present invention.
  • each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.
  • the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognized haplotypes. For each positive identification, the complementary haplotype is added to the list of recognized haplotypes, until the phase information
  • Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R.S. et al.. 1997). 35 Biallelic markers, because they are densely spaced in the human genome and can be genotyped in greater numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium.
  • linkage disequilibrium the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium.
  • Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls.
  • Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.
  • a first group of between 50 and 300 trait positive individuals preferably about 100 individuals, are recruited according to their phenotypes. A similar number of control individuals are included in such studies.
  • typical examples of inclusion criteria include prostate cancer.
  • the general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies ofthe biallelic markers ofthe present invention in both groups.
  • association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and control populations. In a second phase ofthe analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as is the case for EN, a single phase may be sufficient to establish significant associations.
  • a haplotype frequency analysis the frequency ofthe possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined.
  • the haplotype frequency is then compared for distinct populations of trait positive and control individuals.
  • the number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study.
  • the results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.
  • the biallelic markers ofthe present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions.
  • the analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein.
  • the analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis.
  • Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.
  • TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spiel ann S. et al., 1993; Schaid DJ. et al., 1996, Spielmann S. and Ewens W.J., 1998). Such combined tests generally reduce the false - positive errors produced by separate analyses.
  • haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., 1997; Weir, B.S.. 1996). Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation- Maximization (EM) algorithm (see Dempster et al., 1977; Excoffier L. and Slatkin M.. 1995).
  • EM Expectation- Maximization
  • This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown.
  • Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., 1994) or the Arlequin program (Schneider et al., 1997).
  • the EM algorithm is a generalized iterative maximum likelihood approach to estimation and is briefly described below.
  • Equation 2 The successive steps ofthe E-M algorithm can be described as follows: Starting with initial values ofthe of haplotypes frequencies, noted p ⁇ 0 , p , > # ⁇ these initial values serve to estimate the genotype frequencies (Expectation step) and then estimate another set of haplotype frequencies (Maximization step), noted ?, , p 2 , p H ( , these two steps are iterated until changes in the sets of haplotypes frequency are very small.
  • a stop criterion can be that the maximum difference between haplotype frequencies between two iterations is less than 10 " ' .
  • genotype / ' occurs in phenotypey. and where h k and h / constitute genotype /.
  • Each probability is derived according to eq. 1, and eq. 2 described above.
  • ⁇ l
  • linkage disequilibrium between any two genetic positions
  • linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.
  • Linkage disequilibrium between any pair of biallelic markers comprising at least one ofthe biallelic markers ofthe present invention (M Thread M j ) having alleles (a,/b,) at marker M, and alleles (a/b j ) at marker M j can be calculated for every allele combination (asourceda j a supplementb j b usagea, and b benefitb,), according to the Piazza formula:
  • MLE maximum- likelihood estimate
  • ni ⁇ phenotype (a,/adire a a ⁇ )
  • n 2 ⁇ phenotype (a,/adire a b j )
  • n-, ⁇ phenotype (a,/bbri a/a,)
  • n4 ⁇ phenotype (a,/b dislike a/b,) and N is the number of individuals in the sample.
  • This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype. data are available.
  • Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, M, (a/b,) and M, (a/b,), fitting the Hardy- Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above. The estimation of gametic disequilibrium between ai and aj is simply:
  • D aiaj pr(haplotype(a ⁇ ,a - pr(a t ).pr(aj ).
  • pr(aj is the probability of allele a
  • pr(- is the probability of allele ⁇ and where pr(haplotype (arada aj) is estimated as in Equation 3 above.
  • D' a j D a ⁇ aj / max (-pr(a,). pr(a j ) , -pr(b,). pr(b,)) with D a ⁇ aj ⁇ 0
  • D'auy E>a.a j / max (pr(b,). pr(aj) . pr(a,). pr(b,)) with D a ⁇ aj >0
  • Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200. more preferably around 100.
  • Testing for Association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study.
  • a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study.
  • Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used.
  • the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).
  • the p value related to a biallelic marker association is preferably about 1 x 10 "2 or less, more preferably about 1 x 10 "4 or less, for a single biallelic marker analysis and about 1 x 10 "3 or less, still more preferably 1 x 10 "6 or less and most preferably of about 1 x 10 "8 or less, for a haplotype analysis involving two or more markers.
  • Phenotypic Permutation In order to confirm the statistical significance ofthe first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomized with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype ofthe first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination ofthe probability to obtain by chance the tested haplotype.
  • a risk factor in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci
  • F + is the frequency of the exposure to the risk factor in cases and F " is the frequency of the exposure to the risk factor in controls.
  • F ⁇ and F " are calculated using the allelic or haplotype frequencies ofthe study and further depend on the underlying genetic model (dominant, recessive, additive).
  • AR Attributable risk
  • AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype.
  • P ⁇ is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.
  • Example 5 An association between the EV gene and prostate cancer was established. Further details concerning this association study are provided in Example 5. results are briefly summarized below. Two groups of independent individuals were used in this association study in accordance with the invention. The two groups corresponded to 491 affected individuals and 313 control individuals. The affected populations may be subdivided in familial cases and sporadic cases. Other subdivision can be done regarding the diagnosis age of prostate cancer and their familial antecedent of the disease. In the association study described in Example 5, number of biallelic marker haplotypes were shown to be significantly associated with prostate cancer.
  • a first preferred haplotype (HAP1 of Figure 1 or haplotype 3 of Figure 3) consisting of two biallelic markers (99-5595/380 (A19) and 99-5596-197 (A29)) presented a p-value of l . lxl 0 "9 and an odd-ratio of 22 for the sporadic prostate cancer.
  • This haplotype is significant with sporadic prostate cancer, and more significant with sporadic cases under 65 years old.
  • haplotypes 3 and 4 of Figure 3 A second preferred haplotype (HAP8 of Figure 1 or haplotype 4 of Figure 4) consisting of two biallelic markers (99-23437/347 (A25) and 99-5596-197 (A29)) had a p-value of 2.6x10 "7 and an odd ratio of 3.15 with informative sporadic cases. Phenotypic permutation tests confirmed the statistical significance of these results.
  • haplotypes haplotypes 3 and 4 of Figure 3
  • the invention concerns the haplotypes associated with sporadic prostate cancer comprising at least two biallelic markers selected from the group consisting of 99-5595/380 (A19), 99- 23437/347 (A25) and 99-5596-197 (A29).
  • the invention concerns haplotypes associated with spoaradic prostate cancer which comprises the biallelic marker 99-5596-197 (A29).
  • a third preferred haplotype consisting of three biallelic markers (99-5604/376 (A5), 99-23460/199 (A21) and 99-5590/99 (A28)) presented a p-value of 3.7xl0°and an odd-ratio of 2.32 with prostate cancer familial cases.
  • Another preferred haplotype consisting of three biallelic markers (99-5605/90 (A3), 99-23460/199 (A21) and 99-5590/99 (A28)) presented a p-value of 2.1xl0 "5 and an odd-ratio of 2.43 with prostate cancer familial cases.
  • a fourth preferred haplotype (HAP24 of Figure 2 or haplotype 2 of Figure 3) consisting of four biallelic markers (99-23452/306 (A4), 99-23440/274 (A6), 99-15798/86 (A22) and 99-5590/99 (A28)) presented a p-value of lxlO "6 and an odd-ratio of 2.73 with prostate cancer familial cases.
  • haplotypes (haplotypes 1 and 2 of Figure 3) can therefore be considered to be highly significantly associated with prostate cancer, and more particularly familial prostate cancer.
  • the invention concerns the haplotypes associated with familial prostate cancer comp ⁇ sing at least three biallelic markers selected from the group consisting of 99-5605/90 (A3), 99-5604/376 (A5). 99-23460/199 (A21 ), 99-23452/306 (A4). 99-23440/274 (A6), 99- 15798/86 (A22). and 99- 5590/99 (A28)
  • This information is extremely valuable
  • the knowledge ot a potential genetic predisposition to prostate cancer, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy of prostate cancer and to the development of new therapeutic and diagnostic tools
  • the present invention then also concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers A l to A78 and which are expected to present similar characteristics in terms of their respective association with a given trait
  • the invention concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers A 19, A25, and A29
  • the invention concerns biallelic markers which are in linkage disequilibrium with the specific biallelic markers A3.
  • Mutations in the FN gene which are responsible for a detectable phenotype or trait may be identified by comparing the sequences ofthe EN gene from trait positive and control individuals Once a positive association is confirmed with a biallelic marker of the present invention, the identified locus can be scanned for mutations In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions ofthe EN gene are scanned for mutations In a preferred embodiment the sequence of the EN gene is compared in trait positive and control individuals Preferably, trait positive individuals carry the haplotype shown to be associated with the trait and trait negative individuals do not carry the haplotype or allele associated with the trait
  • the detectable trait or phenotype may comprise a variety of manifestations of altered EN function, including susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer
  • the mutation detection procedure is essentially similar to that used for biallelic marker identification
  • the method used to detect such mutations generally comprises the following steps
  • said biallelic marker is selected from the group consisting of A l to A78. and the complements thereof In a preferred embodiment, said biallelic marker is selected from the group consisting of A l 9 A25. and A29 and the complements thereof In a preferred embodiment said biallelic marker is selected from the group consisting of A3, A5, A4, A6. A21. A22. and A28.
  • candidate polymorphisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format
  • Polymo ⁇ hisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results
  • Polymo ⁇ hisms are considered as candidate "trait-causing " mutations when they exhibit a statistically significant correlation with the detectable phenotype
  • the biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time
  • the trait analyzed using the present diagnostics may be any detectable trait, including susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer Such a diagnosis can be useful in the staging, monitoring, prognosis and/or prophylactic or curative therapy of prostate cancer
  • the diagnostic techniques of the present invention may employ a variety of methodologies to determine whether a test subject has a biallelic marker pattern associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm
  • nucleic acid sample from the individual and determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype. indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular EN polymo ⁇ hism or mutation (trait-causing allele)
  • a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described above in "Methods Of Genotyping D ⁇ A Samples For Biallelic markers
  • the diagnostics may be based on a single biallelic marker or a on group of biallelic markers
  • a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more ofthe biallelic markers Al to A78 is determined
  • a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymorphisms associated with a detectable phenotype have been identified
  • the amplification products are sequenced to determine whether the individual possesses one or more EN polymo ⁇ hisms associated with a detectable phenotype
  • the primers used to generate amplification products may comprise the primers listed in Table 1
  • the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more EN polymo ⁇ hisms associated with a detectable phenotype resulting from a mutation or a polymo ⁇ hism in the EN gene
  • the primers used in the microsequencing reactions may include the primers listed in Table 3
  • the nucleic acid sample is contacted with one or more allele specific
  • the probes used in the hybridization assay may include the probes listed in Table 2
  • the nucleic acid sample is contacted with a second EN oligonucleotide capable of producing an amplification product when used with the allele specific oligonucleotide in an amplification reaction
  • the presence of an amplification product in the amplification reaction indicates that the individual possesses one or more EN alleles associated with a detectable phenotype
  • the identity of the nucleotide present at. at least one biallelic marker selected from the group consisting of A l to A78 and the complements thereof, preferably A 19, A25. and A29. and the complements thereof, is determined and the detectable trait is cancer, more preferably prostate cancer, more particularly sporadic prostate cancer
  • the identity of the nucleotide present at. at least one. biallelic marker selected from the group consisting of Al to A78 and the complements thereof, preferably A3. A5. A4. A6. A21. A22, and A28, and the complements thereof, is determined and the detectable trait is cancer, more preferably prostate cancer, more particularly familial prostate cancer Diagnostic kits comprise any of the polynucleotides ofthe present invention
  • Diagnostics which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects
  • Clinical drug trials represent another application for the markers of the present invention
  • One or more markers indicative of response to an agent acting against prostate cancer or to side effects to an agent acting against prostate cancer may be identified using the methods described above Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result ofthe inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems
  • the invention also concerns a method for the treatment of cancer or prostate cancer comprising the following steps - selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably EN-related markers, associated with cancer or prostate cancer:
  • said biallelic marker is selected from the group consisting of Al to A78. and the complements thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A 19, A25, and A29. and the complements thereof, more preferably the biallelic marker A7 and the complement thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A3, A5, A4. A6. A21. A22. and A28, and the complements thereof.
  • the prophylactic administration of a treatment serves to prevent, attenuate or inhibit the growth of cancer cells.
  • Another embodiment of the present invention consists of a method for the treatment of cancer or prostate cancer comprising the following steps:
  • said biallelic marker is selected from the group consisting of A 1 to A78. and the complements thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A 19. A25. and A29, and the complements thereof, more preferably the biallelic marker A7 and the complement thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A3. A5, A4. A6, A21 , A22. and A28, and the complements thereof.
  • the present invention concerns a method for the treatment of cancer or prostate cancer comprising the following steps:
  • said biallelic marker is selected from the group consisting of A l to A78. and the complements thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A 19. A25, and A29, and the complements thereof, more preferably the biallelic marker A7 and the complement thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A3. A5. A4. A6, A21. A22. and A28. and the complements thereof. To enlighten the choice of the appropriate beginning of the treatment of cancer or prostate cancer, the present invention also concerns a method for the treatment of cancer or prostate cancer comprising the following steps:
  • said biallelic marker is selected from the group consisting of A l to A78. and the complements thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A 19, A25, and A29, and the complements thereof, more preferably the biallelic marker A7 and the complement thereof. In a preferred embodiment, said biallelic marker is selected from the group consisting of A3, A5, A4. A6, A21 , A22, and A28, and the complements thereof. In particular embodiments, the individual is selected by genotyping one or more biallelic markers ofthe present invention.
  • Recombinant Vectors The term “vector "" is used herein to designate either a circular or a linear D ⁇ A or R ⁇ A molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.
  • the present invention encompasses a family of recombinant vectors that comprise a regulatory polynucleotide derived from the EN genomic sequence, or a coding polynucleotide from the EN genomic sequence. Consequently, the present invention further deals with a recombinant vector comprising either a regulatory polynucleotide comprised in the nucleic acids of S ⁇ Q ID No 1 or a polynucleotide comprising the EN coding sequence or both.
  • a recombinant vector of the invention may comprise any ofthe polynucleotides described herein, including regulatory sequences and coding sequences, as well as any EN primer or probe as defined above. More particularly, the recombinant vectors ofthe present invention can comprise any of the polynucleotides described in the "Genomic Sequences Of tThe EN Gene” section, the “EN cDNA Sequences ' “ section, the “Coding Regions " section, the "Polynucleotide constructs "” section, and the "Oligonucleotide Probes And Primers” section.
  • a recombinant vector of the invention is used to amplify the inserted polynucleotide derived from a EN genomic sequence of S ⁇ Q ID No 1 or a EN cD ⁇ A, for example the cD ⁇ A of S ⁇ Q ID No 2 in a suitable cell host, this polynucleotide being amplified at every time that the recombinant vector replicates.
  • a second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising either a regulatory polynucleotide or a coding nucleic acid of the invention, or both.
  • expression vectors are employed to express the EN polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against the EN protein.
  • the expression vectors are used for constructing transgenic animals and also for gene therapy. Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression ofthe genes of interest in host cells.
  • Dominant drug selection markers for establishing permanent, stable cell clones expressing the products are generally included in the expression vectors ofthe invention, as they are elements that link expression ofthe drug selection markers to expression ofthe polypeptide.
  • the present invention relates to expression vectors which include nucleic acids encoding a EN protein, or variants or fragments thereof, under the control of a regulatory sequence selected among the EN regulatory polynucleotides. or alternatively under the control of an exogenous regulatory sequence.
  • a regulatory sequence selected among the EN regulatory polynucleotides. or alternatively under the control of an exogenous regulatory sequence.
  • An example of an amino acid sequence encoding an EN protein which may be expressed is described in Kornblihtt et al. (1985).
  • Recombinant vectors comprising nucleic acids containing an EN-related biallelic marker are also part of the invention.
  • said biallelic marker is selected from the group consisting of Al to A78. and the complements thereof.
  • a recombinant vector according to the invention comprises, but is not limited to. a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid. a plasmid or even a linear D ⁇ A molecule which may consist of a chromosomal, non- chromosomal, semi-synthetic or synthetic D ⁇ A.
  • a recombinant vector can comprise a transcriptional unit comprising an assembly of:
  • Enhancers are cis-acting elements of D ⁇ A. usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
  • a structural or coding sequence which is transcribed into mRNA and ev entually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in ( 1 ).
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell
  • a recombinant protein when expressed without a leader or transport sequence, it may include a N-terminal residue This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product
  • recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the pe ⁇ plasmic space or the extracellular medium
  • preferred vectors will comprise an origin of replication in the desired host,
  • EN polypeptide encoded by the nucleotide sequence of S ⁇ Q ID ⁇ os 1. 2 and 3 or fragments or variants thereof may be useful in order to correct a genetic defect related to the expression of the gene in a host organism or to the production of a biologically inactive EN protein
  • the present invention also deals with recombinant expression vectors mainly designed for the in vivo production ofthe EN polypeptide encoded by any the nucleotide sequences of S ⁇ Q ID No 1 or 2 or fragments or variants thereof by the introduction of the appropriate genetic material in the organism of the patient to be treated
  • This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue
  • the suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression ofthe nucleic acid in the targeted cell
  • it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell such as, for example, a human or a viral promoter
  • a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed
  • the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted
  • Promoter regions can be selected from any desired gene using, for example. CAT
  • Preferred bacterial promoters are the Lad, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR PL and t ⁇ promoters (EP 0036776).
  • Eukaryotic promoters include CMV immediate early. HSV thymidine kinase. early and late SV40. LTRs from retrovirus, and mouse metallothionine-L Selection of a convenient vector and promoter is well within the level of ordinary skill in the art
  • polyadenylation signal to effect proper polyadenylation of the gene transcript
  • the nature ofthe polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals
  • a terminator can serve to enhance message levels and to minimize read through from the cassette into other sequences
  • the vector containing the appropriate DNA sequence as described above, and a polynucleotide encoding an EN polypeptide selected from the group consisting of S ⁇ Q ID No 1 and a fragment or a variant thereof, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide
  • BAC bacterial artificial chromosome
  • a preferred BAC vector consists of pBeloBACl 1 vector that has been described by Kim et al ( 1996) BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or Hindlll sites in the vector Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods After the construction of a BAC library in E coli.
  • One specific embodiment for a method for delivering a protein or peptide to the interior ot a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect
  • a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect
  • COS cells (ATCC N°CRL1650: N°CRL1651 ), Sf-9 cells (ATCC N°CRL171 1 ), C127 cells (ATCC N° CRL-1804), 3T3 (ATCC N° CRL-6361).
  • CHO ATCC N° CCL-61
  • human kidney 293. ATCC N° 45504: N° CRL-1573
  • BHK ECACC N° 84100501 : N° 841 1 1301 ).
  • Other mammalian host cells Other mammalian host cells.
  • Preferred ES cell lines are the following: ES-E14TG2a (ATCC n° CRL-1821 ), ES-D3 (ATCC n° CRL1934 and n° CRL-1 1632). YS001 (ATCC n° CRL-1 1776), 36.5 (ATCC n° CRL- 1 1 1 16). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.
  • a transgenic animal according the present invention comprises any one ofthe polynucleotides. the recombinant vectors and the cell hosts described in the present invention. More particularly, the transgenic animals of the present invention can comprise any of the poly nucleotides described in the "Genomic Sequences Of tThe EN Gene " section, the "EN cDNA Sequences " section, the ' Coding Regions” section, the "Polynucleotide constructs "' section, the 'Oligonucleotide Probes And Primers " section, the "Recombinant Vectors ' section and the 'Cell Hosts section 5
  • a further transgenic animals according to the invention contains in their somatic cells and/or in their germ line cells a polynucleotide comprising a biallelic marker selected from the group consisting of A l to A78, and the complements thereof
  • these transgenic animals may express a desired polypeptide of interest under the control of regulatory polynucleotides capable of providing good yields in the synthesis of this protein of interest, and preferably a tissue specific expression of this 15 protein of interest
  • the positive cells are isolated, cloned and injected into 3 5 days old blastocysts from 35 mice, such as described by Bradley ( 1987)
  • the blastocysts are then inserted into a female host animal and allowed to grow to term
  • the positive ES cells are brought into contact with embryos at the 2 5 days old 8-16 cell stage (morulae) such as described by Wood et al ( 1993), or by Nagy et al ( 1993). the ES cells being internalized to colonize extensively the blastocyst including the cells which will give rise to the germ line
  • the offspring of the female host are tested to determine which animals are transgenic e g include the inserted exogenous DNA sequence and which are wild-type
  • the present invention also concerns a method for screening substances or molecules that are able to interact with the regulatory sequences of the FN gene, such as for example promoter or enhancer sequences
  • the labeled target nucleotide sequence is brought into contact with either a total nuclear extract from cells containing transcription factors, or with different candidate molecules to be tested.
  • the interaction between the target regulatory sequence ofthe EN gene and the candidate molecule or the transcription factor is detected after gel or capillary electrophoresis through a retardation in the migration.
  • Another subject ofthe present invention is a method for screening molecules that modulate the expression ofthe F ⁇ protein.
  • Such a screening method comprises the steps of: a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the F ⁇ protein or a variant or a fragment thereof, placed under the control of its own promoter; b) bringing into contact the cultivated cell with a molecule to be tested; c) quantifying the expression of the F ⁇ protein or a variant or a fragment thereof.
  • the nucleotide sequence encoding the F ⁇ protein or a variant or a fragment thereof comprises an allele of at least one of the biallelic markers A l to A78, and the complements thereof.
  • the present invention also concerns a method for screening substances or molecules that are able to increase, or in contrast to decrease, the level of expression of the EN gene. Such a method may allow the one skilled in the art to select substances exerting a regulating effect on the expression level of the EN gene and which may be useful as active ingredients included in pharmaceutical compositions for treating patients suffering from cancer, and more particularly prostate cancer.
  • polynucleotides encoding a detectable protein there may be cited polynucleotides encoding beta galactosidase.
  • arrays means a one dimensional, two dimensional, or multidimensional arrangement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mR ⁇ As capable of hybridizing thereto
  • the arrays may contain a plurality of nucleic acids derived from genes whose expression levels are to be assessed
  • the arrays may include the EN genomic D ⁇ A.
  • EN cDNA probes labeled with an appropriate compound such as biotin. digoxigenin or fluorescent dye. are synthesized from the appropriate mR ⁇ A population and then randomly fragmented to an average size of 50 to 100 nucleotides The said probes are then hybridized to the chip After washing as described in Lockhart et al . supra and application of different electric fields (Sosnowsky et al 1997) , the dyes or labeling compounds are detected and quantified Duplicate hybridizations are performed Comparative analysis ofthe intensity ofthe signal originating from cD ⁇ A probes on the same target oligonucleotide in different cD ⁇ A samples indicates a differential expression of EN mR ⁇ A
  • Preferred antisense polynucleotides according to the present invention are complementary to a sequence ofthe mR ⁇ As of EN that contains either the translation initiation codon ATG or a splicing donor or acceptor site
  • the antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression ofthe EN mR ⁇ A in the duplex
  • Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al . ( 1986) and Izant and Weintraub, ( 1984)
  • antisense molecules are obtained by reversing the orientation of the EN coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell.
  • the antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript.
  • Another approach involves transcription of EN antisense nucleic acids in vivo by operably linking D ⁇ A containing the antisense sequence to a promoter in a suitable expression vector.
  • the simplified cycle of a hammerhead ribozyme consists of (1 ) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand: and (3) release of cleavage products, which gives rise to another catalytic cycle.
  • the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous.
  • a preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophihc groups or to use liposomes as a convenient vector.
  • Preferred antisense ribozymes according to the present invention are prepared as described by Sczakiel et al.(l 995), the specific preparation procedures being referred to in said article being herein incorporated by reference.
  • a portion of the EN genomic D ⁇ A can be used to study the effect of inhibiting EN transcription within a cell.
  • homopurine sequences were considered the most useful for triple helix strategies.
  • homopyrimidine sequences can also inhibit gene expression.
  • Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homo ⁇ yrimidine sequences.
  • both types of sequences from the EN genomic D ⁇ A are contemplated within the scope of this invention.
  • the sequences ofthe EN genomic D ⁇ A are first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting EV expression.
  • oligonucleotides containing the candidate sequences are assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which express the EN gene. 5
  • the oligonucleotides can be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation. D ⁇ A ⁇ -Dextran. electroporation. liposome-mediated transfection or native uptake.
  • Treated cells are monitored for altered cell function or reduced EN expression using techniques such as Northern blotting. RNase protection assays, or PCR based strategies to monitor 10 the transcription levels ofthe EN gene in cells which have been treated with the oligonucleotide.
  • the natural (beta) anomers of the oligonucleotide units can be 15 replaced with alpha anomers to render the oligonucleotide more resistant to nucleases.
  • an intercalating agent such as ethidium bromide, or the like, can be attached to the 3' end of the alpha oligonucleotide to stabilize the triple helix.
  • nucleic acid codes of the invention encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one ofthe following: a) a contiguous span of at least 12, 15. 18, 20. 25. 30. 35. 40, 50. 60. 70. 80. 90, 100. 150. 200, 500. or 1000 nucleotides of SEQ ID No 1. wherein said contiguous span comprises at least 1 ofthe following nucleotide positions of SEQ ID No 1 : 1 -2000. 2154-2986. 31 16-4350, 4489-5847. 5980- 25 6959, 7098-9473, 9633-12525. 12718-13485.
  • a contiguous span of at least 12. 15. 18. 20. 25. 30. 35. 40. 50. 60. 70. 80. 90, 100. 150, 200. or 500 nucleotides. to the extent that such lengths are consistent with the specific sequence, of a sequence selected from the group consisting of SEQ ID Nos. 4 to 27. and the complements thereof, optionally wherein said contiguous span comprises either allele 1 or allele 2 of a EN-related biallelic marker selected from the group consisting of A20 to A30 and A64 to A78: and sequences complementary to all ofthe preceding sequences. Homologous sequences refer to a sequence having at least 99%. 98%.
  • nucleic acid codes ofthe invention can be represented in the traditional single character format (See the inside back cover of Stryer. Lubert. Biochemistry. 3 rd edition. W. H Freeman & Co., New York.), or in any other format or code which records the identity ofthe nucleotides in a sequence.
  • nucleic acid codes ofthe invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium.
  • a skilled artisan can readily adopt any ofthe presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more ofthe nucleic acid codes ofthe invention.
  • Another aspect ofthe present invention is a computer readable medium having recorded thereon at least 2. 5, 10, 15. 20, 25, 30. or 50 nucleic acid codes of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media may be a hard disc, a floppy disc, a magnetic tape. CD-ROM. DVD. RAM. or ROM as well as other types of other media known to those skilled in the art.
  • the computer system further includes one or more data retrieving devices for reading the data stored on the data storage components
  • the data retrieving device may represent for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, a hard disk drive, a CD-ROM drive, a DVD drive, etc
  • the data storage component is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc containing control logic and/or data recorded thereon
  • the computer system may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device
  • Software for accessing and processing the nucleotide sequences ofthe nucleic acid codes ofthe invention may reside in main memory during execution
  • Another aspect ofthe present invention is a method for determining the level of homology between a nucleic acid code ofthe invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program
  • the computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters.
  • the method may be implemented using the computer systems described above. The method may also be performed by reading 2. 5. 10. 15. 20, 25, 30.
  • the computer program may be a computer program which compares the nucleotide sequences ofthe nucleic acid codes ofthe present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code ofthe invention differs from a reference nucleic acid sequence at one or more positions.
  • a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code ofthe invention.
  • the computer program may be a program which determines whether the nucleotide sequences ofthe nucleic acid codes ofthe invention contain one or more biallelicmarker or single nucleotide polymo ⁇ hisms (SNP) with respect to a reference nucleotide sequence.
  • SNP single nucleotide polymo ⁇ hisms
  • These single nucleotide polymo ⁇ hisms may each comprise a single base substitution, insertion, or deletion
  • the biallelic markers may each comprise nucleotide substitutions, insertions, or deletions of 1 to 10 contiguous nucleotides, preferably 1 to 5 contiguous nucleotides.
  • Another aspect ofthe present invention is a method for determining the level of homology between a polypeptide code ofthe invention and a reference polypeptide sequence, comprising the steps of reading the polypeptide code ofthe invention and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.
  • another aspect ofthe present invention is a method for determining whether a nucleic acid code ofthe invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program.
  • the computer program is a program which identifies single nucleotide polymo ⁇ hisms.
  • the method may be implemented by the computer systems described above. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30. or 50 ofthe nucleic acid codes ofthe invention and the reference nucleotide sequences through the use ofthe computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program.
  • the computer based system may further comprise an identifier for identifying features within the nucleotide sequences ofthe nucleic acid codes ofthe invention.
  • An "identifier" refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of the invention.
  • the nucleic acid codes ofthe invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, they may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art. such as DB2. SYBASE, or ORACLE.
  • many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide sequences to be compared to the nucleic acid codes ofthe invention.
  • the following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes ofthe invention.
  • the programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group). Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al. 1990), FASTA (Pearson and Lipman. 1988), FASTDB (Brutlag et al..
  • Catalyst (Molecular Simulations Inc.). Catalyst/SHAPE (Molecular Simulations Inc.). Cerius 2 .DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.). Insight II. (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.). CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi. (Molecular Simulations Inc.), QuanteMM. (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.).
  • Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database. Derwents * s World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.
  • the pellet ot white cells was lysed overnight at 42°C with 3 7 ml of lysis solution composed of
  • the pool was constituted by mixing equivalent quantities of DNA from each individual
  • the primers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.
  • 5 Primers from the columns labeled "Position range of amplification primer in SEQ ID No 1 ,” and “Position range of amplification primer” contain the following additional PU 5 ' sequence: TGTAAAACGACGGCCAGT; and primers from the columns labeled "Complementary position range of amplification primer in SEQ ID No 1, " and "Complementary position range of amplification primer” contain the following RP 5 " sequence: CAGGAAACAGCTATGACC.
  • the 10 primer containing the additional PU 5 ' sequence is listed in SEQ ID No 28.
  • the primer containing the additional RP 5 " sequence is listed in SEQ ID No 29.
  • DNA amplification was performed on a Genius II thermocycler. After heating at 95°C for 15 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95°C, 54°C for 1 min, and 30 sec at 72°C. For final elongation, 10 min at 72°C ended the amplification.
  • the quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).
  • Example 2 The sequencing of the amplified DNA obtained in Example 2 was carried out on ABI 377 sequencers. The sequences ofthe amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of 25 the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software (2.1.2 version)).
  • sequence data were further evaluated to detect the presence of biallelic markers within the amplified fragments.
  • the polymo ⁇ hism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as 30 described previously.
  • BM refers to "biallelic marker " '. All l and all2 refer respectively to allele 1 and allele 2 of the biallelic marker.
  • the biallelic markers identified in example 3 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 1.
  • Amplification from genomic DNA of individuals was performed by PCR as described above for the detection ofthe biallelic markers with the same set of PCR primers (Table 1 ).
  • the preferred primers used in microsequencing were about 19 nucleotides in length and hybridized just upstream ofthe considered polymo ⁇ hic base. According to the invention, the primers used in microsequencing are detailed in Table 3.
  • the microsequencing reaction mixture was prepared by adding, in a 20 ⁇ l final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G). 1.25 ⁇ l Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCL). and the two appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymo ⁇ hic site of each biallelic marker tested, following the manufacturer's recommendations. After 4 minutes at 94°C, 20 PCR cycles of 15 sec at 55°C. 5 sec at 72°C. and 10 sec at 94°C were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The unincorporated dye terminators were then removed by ethanol precipitation.
  • the software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous
  • the software identifies significant peaks (according to shape and height criteria) Among the significant peaks, peaks corresponding to the targeted site are identified based on their position When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio
  • haplotype frequency estimation can be derived using the E-M algorithm (see above). It has to be noted that all of these approaches are applied to markers under Hardy- Weinberg equilibrium, and only these markers are included.
  • the haplo-max test procedure is based on the haplotype frequency difference of each 0 haplotype between the two groups. For one combination of marker with h haplotypes. h differences of haplotype frequencies can be compared via a Pearson chi-square statistic ( 1 degree of freedom). The haplo-max test selects the difference showing the maximum positive (Max-M) or negative (Max-S) test value between cases and controls, rejecting test values based on rare haplotype frequencies (with an estimated number of haplotypes inferior to 10). Here, for one combination of 5 marker, there is one Max-M and one Max-S test value.
  • significance thresholds taking into account the multiple testing procedure due to selection of the maximum test value can be arbitrarily set.
  • HAP 1 haplotype and prostate cancer were still more significant in the sporadic cases under 65 years old with a p-value of 2x10 l j (see Figure 3, haplotype 3)
  • haplotypes ofthe figure 1 are also highly significant, namely HAP2. HAP3. HAP4, HAP5, HAP6. and HAP7 These haplotypes presented p-value comprised in the range between 2 2x10 s and 8 3x10 "3 They often comprised the biallelic marker (99-5596-197 (A29) allele A) Haplotype HAP8 ofthe figure 1 had a highly significant p value in the informative sporadic population (2 6x10 " ') (see Figure 3. haplotype 4)
  • Haplotype no 9 (HAP9) of the figure 1 consisting of three biallelic markers (99-23444/203 (A18) allele G. 99-5595/380 (A 19) allele A and 99-5596- 197 (A29) allele A) had a p-value of 3x10 8 and an odd ratio of 18 64 Estimated haplotype frequencies were 6 5 % in the cases and 0 4% in the controls
  • the three-markers haplotypes HAP 10 to HAP 17 and the four-markers haplotypes HAP20 to HAP28 ofthe figure 1 also showed very significant association
  • the haplotypes HAP10 to HAP17 and HAP20 to HAP28 all comprise the biallelic marker 99-5596-197 (A29)
  • haplotypes HAP1 and HAP9 ofthe figure 1 are both strongly associated with sporadic prostate cancer They can be used in diagnosis of prostate cancer
  • FIG. 2 shows the most significant haplotypes obtained with the familial cases.
  • Two three-markers haplotypes namely HAP9 and HAP 10. showed a highly significant association with familial prostate cancer.
  • the haplotype HAP9 consisting of three biallelic markers (99-5605/90 (A3) allele G. 99-23460/199 (A21 ) allele C and 99-5590/99 (A28) allele T) presented a p-value of 2.1 x10 "" and an odd-ratio of 2.43.
  • Estimated haplotype frequencies were 16.8% in the familial cases and 7.6 % in the controls.
  • haplotype HAP10 consisting of three biallelic markers (99-5604/376 (A5) allele G, 99-23460/199 (A21 ) allele C and 99-5590/99 (A28) allele T) presented a p-value of 3.7x10° and an odd-ratio of 2.32.
  • Estimated haplotype frequencies were 17.1 % in the familial cases and 8.2 % in the controls.
  • haplotype 1 ten other three-markers haplotypes are also significant, namely HAP1 1 to HAP20.
  • These haplotypes presented p-value comprised in the range between 8.3x10° and 9.6x10 "4 .
  • the four-markers haplotypes HAP 22 to HAP 33 showed a highly significant association with familial prostate cancer and presented p-values comprised in the range between 3.2x10 " ' and 9.5x 10 "6 .
  • One preferred haplotype HAP22 consisting of the four biallelic markers (99-23452/306 (A4) allele G. 99-5582/71 (A7) allele G. 99-15798/86 (A22) allele T and 99-5590/99 (A28) allele T) presented a p-value of 3.2x10 " ' and an odd-ratio of 2.82.
  • Estimated haplotype frequencies were 18.6 % in the familial cases and 7.5 % in the controls.
  • An other preferred haplotype HAP 24 consisting of the four biallelic markers (99-23452/306 (A4) allele G. 99-23440/274 (A6) allele A, 99-15798/86 (A22) allele T and 99-5590/99 (A28) allele T) presented a p-value of lxl 0 "6 and an odd-ratio of 2.73. Estimated haplotype frequencies were 18.6 % in the familial cases and 7.7 % in the controls.
  • the haplotypes HAP 10 and HAP24 are the more preferred haplotype of the invention. It can be used in diagnosis of prostate cancer and more particularly familial prostate cancer.
  • the statistical significance ofthe results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 1000 times on a computer. For this computer simulation, data from the affected and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the case-control populations used to produce the data summarized in Figure 2.
  • This experiment was reiterated 1000 times and the results are shown in Figure 3.

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Abstract

L'invention cocnerne la séquence génomique du gène de Fibronectine. Elle porte également sur des marqueurs bialléliques du gène de Fibronectine et l'association établie entre ces marqueurs et le cancer, notamment le cancer de la prostate. Elle se rapporte encore à un moyen pour déterminer la prédisposition de sujets au cancer ainsi qu'à un moyen pour le diagnostic du cancer et pour le pronostic/la détection d'une éventuelle réponse thérapeutique aux agents agissant sur le cancer.
PCT/IB2000/000431 1999-03-29 2000-03-28 Gene de fibronectine humaine associe au cancer de la prostate et marqueurs bialleliques WO2000058509A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2008046510A1 (fr) * 2006-10-16 2008-04-24 Bayer Healthcare Ag Fn1 utilisé comme biomarqueur, cible thérapeutique et diagnostique
US9938581B2 (en) 2009-04-17 2018-04-10 Avellino Co., Ltd. Primers for diagnosing Avellino corneal dystrophy
US11268146B2 (en) 2009-04-17 2022-03-08 Avellino Co., Ltd. Primers for diagnosing Avellino corneal dystrophy
US9970051B2 (en) 2010-10-01 2018-05-15 Avellino Co., Ltd. System for diagnosing Avellino corneal dystrophy
EP2925895A4 (fr) * 2013-03-15 2016-08-17 Avellino Lab Usa Inc Procédés servant à un meilleur isolement des matrices d'adn génomique destinés à la détection d'allèles
US9856516B2 (en) 2013-03-15 2018-01-02 Avellino Labs Usa, Inc. Methods for improved isolation of genomic DNA templates for allele detection
EP3486328A1 (fr) * 2013-03-15 2019-05-22 Avellino Lab USA, Inc. Procédés servant à un meilleur isolement des matrices d'adn génomique destinés à la détection d'allèles
US10889850B2 (en) 2013-03-15 2021-01-12 Avellino Lab Usa, Inc. Methods for improved isolation of genomic DNA templates for allele detection
EP3825413A1 (fr) * 2013-03-15 2021-05-26 Avellino Lab USA, Inc. Procédés servant à un meilleur isolement des matrices d'adn génomique destinées à la détection d'allèles
US11525160B2 (en) 2013-11-15 2022-12-13 Avellino Lab Usa, Inc. Methods for multiplex detection of alleles associated with ophthalmic conditions
US11987809B2 (en) 2015-11-13 2024-05-21 Avellino Lab Usa, Inc. Methods for the treatment of corneal dystrophies

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