WO2010019690A1 - Polymorphismes associés à un cancer colorectal en développement, leurs méthodes de détection et d'utilisations - Google Patents

Polymorphismes associés à un cancer colorectal en développement, leurs méthodes de détection et d'utilisations Download PDF

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WO2010019690A1
WO2010019690A1 PCT/US2009/053582 US2009053582W WO2010019690A1 WO 2010019690 A1 WO2010019690 A1 WO 2010019690A1 US 2009053582 W US2009053582 W US 2009053582W WO 2010019690 A1 WO2010019690 A1 WO 2010019690A1
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seq
crc
polynucleotide
nos
colorectal cancer
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PCT/US2009/053582
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Albert De La Chapelle
Stephan M. Tanner
Laura Valle
Boris Pasche
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The Ohio State University Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C12Q2600/156—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

  • This invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions involving biomarkers for colorectal cancer (CRC). Certain aspects of the invention include application in diagnostics, therapeutics, and prognostics of CRC.
  • CRC colorectal cancer
  • TGF- ⁇ transforming growth factor ⁇
  • TGF ⁇ Rl type I receptor gene
  • TGF ⁇ Rl is a notable candidate for a gene that, when its expression is reduced, causes predisposition to CRC or acts as a modifier of other genes resulting in a predisposition.
  • ASE results in reduced expression, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases.
  • a method for assessing a pathological condition in a subject comprising measuring the expression of one or more alleles from the subject, wherein a reduction in the expression of the alleles from the subject compared to the expression of the alleles in a healthy control is indicative of a colorectal cancer (CRC) or a predisposition to CRC.
  • the biomarker comprises transforming growth factor ⁇ receptor type 1 (TGF ⁇ Rl).
  • Figure 1 TGF ⁇ Rl allele-specific expression (ASE) distribution in 138 CRC patients and 105 controls studied by SNaPshot.
  • the ASE cut-off value of 1.5 chosen to categorize the cases is indicated, together with its associated P-value obtained from comparing the proportions of cases (29/138) and controls (3/105) above the indicated value.
  • Figures 2A and 2B ASE determination in two ASE CRC probands.
  • Figure 2A ASE detection in blood DNA by SNaPshot.
  • the ASE ratio was calculated by normalizing the ratio between the peak areas of the two alleles in cDNA with the same parameters in genomic DNA (gDNA). In both examples, the transcript from the "a" allele is reduced with respect to the other allele.
  • Figure 2B Semiquantitative RT-PCR of the cDNA from monochromosomal hybrids of the same two patients.
  • Human TGF ⁇ Rl expression (amplicon size 135 bp) was assessed and mouse Gpi used as control (176 bp).
  • the values shown below the gel represent the ratios of the densitometric values of human TGF ⁇ Rl versus mouse Gpi, showing reduced expression of human TGF ⁇ Rl in the hybrids that contain the "a" allele.
  • Figures 3A and 3C Analysis of SMAD-mediated TGF- ⁇ signaling in lymphoblastoid cell lines from ASE CRC patients and non-ASE healthy controls:
  • FIG. 3A SMAD2 and phosphorylated SMAD2 (pSMAD2) expression was assessed by Western blotting in lymphoblastoid cell lines from ASE patients (P-I, P-5, P- 14) and non-ASE controls (C-I, C-2 and C-3), after exposure to TGF- ⁇ (100 pM), at various time points from 0 to 16 hours and using ⁇ -actin as a loading control. In all three ASE cases, lower constitutive pSMAD2 was observed when compared with non-ASE controls. The differences in pSMAD2 expression between ASE and non-ASE cell lines were further enhanced after exposure to TGF- ⁇ .
  • Figure 3B SMAD2 and p-SMAD2 expression 1 hour after exposure to different TGF- ⁇ concentrations. The effect shown in Figure 3A also occurs at low concentrations of TGF- ⁇ (5pM).
  • Figure 3C pSMAD3 detection in nuclear extracts from three ASE patients and three non-ASE controls after exposure to TGF- ⁇ l.
  • the three non-ASE lymphoblastoid cell lines had pSMAD3 expression in the nucleus while nuclear pSMAD3 expression was undetectable in two ASE cases (P-I and P-14) and barely detectable in one case (P-5).
  • Figure 4A Diagram of the TGFBRl genomic region. The uppermost line depicts the 96.5 kb region sequenced in six ASE patients (four monochromosomal hybrids and four diploid DNAs). Shown are the locations of the 2-bp CA deletion upstream of exon 1, the 9 A/6 A polymorphism in exon 1, and the four SNPs in the 3'-UTR used for ASE determinations.
  • FIG. 4C Two major haplotypes identified in ASE patients are shown ("GAAGAGCATA” disclosed as [SEQ ID NO: 5]).
  • Figure 5 ASE evaluation strategy in cases and controls and number of patients at each step.
  • Figure 6 ASE inheritance and co-segregation with CRC.
  • ASE values are framed and in red if over 1.5.
  • the haplotypes inferred by MERLIN from genotyping data of 35 SNPs are represented as colored bars.
  • each colored bar represents a different haplotype, and in all of them, the red one corresponds to the down-expressed allele in the proband.
  • two borderline ASE values «1.4
  • have not been marked as positive in red both occur in individuals (red asterisks) who carry their corresponding proband's affected allele.
  • Figure 8 Table 2. Sensitivity, specificity and Youden's index for different ASE cutoff values.
  • Figure 9 Table 3. Germline and tumor characteristics of informative colorectal cancer patients according to their ASE status.
  • Figure 11 Table 5. SNPs located in the TGF ⁇ Rl region used for haplotype inference by MERLIN and PHASE.
  • Figure 12 Table 6. Characteristics of the family members represented in Figure 6, showing their cancer affection, tumor micro satellite instability status, expression of the DNA mismatch repair proteins, current age or age at death and ASE value.
  • Figure 13 rs334348 [SEQ ID NO:1], rs334349 [SEQ ID NO:2], rsl590 [SEQ ID NO:3] and rs7871490 [SEQ ID NO:4].
  • a nucleic acid sequence at which more than one sequence is possible in a population is referred to herein as a "polymorphic site.”
  • Polymorphic sites can allow for differences in sequences based on substitutions, insertions, or deletions. Such substitutions, insertions, or deletions can result in frame shifts, the generation of premature stop codons, the deletion or addition of one or more amino acids encoded by a polynucleotide, alter splice sites, and affect the stability or transport of MRNA.
  • a polymorphic site is a single nucleotide in length, the site is referred to as a single nucleotide polymorphism ("SNP").
  • SNPs are the most common form of genetic variation responsible for differences in disease susceptibility. SNPs can directly contribute to or, more commonly, serve as markers for many phenotypic endpoints such as disease risk differences between patients.
  • the instant invention concerns the identification of genetic factors that predispose individuals to colorectal cancer (CRC), with a focus on candidate genes and specifically, nucleic acid fragments of genes having single nucleotide polymorphisms ("SNPs").
  • CRC colorectal cancer
  • SNPs single nucleotide polymorphisms
  • the invention provides isolated polynucleotides containing SNPs located within sequences selected from the group consisting of sequences identified by Sequence Identification Numbers ("SEQ ID NOs.") 1-4 and the complements of the sequences identified by SEQ ID NOs: 1-4 as well as vectors, recombinant host cells, transgenic animals, and compositions containing such polynucleotides.
  • the invention also provides methods of diagnosing a susceptibility to colorectal cancer (CRC) in an individual, by detecting one or more at-risk alleles of SNPs associated with colorectal cancer (CRC).
  • the invention provides methods of diagnosing a susceptibility to colorectal cancer (CRC) in an individual by detecting one or more haplotypes associated with colorectal cancer (CRC).
  • SNP refers to a single nucleotide polymorphism at a particular position in the human genome that varies among a population of individuals. As used herein, a SNP may be identified by its name or by location within a particular sequence.
  • nucleotide sequences disclosed by the SEQ ID NO. encompass the complements of the nucleotide sequences.
  • SNP encompasses any allele among a set of alleles.
  • allele refers to a specific nucleotide among a selection of nucleotides defining a SNP.
  • At-risk allele refers to an allele that is associated with colorectal cancer (CRC).
  • haplotype refers to a combination of particular alleles from two or more
  • At-risk haplotype refers to a haplotype that is associated with colorectal cancer (CRC).
  • polynucleotide refers to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • Polynucleotides may have any three-dimensional structure including single-stranded, double- stranded and triple helical molecular structures, and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, short interfering nucleic acid molecules (siNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.
  • the terms "individual,” “host,” and “subject” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human.
  • the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
  • a polynucleotide of the present invention can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of a sequence selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4, polynucleotides can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y, 1989).
  • a polynucleotide can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the polynucleotide so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to all or a portion of a polynucleotide can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • allele specific expression refers to the differential expression of the two alleles from the two chromosome copies in a mammalian cell and implies thereby that one allele is lower expression than the other.
  • hybridization refers to the process of binding, annealing, or base-pairing between two single- stranded nucleic acids.
  • stringency of hybridization is determined by the conditions of temperature and ionic strength. Nucleic acid hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which the hybrid is 50% denatured under defined conditions. Equations have been derived to estimate the Tm of a given hybrid; the equations take into account the G+C content of the nucleic acid, the length of the hybridization probe, etc. (e.g., Sambrook et al., 1989).
  • hybridizations are generally carried out in solutions of high ionic strength (6x SSC or 6x SSPE) at a temperature that is about 2025 0 C below the Tm. If the sequences to be hybridized are not identical, then the hybridization temperature is reduced 1-1.5 0 C for every 1% of mismatch.
  • the washing conditions should be as stringent as possible (i.e., low ionic strength at a temperature about 12-2O 0 C below the calculated Tm).
  • highly stringent conditions typically involve hybridizing at 68 0 C in 6x SSC/5x Denhardt's solution/1.0% SDS and washing in 0.2x SSCVO.1 % SDS at 65 0 C.
  • the optimal hybridization conditions generally differ between hybridizations performed in solution and hybridizations using immobilized nucleic acids. One skilled in the art will appreciate which parameters to manipulate to optimize hybridization.
  • nucleic acid refers to sequences of linked nucleotides.
  • the nucleotides may be deoxyribonucleotides or ribonucleotides, they may be standard or non-standard nucleotides; they may be modified or derivatized nucleotides; they may be synthetic analogs.
  • the nucleotides may be linked by phosphodiester bonds or non- hydrolyzable bonds.
  • the nucleic acid may comprise a few nucleotides (i.e., oligonucleotide), or it may comprise many nucleotides (i.e., polynucleotide).
  • the nucleic acid may be single- stranded or double- stranded.
  • Probes based on the sequence of a polynucleotide of the invention can be used to detect transcripts or genomic sequences.
  • a probe may comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding a protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding a protein has been mutated or deleted.
  • ASE inherited allele-specific expression
  • TGF- ⁇ transforming growth factor beta
  • TGF ⁇ Rl transforming growth factor beta type I receptor gene
  • CRC familial colorectal cancers
  • the present invention provides isolated polynucleotides comprising a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS.: 1-4 and the complements of sequences identified by SEQ ID NOS.: 1-4; wherein the presence of a particular allele of a SNP (a particular nucleotide base) is indicative of a propensity to develop colorectal cancer (CRC) or otherwise may be used to identify an at-risk individual.
  • CRC colorectal cancer
  • the polynucleotide is selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4. In another embodiment, the polynucleotide comprises at least a portion of a sequence selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4.
  • the present invention also relates to isolated polynucleotides comprising a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4, which hybridize, are complementary, or are partially complementary to a nucleotide sequence present in a test sample.
  • an isolated polynucleotide is selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4, which hybridizes, is complementary, or is partially complementary to a nucleotide sequence present in a test sample.
  • an isolated polynucleotide comprises at least a portion of a sequence selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4, which hybridizes, is complementary, or is partially complementary to a nucleotide sequence present in a test sample.
  • the SNP is located within SEQ ID NO: 1 or the complement of SEQ ID NO: 1.
  • the SNP is located within SEQ ID NO: 2 or the complement of SEQ ID NO: 2.
  • the SNP is located within SEQ ID NO: 3 or the complement of SEQ ID NO: 3.
  • the SNP is located within SEQ ID NO: 4 or the complement of SEQ ID NO: 4.
  • the present invention also provides isolated polynucleotides comprising one or more haplotypes selected from the group consisting of the haplotypes identified in Figure 4C which are indicative of a propensity to develop colorectal cancer (CRC).
  • haplotypes selected from the group consisting of the haplotypes identified in Figure 4C which are indicative of a propensity to develop colorectal cancer (CRC).
  • the invention also provides polypeptides encoded by a polynucleotide, wherein the polynucleotide comprises a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4.
  • a polypeptide is encoded by a polynucleotide, wherein the polynucleotide is selected from the group consisting of sequences identified by SEQ ID NOs: 1-4 and the complements of sequences identified by SEQ ID NOs: 1-4.
  • a polypeptide is encoded by a polynucleotide, wherein the polynucleotide comprises at least a portion of the sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4. Also contemplated are antibodies that bind such polypeptides. [0072]
  • the present invention also provides polypeptides encoded by a polynucleotide, wherein the polynucleotide comprises a haplotype selected from the group consisting of the haplotypes identified in Figure 4C.
  • the invention also provides a vector comprising a haplotype identified in Figure 4C or a SNP located within a sequence selected from the group consisting of the sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4; operably linked to a regulatory sequence.
  • compositions and kits are contemplated which contain the polynucleotides, proteins and/or antibodies of the present invention.
  • One application of the current invention involves prediction of those at higher risk of developing colorectal cancer (CRC). Diagnostic tests that define genetic factors contributing to colorectal cancer (CRC) may be used together with, or independent of, the known clinical risk factors to define an individual's risk relative to the general population. Means for identifying those individuals at risk for colorectal cancer (CRC) should lead to better prophylactic and treatment regimens, including more aggressive management of the current clinical risk factors.
  • the present invention includes methods of diagnosing a susceptibility to colorectal cancer (CRC) in an individual, comprising detecting polymorphisms in nucleic acids of specific genes or gene segments, wherein the presence of the polymorphism in the nucleic acid is indicative of a susceptibility to colorectal cancer (CRC).
  • CRC colorectal cancer
  • the present invention includes methods of diagnosing colorectal cancer (CRC) or a susceptibility to colorectal cancer (CRC) in an individual, comprising determining the presence or absence of particular alleles of SNPs contained in SEQ ID NOS: 1-4.
  • methods comprise screening for one of the at-risk alleles associated with colorectal cancer (CRC) shown in Figure 4C.
  • the SNP is located within SEQ ID NO: 1 or the complement of SEQ ID NO: 1.
  • the SNP is located within SEQ ID NO: 2 or the complement of SEQ ID NO: 2.
  • the SNP is located within SEQ ID NO: 3 or the complement of SEQ ID NO: 3.
  • the SNP is located within SEQ ID NO: 4 or the complement of SEQ ID NO: 4.
  • the invention provides a method of detecting the presence of a polynucleotide in a sample containing a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4, wherein the method comprises contacting the sample with an isolated polynucleotide comprising a sequence (or a portion of a sequence) selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4, under conditions appropriate for hybridization, and assessing whether hybridization has occurred between the polynucleotide in the sample and the isolated polynucleotide; wherein if hybridization has occurred, a certain polynucleotide containing a particular allele of a SNP associated (or not associated) with colorectal cancer (CRC) is present in the sample.
  • CRC colorectal cancer
  • the isolated polynucleotide is completely complementary to the polynucleotide present in the sample. In other embodiments of the above method, the isolated polynucleotide is partially complementary to the polynucleotide present in the sample. In other embodiments, the isolated polynucleotide is at least 80% identical to the polynucleotide present in the sample and capable of selectively hybridizing to the polynucleotide. If desired, amplification of the polynucleotide present in the sample can be performed using known methods in the art.
  • the present invention further provides a method for assaying a sample for the presence of a first polynucleotide which is at least partially complementary to a part of a second polynucleotide wherein the second polynucleotide comprises a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4 comprising: a) contacting the sample with the second polynucleotide under conditions appropriate for hybridization, and b) assessing whether hybridization has occurred between the first and the second polynucleotide, wherein if hybridization has occurred, the first polynucleotide is present in the sample.
  • the presence of the first polynucleotide is indicative of colorectal cancer (CRC) or the propensity to develop colorectal cancer (CRC).
  • CRC colorectal cancer
  • CRC propensity to develop colorectal cancer
  • the second polynucleotide is completely complementary to a part of the sequence of the first polynucleotide.
  • the method further comprises amplification of at least part of the first polynucleotide.
  • the second polynucleotide is 99 or fewer nucleotides in length and is either: (a) at least 80% identical to a contiguous sequence of nucleotides in the first polynucleotide or (b) capable of selectively hybridizing to the first polynucleotide.
  • Also contemplated by the invention is a method of assaying a sample for the presence of a polypeptide associated with colorectal cancer (CRC) encoded by a polynucleotide, wherein the polynucleotide comprises an allele of a SNP associated with colorectal cancer (CRC) located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4, the method comprising contacting the sample with an antibody that specifically binds to the polypeptide.
  • CRC colorectal cancer
  • the presence of a polypeptide associated with colorectal cancer (CRC) in a sample encoded by a polynucleotide is assayed by contacting the sample with an antibody that specifically binds to the polypeptide.
  • the presence of a polypeptide associated with colorectal cancer (CRC) in a sample encoded by a polynucleotide (comprising at least a portion of a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4) is assayed by contacting the sample with an antibody that specifically binds to the polypeptide.
  • CRC colorectal cancer
  • the present invention also contemplates a reagent for assaying a sample for the presence of a first polynucleotide comprising a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4, the reagent comprising a second polynucleotide comprising a contiguous nucleotide sequence which is at least partially complementary to a part of the first polynucleotide.
  • the second polynucleotide is completely complementary to a part of the first polynucleotide.
  • the present invention also encompasses a reagent kit for assaying a sample for the presence of a first polynucleotide comprising a SNP located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4, comprising in separate containers: a) one or more labeled second polynucleotides comprising a sequence selected from the group consisting of the sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4; and b) reagents for detection of the label.
  • kits are contemplated containing polynucleotides which can be used to assay samples for the presence of polynucleotides containing an allele of a SNP associated (or not associated) with colorectal cancer (CRC) located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4.
  • Kits are also contemplated which contain antibodies which can be used to assay samples for the presence of proteins associated (or not associated) with colorectal cancer (CRC) that are encoded by the polynucleotides containing an allele of a SNP associated (or not associated) with colorectal cancer (CRC).
  • Other methods of diagnosing a susceptibility to colorectal cancer (CRC) in an individual comprise determining the expression or composition of a polypeptide in a control sample encoded by a polynucleotide containing an allele of a SNP not associated with colorectal cancer (CRC) and comparing it with the expression or composition of a polypeptide in a test sample encoded by the same polynucleotide except containing an allele of a SNP associated with colorectal cancer (CRC), wherein the presence of an alteration in expression or composition of the polypeptide in the test sample compared to the control sample is indicative of a susceptibility to colorectal cancer (CRC).
  • CRC susceptibility to colorectal cancer
  • the invention also relates to a method of diagnosing colorectal cancer (CRC) or a susceptibility to colorectal cancer (CRC) in an individual, comprising determining the presence or absence in the individual of certain haplotypes.
  • methods comprise screening for one of the at-risk haplotypes shown in Figure 4C.
  • the present invention encompasses a method for diagnosing a susceptibility to colorectal cancer (CRC) in an individual, or a method of screening for individuals with a susceptibility to colorectal cancer (CRC), comprising detecting a haplotype associated with colorectal cancer (CRC) selected from the group consisting of the haplotypes shown in Figure 4C.
  • the presence or absence of the haplotype may be determined by various methods, including, for example, using enzymatic amplification of nucleic acid from the individual, electrophoretic analysis, restriction fragment length polymorphism analysis and/or sequence analysis.
  • a method of diagnosing a susceptibility to colorectal cancer (CRC) in an individual, or for screening individuals for a susceptibility to colorectal cancer (CRC) comprising: a) obtaining a polynucleotide sample from the individual; and b) analyzing the polynucleotide sample for the presence or absence of a haplotype, comprising a haplotype shown in Figure 4C, wherein the presence of the haplotype corresponds to a susceptibility to colorectal cancer (CRC).
  • a method of determining the susceptibility to colorectal cancer (CRC) in an individual comprising detecting multiple SNPs identified in Figure 4C.
  • the method of determining the susceptibility to colorectal cancer (CRC) in an individual comprises detecting multiple SNPs identified in one or more of: SEQ ID NOS: 1, 2, 3 and/or 4.
  • the presence of a first polynucleotide in a sample containing one or more at-risk alleles in Figure 4C is assayed for by contacting the sample with probe polynucleotides that are complementary to the first polynucleotide.
  • at least one SNP is located within SEQ ID NO: 1 or the complement of SEQ ID NO: 1.
  • at least one SNP is located within SEQ ID NO: 2 or the complement of SEQ ID NO: 2.
  • at least one SNP is located within SEQ ID NO: 3 or the complement of SEQ ID NO: 3.
  • at least one SNP is located within SEQ ID NO: 4 or the complement of SEQ ID NO: 4.
  • the invention pertains to a method of identifying a gene associated with colorectal cancer (CRC) comprising: (a) identifying a gene containing a SNP that is located within a sequence selected from the group consisting of sequences identified by SEQ ID NOS: 1-4 and the complements of sequences identified by SEQ ID NOS: 1-4; and (b) comparing the expression of the gene in an individual having the at-risk allele with the expression of the gene in an individual having the non-risk allele for differences indicating that the gene is associated with colorectal cancer (CRC).
  • CRC colorectal cancer
  • the invention pertains to a method of identifying a gene associated with colorectal cancer (CRC) comprising: (a) identifying a gene containing an at-risk haplotype identified in Figure 4C; and (b) comparing the expression of the gene in an individual having the at-risk haplotype with the expression of the gene in an individual not having the at-risk haplotype for differences indicating that the gene is associated with colorectal cancer (CRC).
  • CRC colorectal cancer
  • the isolated nucleic acid can be from about 3 to 101 nucleotides in length.
  • the isolated nucleic acid being a length selected from the group of from about 5 to 101, from about 7 to 101, from about 9 to 101, from about 15 to 101, from about 20 to 101, from about 25 to 101, from about 30 to 101, from about 40 to 101, from about 50 to 101, from about 60 to 101, from about 70 to 101, from about 80 to 101, from about 90 to 101, and from about 99 to 101 nucleotides in length.
  • the SNP is selected from the group of rs334338, rs334349, rsl590 and rs7871490.
  • a polynucleotide useful to predict colorectal cancer (CRC) risk comprising a complement to a sequence selected from the group of SEQ ID NOs: 1-4.
  • the complement can be from about 3 to 101 nucleotides in length.
  • the complement can be a length selected from the group of from about 5 to 101, from about 7 to 101, from about 9 to 101, from about 15 to 101, from about 20 to 101, from about 25 to 101, from about 30 to 101, from about 40 to 101, from about 50 to 101, from about 60 to 101, from about 70 to 101, from about 80 to 101, from about 90 to 101, and from about 99 to 101 nucleotides in length.
  • the polynucleotide has a Single Nucleotide Polymorphism (SNP) selected from the group of rs334338, rs334349, rsl590 and rs7871490.
  • the complement can be an allele-specific probe or primer.
  • the complement can be from about 3 to 101 nucleotides in length.
  • a method of distinguishing patients having an increased susceptibility to colorectal cancer (CRC) from patients who do not includes the step of detecting at least one Single Nucleotide Polymorphism (SNP) in any of SEQ ID NOs: 1-4 in a nucleic acid sample from the patients, wherein the presence or absence of the SNP can be used to assess increased susceptibility to CRC.
  • SNP Single Nucleotide Polymorphism
  • the presence of the SNP is an indication that patients have an increased susceptibility to CRC. In certain other embodiments, the presence of the SNP is an indication that patients have a decreased susceptibility to CRC.
  • the method includes distinguishing patients where the SNP is selected from the group of rs334338, rs334349, rsl590 and rs7871490.
  • the method includes determining the colorectal cancer (CRC) risk in a patient, comprising the step of identifying one or more Single Nucleotide Polymorphism (SNP) in any of SEQ ID NOS: 1-4 in a nucleic acid sample from the patient.
  • CRC colorectal cancer
  • the method includes determining CRC risk, where the presence of the SNP is an indication that the patient has a risk of CRC.
  • the method includes determining the CRC risk, where the presence of the SNP is an indication that the patient does not have a risk of CRC.
  • the method includes determining the CRC risk, where the SNP is selected from the group of rs334338, rs334349, rsl590 and rs7871490.
  • the method includes of detecting CRC, where the genetic material is combined with one or more polynucleotide probes capable of hybridizing selectively to a SNP in any of SEQ ID NOS: 1-4.
  • the method includes detecting CRC, where the probes are oligonucleotides capable of priming polynucleotide synthesis in a polymerase chain reaction.
  • the method includes detecting CRC, where the genetic material comprises one or more of: DNA, RNA and/or where the genetic material is amplified.
  • nucleotides present in one or several of the SNP markers in an individual's nucleic acid can be done by any method or technique capable of determining nucleotides present at a polymorphic site.
  • nucleotides present in SNP markers can be determined from either nucleic acid strand or from both strands.
  • Each of the 4 hybrid clones contained either the maternal or paternal copy of chromosome 9, plus the mouse genome (10).
  • ASE determination in the diploid samples indicated that the expression of one allele (a) was reduced compared to the other allele (b).
  • the densitometric values of the RT- PCR of human TGF ⁇ Rl were compared with the corresponding values for mouse Gpi (10).
  • One allele (a) showed reduced expression in both patients.
  • TGF- ⁇ lymphoblastoid cell lines from four ASE patients and four non-ASE healthy controls were exposed to TGF- ⁇ (10), which binds TGF ⁇ R2 and leads to the formation of the TGF ⁇ R2/TGF ⁇ Rl/TGF- ⁇ heteromeric complex.
  • TGF- ⁇ binds TGF ⁇ R2 and leads to the formation of the TGF ⁇ R2/TGF ⁇ Rl/TGF- ⁇ heteromeric complex.
  • pSMAD2 phosphorylated SMAD2
  • SMAD3 The phosphorylation of SMAD3 is an essential step in signal transduction by TGF- ⁇ for inhibition of cell proliferation (13). Furthermore, SMADd3-deficient mice are prone to colon cancer development (14, 15).
  • TGF ⁇ Rl ASE an antibody targeting the Ser423/425 site in SMAD3 (10, 16). Constitutive levels of pSMAD3 were detectable in the lymphoblastoid cell lines of three non-ASE controls while pSMAD3 was barely detectable in one ASE case (Figure 3C).
  • a GCG trinucleotide variable number of tandem repeat polymorphism occurs in exon 1 of TGF ⁇ Rl.
  • the most common allele contains 9 repeats leading to a stretch of 9 alanines (9A) in the signal peptide of the receptor protein.
  • the second most common allele has 6 repeats (6A) and occurs in approximately 14% of all individuals in most Caucasian populations (6).
  • the 6 A allele has been associated with a low-level but statistically significant predisposition to several forms of cancer (17-20). Recent studies suggest that the association of 6A with colon cancer is either weak (O.R.1.2, 95% CI: 1.01-1.43) (17) or borderline significant (O.R.I.13, CI: 0.98-1.30) (21).
  • the mutations were: c.634G>A (p.Gly212Asp) in one tumor, and c.682_685delAAG (p.delGlu228) in two tumors. These mutations occurred in exon 4 which encodes the kinase domain of the protein. LOH analyses and exon 4 sequencing in 49 tumors of CRC patients without ASE showed that none of these tumors had evidence of somatically- acquired mutations, and 5 showed LOH (Table 3 ( Figure 9)).
  • TGF ⁇ Rl ASE of TGF ⁇ Rl contributes to CRC development.
  • the TGF- ⁇ pathway is strongly involved in the carcinogenesis of colon and other cancers and its signaling is dependent on the integrity of both of its receptors (TGF ⁇ Rl and TGF ⁇ R2) (22, 23).
  • TGF ⁇ Rl and TGF ⁇ R2 TGF ⁇ R2 and TGF ⁇ R2
  • the inventors also investigated what proportion of all CRC is attributable to ASE of TGF ⁇ Rl. From the available data of the present case-control study we estimated the population attributable risk (PAR). If ASE occurs in 21% of cases and 3% of controls, the estimated PAR is 18.7% (CI: 10.8-25.8). If ASE occurs in 12% of cases and 1.5% of controls the estimated PAR is 10.6% (CI: 6.0-14.9). These numbers are estimates, representing the Caucasian-dominated population of Central Ohio, and are heavily dependent on the relevant allele frequencies which may show strong inter-ethnic variation. We nevertheless conclude that ASE of TGFBRl is a major contributor to the genetic predisposition to CRC.
  • Controls were obtained from the Ohio State University Division of Human Genetics collection of samples.
  • Polymorphisms in the cDNA of the gene were used as markers to distinguish and measure the expression of the two alleles, using the SNaPshot (PE Applied Biosystems, Foster City, CA) technique as described (34).
  • SNaPshot PE Applied Biosystems, Foster City, CA
  • ASE ratio calculations were performed as described (34). Briefly, the ratio of the two alleles in the cDNA of the transcript was normalized with the ratio of the two alleles in genomic DNA, applying the following formula: cDNA (peak area common allele / peak area rare allele) divided by gDNA (peak area common allele / peak area rare allele).
  • cDNA peak area common allele / peak area rare allele
  • gDNA peak area common allele / peak area rare allele
  • LOH was determined by the same SNaPshot methodology, markers and cut-off values as the ASE evaluation.
  • the normalized ratio was calculated as: tumor DNA (peak area common allele / peak area rare allele) divided by germline DNA (peak area common allele / peak area rare allele). All primers and PCR conditions are available upon request.
  • Hybrid cell lines were cultured under selective pressure using Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% HAT supplement, 1% Geneticin, 1% penicillin- streptomycin and 1% Minimum Essential Medium - Non Essential Amino Acids solution (MEM-NEAA), all from Gibco (Invitrogen).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • HAT supplement HAT supplement
  • Geneticin 1%
  • penicillin- streptomycin 1%
  • Minimum Essential Medium -N Essential Amino Acids solution MEM-NEAA
  • TBFBRl transcript semi-quantitative RT-PCR was performed for each clone.
  • Human TGF ⁇ Rl forward and reverse primers were designed in different exons (exons 7 and 8, respectively), creating a 135 bp amplicon.
  • Murine Gpi was used as amplification control (size 176 bp), taking advantage of the fact that the hybrid cell lines carried all mouse chromosomes. After separation in a 2.5% agarose gel the relative amount of human product was quantified by comparing the densitometrically determined TGFBRl -human/Gpi-mouse ratios.
  • PCR products were incubated with ExoS AP-IT (USB Corp., Cleveland, OH) for 1 hour at 37 0 C and for 15 minutes at 75 0 C. All products were sequenced in both directions using the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 and the Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, CA).
  • a 96.5 kb genomic region was studied for mutations by sequencing.
  • the analyzed region extends from the end of the COL15A1 gene (35 kb upstream of the first exon of TGF ⁇ Rl) to 12.5 kb downstream of the TGF ⁇ Rl 3'-UTR.
  • the entire region was divided into 18 overlapping amplicons of 1.7 to 10 kb. Each fragment was PCR amplified using the Expand Long Template PCR System (Roche Applied Science, Mannheim, Germany).
  • the long- range PCR amplification products were cloned into chemically competent TOPlO cells (Invitrogen, Carlsbad, CA) following a standard cloning protocol and using a pBluescript- modified vector. Clones were analyzed by restriction enzyme digestion and positive clones were sequenced. Forward and reverse sequencing was performed with primers separated by 400 bp creating overlapping amplicons. For each of the 18 large amplicons at least three clones were sequenced for the monochromosomal hybrids and at least six clones for the diploid samples. Only the changes common to all clones were considered, so that artifacts produced by the long-range PCR amplification were eliminated from the analysis.
  • the expected PCR bands were excised from 2.0% agarose gels, purified using the QIAquick Gel Extraction Kit (Qiagen, Germantown, MD), and then sequenced with a dGTP BigDyeTM Terminator Version 3.0 Ready Reaction Cycle Sequencing Kit on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA).
  • Lymphobastoid cell lines were grown in RPMI with 20% FBS, 2% antibiotic- antimycotic solution (all from Gibco, Invitrogen), and 0.33% Tylosin (Sigma) at 37 0 C, 5% CO2 and -85% humidity. After overnight serum starvation (0.1% FBS) TGF- ⁇ l (R&D Systems, Minneapolis, Minn) was added to the medium to a final concentration of 10OpM. Cells were harvested at different time points: before the addition of TGF- ⁇ (time 0) and after 1, 4, 8 and 16 hours.
  • lymphoblastoid cell nuclear extracts were isolated by using NE-PER nuclear and cytoplasmic extraction kit (cat # 78833) (Pierce, Rockford, IL-61105). Fifteen micrograms of protein from each sample were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane.
  • Rabbit anti-pSmad3 (Ser423/425) (pSMAD3) was a gift from Dr. Koichi Matsuzaki, Kanzai Medical University, Osaka, Japan.
  • Mouse anti-Histone 1 sc-8030 was used as a loading control and purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • the present invention provides methods for assessing the genetic predisposition of a subject to develop colorectal cancer and potentially other cancers.
  • the detection method is based upon the differential expression of alleles (or presence of their underlying haplotypes) of TGF ⁇ Rl in normal somatic cells (such as white blood cells).
  • the differential allelic expression was termed allele specific expression (ASE).
  • ASE allele specific expression
  • TGF ⁇ Rl alleles or their underlying haplotypes are lower expressed in people with CRC and that this lowered expression is heritable. These risk-alleles (or their underlying haplotypes) are more common in CRC cancer patients than normal controls.
  • ASE in a sample of cells from a subject may be used to predict relative risk to develop CRC and potentially other cancers, and ultimately the prognosis, for that subject.
  • a biomarker ASE of TGF ⁇ Rl provides information as to the risk that someone will be more likely to develop CRC and potentially other cancers.
  • One embodiment includes measuring ASE in a sample of cells from an unaffected subject.
  • the ASE values may then be used to generate a risk score that is predictive of predisposition to cancer.
  • the ASE may be measured by a variety of techniques that are well known in the art. Quantifying the total or allelic levels of the messenger RNA (mRNA) of TGF ⁇ Rl may be used to define the level of total mRNA or the level of ASE. Alternatively, quantifying the levels of the protein product of TGF ⁇ Rl may be used to measure the expression of the biomarker. Additional information regarding the methods discussed below may be found in Ausubel et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or Sambrook et al. (1989) .Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. One skilled in the art will know which parameters may be manipulated to optimize detection of the mRNA or protein of interest.
  • ASE ratio calculations may be performed as described (S3). Briefly, the ratio of the two alleles in the cDNA of the transcript was normalized with the ratio of the two alleles in genomic DNA, applying the following formula: cDNA (peak area common allele / peak area rare allele) divided by gDNA (peak area common allele / peak area rare allele).
  • cDNA peak area common allele / peak area rare allele
  • gDNA peak area common allele / peak area rare allele
  • a nucleic acid microarray may also be used to quantify the differential expression of the biomarker.
  • Microarray analysis may be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology (Santa Clara, CA) or the Microarray System from Incyte (Fremont, CA).
  • Affymetrix GeneChip® technology Santa Clara, CA
  • the Microarray System from Incyte Fremont, CA
  • single- stranded nucleic acids e.g., cDNAs or oligonucleotides
  • the arrayed sequences are then hybridized with specific nucleic acid probes from the cells of interest.
  • Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
  • the RNA may be amplified by in vitro transcription and labeled with a marker, such as biotin.
  • the labeled probes are then hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove the non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
  • the raw fluorescence intensity data in the hybridization files are generally preprocessed with the robust multichip average (RMA) algorithm to generate expression values.
  • RMA robust multichip average
  • Quantitative real-time PCR may also be used to measure the differential expression of the biomarker.
  • the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction.
  • the amount of PCR product is followed cycle -by-cycle in real time, which allows for determination of the initial concentrations of mRNA.
  • the reaction may be performed in the presence of a fluorescent dye, such as SYBR Green, which binds to double- stranded DNA.
  • the reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified.
  • a non-limiting example of a fluorescent reporter probe is a TagMan® probe (Applied Biosystems, Foster City, CA).
  • the fluorescent reporter probe fluoresces when the quencher is removed during the PCR extension cycle.
  • Muliplex QRT-PCR may be performed by using multiple gene- specific reporter probes, each of which contains a different fluorophore. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. To minimize errors and reduce any sample-to- sample variation, QRT-PCR is typically performed using a reference standard. The ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
  • Suitable reference standards include, but are not limited to, mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and beta-actin.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • beta-actin beta-actin
  • Immunohistochemical staining may also be used to measure the differential expression of the biomarker.
  • This method enables the localization of a protein in the cells of a tissue section by interaction of the protein with a specific antibody.
  • the tissue may be fixed in formaldehyde or another suitable fixative, embedded in wax or plastic, and cut into thin sections (from about 0.1 mm to several mm thick) using a microtome.
  • the tissue may be frozen and cut into thin sections using a cryostat.
  • the sections of tissue may be arrayed onto and affixed to a solid surface (i.e., a tissue microarray).
  • the sections of tissue are incubated with a primary antibody against the antigen of interest, followed by washes to remove the unbound antibodies.
  • the primary antibody may be coupled to a detection system, or the primary antibody may be detected with a secondary antibody that is coupled to a detection system.
  • the detection system may be a fluorophore or it may be an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can convert a substrate into a colorimetric, fluorescent, or chemiluminescent product.
  • the stained tissue sections are generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for the biomarker.
  • An enzyme-linked immunosorbent assay may be used to measure the differential expression of the biomarker.
  • an ELISA assay There are many variations of an ELISA assay. All are based on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate.
  • the original ELISA method comprises preparing a sample containing the biomarker proteins of interest, coating the wells of a microtiter plate with the sample, incubating each well with a primary antibody that recognizes a specific antigen, washing away the unbound antibody, and then detecting the antibody- antigen complexes.
  • the antibody- antibody complexes may be detected directly.
  • the primary antibodies are conjugated to a detection system, such as an enzyme that produces a detectable product.
  • the antibody- antibody complexes may be detected indirectly.
  • the primary antibody is detected by a secondary antibody that is conjugated to a detection system, as described above.
  • the microtiter plate is then scanned and the raw intensity data may be converted into expression values using means known in the art.
  • An antibody microarray may also be used to measure the differential expression of the biomarker.
  • a plurality of antibodies is arrayed and covalently attached to the surface of the microarray or biochip.
  • a protein extract containing the biomarker proteins of interest is generally labeled with a fluorescent dye.
  • the labeled biomarker proteins are incubated with the antibody microarray. After washes to remove the unbound proteins, the microarray is scanned.
  • the raw fluorescent intensity data maybe converted into expression values using means known in the art.
  • Luminex multiplexing microspheres may also be used to measure the differential expression of the biomarker.
  • These microscopic polystyrene beads are internally color-coded with fluorescent dyes, such that each bead has a unique spectral signature (of which there are up to 100). Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest (i.e., biomarker mRNA or protein, respectively).
  • the target is also tagged with a fluorescent reporter.
  • there are two sources of color one from the bead and the other from the reporter molecule on the target.
  • the beads are then incubated with the sample containing the targets, of which up 100 may be detected in one well.
  • the small size/surface area of the beads and the three dimensional exposure of the beads to the targets allows for nearly solution-phase kinetics during the binding reaction.
  • the captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay.
  • the data from the acquisition files may be converted into expression values using means known in the art.
  • In situ hybridization may also be used to measure the differential expression of the biomarker.
  • This method permits the localization of mRNAs of interest in the cells of a tissue section.
  • the tissue may be frozen, or fixed and embedded, and then cut into thin sections, which are arrayed and affixed on a solid surface.
  • the tissue sections are incubated with a labeled antisense probe that will hybridize with an mRNA of interest.
  • the hybridization and washing steps are generally performed under highly stringent conditions.
  • the probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be detected and visualized under a microscope.
  • each antisense probe may be detected simultaneously, provided each antisense probe has a distinguishable label.
  • the hybridized tissue array is generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for each biomarker.
  • ASE of TGF ⁇ Rl or the presence of underlying haplotypes is measured by SNAPshot technology in RNA or DNA isolated from a peripheral blood sample of an individual; no biopsy material or cancer cells are necessary.
  • RNA or protein may also be extracted from a fixed or wax-embedded sample.
  • the subject will generally be a mammalian subject.
  • Mammals may include primates, livestock animals, and companion animals.
  • Primates may include humans, apes, monkeys, and gibbons;
  • Livestock animals may include horses, cows, goats, sheep, deer and pigs;
  • Companion animals may include dogs, cats, rabbits, and rodents (including mice, rats, and guinea pigs).
  • the subject is a human.
  • haplotypes from the genotype and known haplotype data were performed with the PHASE V2.1.1 program (37,38). For this purpose, only SNPs with a minimum minor allele frequency of 0.05, and a maximum presence of missing values of 10% were chosen. Permutation based test available through the PHASE program was used to test for significant differences in haplotype frequency distributions between cases and controls, with 1000 permutations. This tests the null hypothesis that the case and control haplotypes are a random sample from a common set of haplotype frequencies, versus the alternative that cases are more similar to each other than to controls. Haplotype frequencies of inferred haplotypes were compared between cases and controls by grouping each haplotype versus all the others together, using Fisher's exact test. Haplotype inference and LOD-score calculation in families was performed with MERLIN (39).
  • Receiver operating characteristic (ROC) analysis was performed by estimating sensitivity and specificity for varied ASE cut-off points. Youden's index (40) was calculated for several cut-off values (Table 2 ( Figure 8)). [00191] Odds ratios (OR) and 95% confidence intervals were estimated using unconditional maximum likelihood estimation (WaId) method and with normal approximation. Estimated population attributable risks (PAR) were obtained from a case-control study by using the method described by Armitage et al. (40).
  • kits for predicting the risk for developing CRC comprises a plurality of agents for measuring differential expression of alleles (or presence of their underlying haplotypes) of TGF ⁇ Rl, means for converting the expression data into ASE values or classifying genotypes into haplotypes, and means for generating risk scores that indicate relative risk to develop CRC and potentially other cancers.
  • the agents in the kit for measuring ASE or generate genotypes may comprise a plurality of PCR probes and primers.
  • kits for detecting CRC an individual comprising one or more reagents for detecting 1) one or more microRNAs; 2) one or more target genes of one or more microRNAs; 3) one or more polypeptides expressed by the target genes or 4) a combination thereof.
  • the kit can comprise hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele- specific oligonucleotides, and antibodies that bind to the polypeptide expressed by the target gene.
  • the kit comprises at least contiguous nucleotide sequence that is substantially or completely complementary to a region one or more of the microRNAs.
  • one or reagents in the kit are labeled, and thus, the kits can further comprise agents capable of detecting the label.
  • a method of prevention of colorectal cancer (CRC) morbidity and mortality in the population comprising administering a diagnostic screening to individuals in the population, and if an individual has at least one risk factor selected from the group: an at-risk haplotype for CRC; an at-risk haplotype in the TGF ⁇ Rl gene; an at-risk polymorphism in TGF ⁇ Rl; dysregulation of TGF ⁇ Rl mRNA expression; dysregulation of a TGF ⁇ Rl mRNA isoform; or decreased TGF ⁇ Rl protein expression, the individual then can undergo routine colonoscopy and potentially therapy to prevent CRC from developing or spreading, thereby lowering CRC morbidity and mortality.
  • CRC colorectal cancer

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Abstract

La présente invention porte sur une méthode d'évaluation d'une susceptibilité génétique à un cancer colorectal et potentiellement à d'autres cancers chez un sujet, laquelle méthode comprend la mesure de l'expression spécifique d'un allèle ou de la présence d'haplotypes à risque, une différence de l'expression ou de la présence d'haplotypes à risque étant indicative d'un cancer colorectal (CRC) ou d'une prédisposition à un CRC.
PCT/US2009/053582 2008-08-12 2009-08-12 Polymorphismes associés à un cancer colorectal en développement, leurs méthodes de détection et d'utilisations WO2010019690A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20100267028A1 (en) * 2008-12-24 2010-10-21 Boris Pasche Tgfbr1 HAPLOINSUFFICIENCY MODIFIES RISK FOR CANCER
US8492096B2 (en) * 2008-12-24 2013-07-23 Boris Pasche TGFBR1 expression modifies risk for colorectal cancer
EP2572000B1 (fr) * 2010-05-19 2017-01-11 Signature Diagnostics AG Procédé pour diagnostiquer le cancer colorectal
US20150275307A1 (en) * 2012-10-16 2015-10-01 University Of Utah Research Foundation Compositions and methods for detecting sessile serrated adenomas/polyps
NL2033031B1 (en) * 2022-08-22 2024-03-04 Jiangsu Vocational College Medicine Single nucleotide polymorphism (snp) molecular marker and kit for assessing tumor progression risk of patient with carcinoma of colon and rectum, and use thereof
CN116334223A (zh) * 2023-03-02 2023-06-27 武汉大学 可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用
CN116334223B (zh) * 2023-03-02 2023-12-22 武汉大学 可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用

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