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WO2009117595A2 - Variations de séquence sur un risque de prédiction du chromosome 15 pour le cancer du poumon - Google Patents

Variations de séquence sur un risque de prédiction du chromosome 15 pour le cancer du poumon Download PDF

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WO2009117595A2
WO2009117595A2 PCT/US2009/037694 US2009037694W WO2009117595A2 WO 2009117595 A2 WO2009117595 A2 WO 2009117595A2 US 2009037694 W US2009037694 W US 2009037694W WO 2009117595 A2 WO2009117595 A2 WO 2009117595A2
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seq
nucleic acid
chromosome
lung cancer
subject
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WO2009117595A3 (fr
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Christopher Amos
Margaret Spitz
Richard Houlston
Xifeng Wu
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to the fields of oncology and genetics. More particularly, it concerns methods for diagnosing or determining risk of lung cancer that involve polymorphism analysis of loci on chromosome 15 and analysis of chromosome 15 for deletions and insertions.
  • Lung cancer is frequently cited as a malignancy solely attributable to environmental exposures, primarily cigarette smoke.
  • evidence that genetic factors influence lung cancer susceptibility has been provided by numerous studies since the landmark study of Tokuhata and Lilienfeld (1963) which demonstrated a 2.5-fold higher risk to first-degree relatives of lung cancer cases compared with controls, with a lower relative risk to nonsmoking relatives of cases compared to nonsmoking relatives of controls.
  • Subsequent epidemiological case-control analyses have consistently provided evidence for an approximately two-fold increased risk in relatives of cases (Amos et al., 1999; Jonsson et al., 2004).
  • Direct evidence for a genetic predisposition to lung cancer is provided by the increased risk associated with a number of rare Mendelian cancer syndromes, such as carriers of constitutional tumor protein p53 (TP53) (Hwang et al., 2003) and retinoblastoma gene mutations (Sanders et al., 1989; Kleinerman et al., 2000), as well as in patients with Bloom's syndrome (Takemiya et al., 1987), Werner's syndrome (Yamanaka et al., 1997), and strongly familial lung cancer (Bailey- Wilson et al., 2004).
  • TP53 constitutional tumor protein p53
  • retinoblastoma gene mutations anders et al., 1989; Kleinerman et al., 2000
  • Bloom's syndrome Takemiya et al., 1987
  • Werner's syndrome Yamamiya et al., 1997)
  • strongly familial lung cancer Bailey- Wilson e
  • the invention is in part based on the finding that risk variants for lung cancer include polymorphisms on chromosome 15 and insertions/deletions of chromosome 15. For example, particular SNPs within a region of chromosome 15 have been found to be predictive of risk of lung cancer. Genotyping of these variants can aid in the identification of subjects at risk for the development of lung cancer.
  • embodiments of the present invention generally include methods for diagnosing or predicting the development of lung cancer in a subject, involving determining whether a nucleic acid sequence obtained from the subject exhibits a polymorphism on chromosome 15.
  • polymorphism and “mutation” refer to the condition in which there is a variation in the DNA sequence between some members of a species.
  • mutation is used to denote a polymorphism that results in the gene coding for a nonfunctioning protein or a protein with a substantially altered or reduced function or that additionally contributes to a disease condition.
  • the polymorphism is in the region of 15q25.1 of chromosome 15.
  • the methods involve determining whether a nucleic acid sequence obtained from the subject exhibits a polymorphism of an 117 kb region of chromosome 15 extending from 76593078 bp to 76710000 bp, wherein the polymorphism is indicative of said subject having lung cancer or developing lung cancer compared to a subject or group of subjects without the polymorphism.
  • a wild-type consensus sequence strand of the region of chromosome 15 extending from 76593000 bp to 76710000 bp is provided in SEQ ID NO:37.
  • determining whether a nucleic acid sequence exhibits a polymorphism involves comparing the polymorphism in an 117 kb region of chromosome 15 extending from 76593078 bp to 7671000 bp to a wild type 117 kb region of chromosome 15 extending from 76593078 bp to 7671000 bp.
  • the polymorphism is a single nucleotide polymorphism (SNP).
  • SNP associated with risk is at position 76593078 bp or position 7671000 bp.
  • any other SNP in the region of chromosome 15q25.1 that is associated with these SNPs and shows a standardized disequilibrium coefficient above 0.6 is also predictive of lung cancer risk and is covered by this patent application.
  • the polymorphism may be a mutation in or near a gene that encodes nicotinic acetylcholine receptor alpha subunit 3, nicotinic acetylcholine receptor alpha subunit 5, proteasome alpha 4 subunit isoform 1, or LOC123688 isoform 1, each of which is located on 15q25.1. Determining whether a particular nucleic acid sequence exhibits a polymorphism can be by any method known to those of ordinary skill in the art. Techniques that may be involved in this determination are well-known to those of ordinary skill in the art.
  • Examples of such techniques include allele specific oligonucleotide hybridization, size analysis, sequencing, hybridization, 5' nuclease digestion, single-stranded conformation polymorphism analysis, allele specific hybridization, primer specific extension, and oligonucleotide ligation assays. Additional information regarding these techniques is discussed in the specification below.
  • the distribution of polymorphic patterns in a large number of individuals is determined, and compared with the distribution of polymorphic patterns in subjects that have been matched for age, ethnic origin, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different status markers. Correlations are achieved using any statistical method known in the art, including but not limited to nominal logistic regression or standard least squares regression analysis. In this manner, it is possible to establish statistically significant correlations between particular polymorphic patterns and lung cancer.
  • the methods set forth herein further involve obtaining a biological sample from the subject and testing the biological sample to identify whether a polymorphism is contained within the 117 kb region of chromosome 15.
  • the biological sample may be obtained from any nucleated cell source.
  • Non-limiting examples of cell sources available in clinical practice include nucleated blood cells, buccal cells, any cells present in tissue obtained by biopsy or any other cell collection method.
  • Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine, cerebrospinal fluid, feces, and tissue exudates at the site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art.
  • Determination of the sequence of the extracted nucleic acid at a locus of interest is achieved by any means known in the art, including but not limited to direct sequencing, hybridization with allele-specific oligonucleotides, allele-specif ⁇ c PCR, ligase-PCR, HOT cleavage, denaturing gradient gel electrophoresis (DDGE), and single-stranded conformational polymorphism (SSCP) analysis.
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method, by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; and sequencing using a chip-based technology.
  • DNA from a patient is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • the method further involves amplification of a nucleic acid from said sample.
  • the amplification may or may not involve PCR.
  • the primers are located on a chip.
  • the subject can be any subject, such as a mammal.
  • mammals include mice, rats, rabbits, dogs, cats, sheep, goats, non-human primates (chimpanzees, baboons, monkeys), cows, horses, pigs, and humans.
  • the subject is a human.
  • the human is a subject who has no history of cancer.
  • the subject is a human with a history of a previously treated cancer.
  • the previously treated cancer may be a cancer treated by surgical excision, chemotherapy, and/or radiation therapy.
  • the subject has a history of cancer, such as a non-lung cancer.
  • the subject has one or more risk factors associated with the development of cancer.
  • risk factors for the development of cancer include a history of smoking or a family history of cancer.
  • the method may involve further analyzing the subject for the presence of one or more additional risk factors for the development of cancer.
  • the additional risk factor may be a history of smoking, or a history of cancer that has been previously treated, or a family history of cancer.
  • the method further involves making a decision on the timing and/or frequency of cancer diagnostic testing for said subject.
  • the method may further comprise making a decision on the timing and/or frequency of prophylactic cancer treatment or screening for said subject.
  • the invention also involves methods for determining the need for routine diagnostic testing of a subject for lung cancer comprising analyzing a nucleic acid-containing sample from the subject for the presence of an SNP in a 117 kb region of strong LD on chromosome
  • SNP is at position 76593078 bp or position 7671000 bp.
  • some embodiments concern a method for diagnosing or predicting the development of lung cancer in a subject, involving determining whether a nucleic acid sequence obtained from the subject exhibits an insertion or a deletion in the 15q25.1 region of chromosome 15, wherein the insertion or deletion is indicative of said subject having lung cancer or developing lung cancer compared to a subject without the insertion or deletion.
  • a method for diagnosing or predicting the development of lung cancer in a subject comprising determining whether a nucleic acid sequence obtained from the subject exhibits an insertion or a deletion in the 15q25.1 region of chromosome 15, wherein the insertion or deletion is indicative of said subject having lung cancer or developing lung cancer compared to a subject without the insertion or deletion
  • the insertion comprises an insertion of GCGG (SEQ ID NO:40).
  • the insertion consists of an insertion of GCGG (SEQ ID NO:40).
  • the insertion is at position within 76,700,410 bp of chromosome 15.
  • the insertion comprises an insertion of A (adenosine nucleobase).
  • the insertion may be at position 76,700,126 bp of chromosome 15.
  • the nucleic acid sequence obtained from the subject exhibits a deletion in the 15q25.1 region of chromosome 15.
  • the deletion may comprise a deletion of a nucleic acid sequence comprising SEQ ID NO:40 or SEQ ID NO:43 in the 15q25.1 region of chromosome 15.
  • the deletion is at position 76,700,410 bp to 76,700,417 bp or 76,644,869 bp to 76,644,890 bp of chromosome 15.
  • the present invention also provides isolated nucleic acids comprising the polymorphic positions set forth herein; vectors comprising the nucleic acids; transformed host cells comprising the vectors, and transgenic animals that include the polymorphic positions set forth herein.
  • the invention also provides probes which are useful for detecting these polymorphisms.
  • the present invention also contemplates the preparation of kits for use in accordance with the present invention. Suitable kits include various reagents for use in accordance with the present invention in suitable containers and packaging materials, including tubes, vials, and shrink-wrapped and blow-molded packages. Kits are discussed in greater detail in the specification below.
  • a kit of the present invention may include a gene specific PCR primer pair that anneal to DNA that flank a site within a 117 kb region of chromosome 15 extending from 76593078 bp to 7671000 bp.
  • the primers flank a SNP that is at position 76593078 bp or position 7671000 bp.
  • the kit includes at least one primer that comprises SEQ ID NO:38 or SEQ ID NO:39.
  • the kit includes a first primer comprising SEQ ID NO:38 and a second primer comprising SEQ ID NO:39.
  • the primer or primers may optionally be attached to a chip.
  • the kit may include one or more reagents for DNA amplification.
  • kits that include a chip and a single-stranded nucleic acid sequence attached to the chip, wherein the single-stranded nucleic acid sequence comprises at least 20 contiguous bases of SEQ ID NO:37.
  • the single- stranded nucleic acid may comprise at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more contiguous bases of SEQ ID NO:37.
  • the single-stranded nucleic acid sequence comprises at least 20-200 contigous bases of SEQ ID NO:37.
  • the single-stranded nucleic acid sequence comprises at least 20-100 contiguous bases of SEQ ID NO:37.
  • kits that includes a chip and a single-stranded nucleic acid sequence attached to the chip, wherein the single-stranded nucleic acid sequence includes at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12. SEQ ID NO:13.
  • SEQ ID NO:14 SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
  • kits that includes a PCR primer that anneals to DNA that flank a site within a region of chromosome 15, wherein the primer includes SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51.
  • the kit comprises a primer that consists of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51.
  • the kit may include a single primer or multiple primers.
  • composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • FIG. 1 Results from genome-wide association analysis of directly tested SNPs in the Texas Discovery set using Illumina 300K HumanHap version 1.1 Beadchips.
  • FIG. 2. Genome-wide results from analysis after adjustment for population substructure defined by forming 44 clusters of individuals showing identity by state similarity.
  • FIG. 3 The 15q25.1 locus.
  • the top panel shows SNP single marker association results. Results in blue depict genotyped SNPs while results in red are for imputed SNPs. All known genes and predicted transcripts in the local area are shown. Positions are that of UCSC March 2006 assembly; NCBI build 36.1.
  • the bottom panel shows the LD structure at 15q21.4. Boxes are shaded according to the square of the correlation coefficient (r 2 ), derived from Phase 1 genotypes in Haploview software (v3.2).
  • FIG. 4 Effects of SNPs according to smoking behavior in current, former and never smokers adjusting for age, sex, and packyears of tobacco smoke exposure.
  • the X-axis indicates the extent of exposure, starting with never smokers (UK population, panel C only), followed by former smokers who quit 24 or more years ago, former smokers who quit 15-23 years ago, formers smokers who quit less than 15 years ago and current smokers.
  • Panel B presents data from the Texas discovery set
  • panel presents data from the Texas replication set
  • Panel C presents data from the UK replication set.
  • Suitable tissues include almost any nucleic acid containing tissue.
  • Non- limiting examples include blook (white blood cells) and oral mucosal tissue (e.g., obtained with a swab).
  • the tissue can be obtained using any method known to those of ordinary skill in the art.
  • blood may be collected in heparinized syringes or other appropriate vessel following venipuncture with a hypodermic needle.
  • Oral tissue may advantageously be obtained from a mouth rinse.
  • Oral tissue or buccal cells may be collected with oral rinses or by swabbing of the oral cavity, or any other method known to those of ordinary skill in the art.
  • SNP-ITTM SNP-Identif ⁇ cation Technology
  • Sequenom uses a hybridization capture technology plus MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time-of-Flight mass spectrometry) to detect sequence variation with their MassARRAYTM system.
  • MALDI-TOF Microx Assisted Laser Desorption/Ionization-Time-of-Flight mass spectrometry
  • Promega has the READITTM SNP/Genotyping System (U.S. Patent 6,159,693).
  • DNA or RNA probes are hybridized to target nucleic acid sequences.
  • Probes that are complementary to the target sequence at each base are depolymerized with a proprietary mixture of enzymes, while probes which differ from the target at the interrogation position remain intact.
  • the method uses pyrophosphorylation chemistry in combination with luciferase detection to provide a highly sensitive and adaptable SNP scoring system.
  • Third Wave Technologies has the Invader OSTM method that uses their proprietary Cleavase® enzymes, which recognize and cut only the specific structure formed during the Invader process.
  • the Invader OS relies on linear amplification of the signal generated by the Invader process, rather than on exponential amplification of the target.
  • Invader OS assay does not utilize PCR in any part of the assay.
  • nucleic acid arrays placed on chips As discussed above, one convenient approach to detecting variation involves the use of nucleic acid arrays placed on chips. This technology has been widely exploited by companies such as Affymetrix, and a large number of patented technologies are available. Specifically contemplated are chip-based DNA technologies such as those described by Hacia et al (1996) and Shoemaker et al. (1996). These techniques involve quantitative methods for analyzing large numbers of sequences rapidly and accurately. The technology capitalizes on the complementary binding properties of single stranded DNA to screen DNA samples by hybridization (Pease et al, 1994; Fodor et al, 1991).
  • a DNA array or gene chip consists of a solid substrate to which an array of single-stranded DNA molecules have been attached. For screening, the chip or array is contacted with a single-stranded DNA sample, which is allowed to hybridize under stringent conditions. The chip or array is then scanned to determine which probes have hybridized.
  • a gene chip or DNA array would comprise probes specific for chromosomal changes evidencing the predisposition towards the development of a neoplastic or preneoplastic phenotype.
  • such probes could include PCR products amplified from patient DNA synthesized oligonucleotides, cDNA, genomic DNA, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), chromosomal markers or other constructs a person of ordinary skill would recognize as adequate to demonstrate a genetic change.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • chromosomal markers or other constructs a person of ordinary skill would recognize as adequate to demonstrate a genetic change.
  • a variety of gene chip or DNA array formats are described in the art, for example U.S. Patents 5,861,242 and 5,578,832, which are expressly incorporated herein by reference.
  • a means for applying the disclosed methods to the construction of such a chip or array would be clear to one of ordinary skill in the art.
  • the basic structure of a gene chip or array comprises: (1) an excitation source; (2) an array of probes; (3) a sampling element; (4) a detector; and (5) a signal amplification/treatment system.
  • a chip may also include a support for immobilizing the probe.
  • a target nucleic acid may be tagged or labeled with a substance that emits a detectable signal.
  • the target nucleic acid may be immobilized onto the integrated microchip that also supports a phototransducer and related detection circuitry.
  • a gene probe may be immobilized onto a membrane or filter, which is then attached to the microchip or to the detector surface itself.
  • the immobilized probe may be tagged or labeled with a substance that emits a detectable or altered signal when combined with the target nucleic acid.
  • the tagged or labeled species may be fluorescent, phosphorescent, or otherwise luminescent, or it may emit Raman energy or it may absorb energy.
  • the DNA probes may be directly or indirectly immobilized onto a transducer detection surface to ensure optimal contact and maximum detection.
  • the ability to directly synthesize on or attach polynucleotide probes to solid substrates is well known in the art. See U.S. Patents 5,837,832 and 5,837,860, both of which are expressly incorporated by reference. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate.
  • Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, 1993), the direct covalent attachment of short, 5'-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et ah, 1991), or the precoating of the polystyrene or glass solid phases with poly- L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl- modif ⁇ ed oligonucleotides using bi-functional cross-linking reagents (Running et ah, 1990; Newton et al, 1993).
  • the probes When immobilized onto a substrate, the probes are stabilized and therefore may be used repeatedly.
  • hybridization is performed on an immobilized nucleic acid target or a probe molecule is attached to a solid surface such as nitrocellulose, nylon membrane or glass.
  • nitrocellulose membrane reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), and photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals) capable of forming covalent links with target molecules.
  • PVDF polyvinylidene difluoride
  • PVDF polystyrene substrates
  • polyacrylamide-based substrate other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), and photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals) capable of forming covalent links with target molecules.
  • Binding of the probe to a selected support may be accomplished by any of several means.
  • DNA is commonly bound to glass by first silanizing the glass surface, then activating with carbodiimide or glutaraldehyde.
  • Alternative procedures may use reagents such as 3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane (APTS) with DNA linked via amino linkers incorporated either at the 3' or 5' end of the molecule during DNA synthesis.
  • GOP 3-glycidoxypropyltrimethoxysilane
  • APTS aminopropyltrimethoxysilane
  • DNA may be bound directly to membranes using ultraviolet radiation. With nitrocellose membranes, the DNA probes are spotted onto the membranes.
  • a UV light source (StratalinkerTM, Stratagene, La Jolla, CA) is used to irradiate DNA spots and induce cross-linking.
  • An alternative method for cross-linking involves baking the spotted membranes at 8O 0 C for two hours in vacuum.
  • Specific DNA probes may first be immobilized onto a membrane and then attached to a membrane in contact with a transducer detection surface. This method avoids binding the probe onto the transducer and may be desirable for large-scale production.
  • Membranes particularly suitable for this application include nitrocellulose membrane (e.g., from BioRad, Hercules, CA) or polyvinylidene difluoride (PVDF) (BioRad, Hercules, CA) or nylon membrane (Zeta-Probe, BioRad) or polystyrene base substrates (DNA.BINDTM Costar, Cambridge, MA).
  • a useful technique in working with nucleic acids involves amplification.
  • Methods of amplification of nucleic acids are well-known in the art, and any such method can be applied in the context of the present invention.
  • Amplifications are usually template-dependent, meaning that they rely on the existence of a template strand to make additional copies of the template.
  • Primers short nucleic acids that are capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, are hybridized to the template strand.
  • primers are from ten to thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form generally is preferred.
  • pairs of primers are designed to selectively hybridize to distinct regions of a template nucleic acid, and are contacted with the template DNA "under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
  • hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences.
  • PCR polymerase chain reaction
  • pairs of primers that selectively hybridize to nucleic acids are used under conditions that permit selective hybridization.
  • primer encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • the primers are used in any one of a number of template dependent processes to amplify the target gene sequences present in a given template sample.
  • One of the best known amplification methods is PCR which is described in detail in U.S. Patents 4,683,195, 4,683,202 and 4,800,159, each incorporated herein by reference.
  • primer sequences are prepared which are complementary to regions on opposite complementary strands of the target-gene(s) sequence.
  • the primers will hybridize to form a nucleic-acid : primer complex if the target-gene(s) sequence is present in a sample.
  • An excess of deoxyribonucleoside triphosphates is added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase, that facilitates template-dependent nucleic acid synthesis.
  • the polymerase will cause the primers to be extended along the target-gene(s) sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target-gene(s) to form reaction products, excess primers will bind to the target-gene(s) and to the reaction products and the process is repeated. These multiple rounds of amplification, referred to as "cycles", are conducted until a sufficient amount of amplification product is produced.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et ah, 1989.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641, filed December 21, 1990.
  • LCR ligase chain reaction
  • LCR ligase chain reaction
  • two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut.
  • the two probe pairs will link to form a single unit.
  • bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs.
  • U.S. Patent 4,883,750 incorporated herein by reference, describes a method similar to LCR for binding probe pairs to a target sequence.
  • PCT/US87/00880 also may be used as still another amplification method in the present invention.
  • a replicative sequence of RNA which has a region complementary to that of a target, is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence, which can then be detected.
  • Isothermal Amplification An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]-triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention.
  • An amplification method is described by Walker et al. 1992, incorporated herein by reference.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method, called Repair Chain Reaction (RCR) involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
  • RCR Repair Chain Reaction
  • Cyclic Probe Reaction Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA, which is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products, which are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR, Kwoh et al. (1989), PCT Application WO 88/10315, 1989, (each incorporated herein by reference).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • PCT Application WO 88/10315 PCT Application WO 88/10315
  • RNA guanidinium chloride extraction
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • amplification methods as described in British Patent Application No. GB 2,202,328, and in PCT Application No. PCT/US89/01025, each incorporated herein by reference, may be used in accordance with the present invention.
  • modified primers are used in a PCRTM like, template and enzyme dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA : RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E.
  • dsDNA double-stranded DNA
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Suitable amplification methods include “race” and “one-sided PCR” (Frohman, 1990; Ohara et al., 1989, each herein incorporated by reference). Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the present invention, Wu et al., 1989, incorporated herein by reference).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al., 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized with light exhibiting the appropriate excitatory spectra.
  • the present invention makes use of additional factors in gauging an individual's risk for developing lung cancer.
  • multiple factors including age, ethnicity, smoking history, body mass index, alcohol consumption history, exercise history, and diet to improve the predictive accuracy of the present methods.
  • a history of cancer in a relative, and the age at which the relative was diagnosed with cancer, are also important personal history measures.
  • the inclusion of personal history measures with genetic data in an analysis to predict a phenotype, cancer in this case, is grounded in the realization that almost all phenotypes are derived from a dynamic interaction between an individual's genes and the environment in which these genes act. Those skilled in the art will realize that the personal history measures listed in this paragraph are unlikely to be the only such environmental factors that affect the penetrance of the cancer phenotype.
  • kits for use in accordance with the present invention.
  • Suitable kits include various reagents for use in accordance with the present invention in suitable containers and packaging materials, including tubes, vials, and shrink-wrapped and blow-molded packages.
  • Materials suitable for inclusion in a kit in accordance with the present invention comprises one or more of the following:
  • reagents required to discriminate between the various possible alleles in the sequence domains amplified by PCR or non-PCR amplification e.g., restriction endonucleases, oligonucleotides that anneal preferentially to one allele of the polymorphism, including those modified to contain enzymes or fluorescent chemical groups that amplify the signal from the oligonucleotide and make discrimination of alleles most robust
  • • reagents required to physically separate products derived from the various alleles e.g., agarose or polyacrylamide and a buffer to be used in electrophoresis, HPLC columns, SSCP gels, formamide gels or a matrix support for MALDI-TOF).
  • Controls are healthy individuals seen for routine care at Kelsey-Seybold Clinics; the largest physician group-practice plan in the Houston Metropolitan area (Hudmon et al, 1997). Controls in the Texas discovery and replication sets were frequency matched to cases according to their smoking behavior, age in 5 year categories, ethnicity, and sex. Former smoking controls were further frequency matched to former smoking cases according to the number of years since smoking cessation (in 5 year categories).
  • the Texas replication set comprised 711 Caucasian lung cancer cases and 632 controls. The replication set was not as well matched as the discovery set with respect to age (mean age in cases of 65 and in controls is 57) but had comparable sex distributions (56% male).
  • Genotyping using Illumina bead technology Genotypes were generated by the Center for Inherited Disease Research for 317498 polymorphic tagging SNPs using Illumina HumanHap300 vl.l BeadChips and the Illumina Infmium II assay (Gunderson et al, 2006). Cluster definitions for each SNP were determined using Illumina BeadStudio Genotyping Module v 2.3.41 and the combined intensity data from -90% of study samples. The resulting cluster definition file was used on all study samples to determine genotype calls and quality scores. Genotype calls were made when a genotype yielded a quality score (Gencall value) of 0.25 or higher. Among these markers, 0.46% of calls were missing. 2353 study samples were attempted.
  • a Python script to check reported versus observed sex is available upon request to the author. After all quality control procedures were completed, 2291 samples including 1154 cases and 1137 controls were available for genotyping.
  • Genotyping using Taqman was performed using the TaqMan® Pre-Designed SNP Genotyping Assays (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction and using primer sequences that were provided by the company.
  • a specific Pre-Designed SNP Genotyping Assay Mix (containing probes and primers) was purchased from the company.
  • PCR was performed in a 5 ⁇ l solution containing 5 ng of genomic DNA, 2.5 ⁇ l 2x Genotyping Master Mix, 0.125 ⁇ l 4Ox Pre-Designed Assay Mix, and 2.375 ⁇ l water in a Dual 384-Well GeneAmp® PCR System 9700 (Applied Biosystems).
  • PCR conditions were 10 minutes at 95° C followed by 40 cycles of 95° C for 15 seconds and 60° C for 1 minute. After PCR was completed, the end-point fluorescence was read by an ABI Prism 7900HT Sequence Detection System and the genotype was automatically called by the SDS software (version 2.1). Positive and negative controls were used in each genotyping assay, and 10% of the samples were randomly selected and run in duplicates. The concordance of genotypes was 100%. SNP rsl481847 was refractory to genotyping by Taqman and was dropped from analysis with rs 1481848 replacing it.
  • Genotyping using KASPar Genotyping of UK samples was conducted by KASPar (KBioscience Ltd, Hertfordshire, UK), a fluorescence-based competitive allele specific PCR which utilizes non-labeled primers. Details of the chemistry can be obtained from KBioscience (http ://www.kbioscience. co .uk/chemistry) .
  • Each 3 ⁇ l genotype assay was based on 7.5 ng of DNA dissolved in 1.5 ⁇ l water, 41 nl of primer assay mix containing the common and allele - specific primers (concentration of primers in Assay Mix: common primer 30 ⁇ M, allellel- specific primer and allele2-specific primer 12 ⁇ M), and 9.75 nl of KTaq and 4x reaction mix final MgCl 2 concentration 2.2 mM). Genotype clusters were called using KlusterCaller (KBioscience).
  • Genotyping quality control was tested using duplicate DNA samples within studies and SNP assays, together with direct sequencing of subsets of samples to confirm genotyping accuracy. For all SNPs, 99.61% concordant results were obtained. Genotyping. Genotyping procedures and quality control approaches are described in the supplemental methods. Data was retained from 315860 SNPs from Illumina analysis that had genotyping results in 90% or more subjects, but 410 were monomorphic for analysis in European-descent participants (and hence not informative).
  • Confirmatory genotyping in Texas was conducted on an independent sample of 711 cases and 632 controls using a Taqman genotyping platform for 10 most significant SNPs during the discovery phase.
  • the Texas replication sample comprised independent cases and controls from the discovery set who were from the same study population source but who tended to be more recently enrolled participants with incomplete frequency matching.
  • Genotyping of UK samples was conducted by competitive allele-specific PCR KASPar chemistry (KBiosciences Ltd, Hertfordshire, UK).
  • a test inflation factor ⁇ was calculated by dividing the median of the test statistics by the expected median from a ⁇ distribution with 1 degree of freedom (Devlin et al, 2001).
  • the mean and median of the ⁇ 2 tests in figure 1 were 1.0196 and 0.4675 - very close to the expected values of 1.00 and 0.456.
  • Comparison of the median ⁇ test with its expected value yields a lambda value of 1.025 very close to its expectation and indicating that population substructure if present, was not having any substantial effect upon the discovery stage analyses that are here presented.
  • HelixTree was used for preliminary analyses and to manipulate data initially, which were then transferred to PLINK (Devlin et al, 2001) and Eigenstrat (Price et al, 2006).
  • the inventors evaluated the association of markers with lung cancer risk allowing for potential effects of population substructure by using a Cochrane-Mantel- Haenszel test (de Bakker et al, 2005) in PLINK.
  • Strata were defined according to genetic similarity defined by a nearest neighbor cluster analysis, which identified 44 clusters.
  • a second analysis was performed to allow for substructure effects using the program Eigenstrat (Price et al, 2006). All genetic data from the discovery set were used to obtain correlation matrices among the subjects.
  • SAS/Genetics version 9.1 was used to conduct association tests to test for Hardy- Weinberg equilibrium and to perform haplotype analyses.
  • Logistic regression, implemented in SAS version 9.1 was used to perform analyses adjusting for smoking and other covariates.
  • Joint analysis of data generated from multiple phases was conducted using standard methods for combining raw data based on the Mantel-Haenszel method. Cochran's Q statistic was used to test for heterogeneity.
  • Haploview software (v3.2) was used to infer the LD structure of the genome in the regions containing loci associated with disease risk (Barrett et al, 2005).
  • deBakker de Bakker et al, 2005
  • haplotype frequencies from HapMap, release 21, build 35.
  • P values combining data from the discovery phase as well as the two replication phases were obtained following the procedure outlined by Skol et al. (2006).
  • the joint test statistics were obtained by comparing allele frequencies in cases versus controls from all studies according to their sample sizes (results from the two replication phases were combined prior to joint analysis).
  • the pointwise P value so derived can be adjusted for multiple testing using a Bonferroni approach, by multiplying the pointwise P value by the number of tests (results not shown).
  • the CaTS software could not provide a result because of numerical overflow, and these results were indicated by > 1 x 10 "5 , which was the least significant P value obtained before the overflow.
  • P values from the replication phase were provided only by combining results from the Texas and UK studies adjusting for center effects using a Cochrane-Mantel-Haenszel procedure, implemented in SAS.
  • GWA genome-wide association study of histologically confirmed non-small cell lung cancer (NSCLC) was conducted to identify common low penetrance alleles influencing lung cancer risk.
  • NSCLC non-small cell lung cancer
  • Illumina HumanHap300 vl.l BeadChips we genotyped 317498 tagging SNPs in a series of 1154 ever-smoking lung cancer cases and 1137 ever-smoking controls (Texas Discovery Series; Table 2).
  • Tables 2A-D Summary of 10 Fast-Track SNPs Analyzed in Discovery and Replication Studies
  • Top SNPs provided for Illumina Hap300 vl.l platform. Nearest known transcribed sequence, as reported by Illumina. Top line is P value from chi-square test for alleles, bottom line is P value from Armitage-Doll Trend test, 4 P values from joint analysis of data from all stages. P values indicated as > 1 x
  • AdjOdds Ratio column was adjusted for age, sex and packyears.
  • AdjOdds Ratio column was adjusted for age, sex, packyears and centers.
  • the SNPs rsl051730 and rs8034191 map to a 100kb region of strong LD on chromosome 15 extending from 76593078 bp to 7671000bp (FIG. 3).
  • Three genes map to this region: the nicotinic acetylcholine receptor alpha subunits 3 and 5 (CHRNA3, CHRNA5) and proteasome alpha 4 subunit isoform 1 (PSMA4) plus the hypothetical gene LOC123688 isoform 1.
  • Table 6 Haplotype Results for the 9 Markers Spanning the Interval Between rs3743079 and rsl051730 [Markers that are reported are rs8034191 (position 76593078), rs3885951 (76612972), rs2036534 (76614003), rs4275821, (76636596), rs6495306, (76652948), rs680244 (76658343), rs621849 (76659916), rsl051730 (7671000), rs8192475 (76698285)].
  • nicotinic acetylcholine receptor pathway in both the etiology and progression of lung cancer (Zhang et al, 2007; Lam et al, 2007; Minna, 2003). Specifically, nicotine has been reported to promote cancer cell proliferation, survival, migration/invasion and tumor angiogenesis through the acetylcholine receptor pathway.
  • the nicotinic acetylcholine receptor may also be a key player in nicotine- mediated suppression of apoptosis in lung cancer cells (Lam et al, 2007).
  • CHRNA3 is perhaps the more attractive candidate as a susceptibility gene for lung cancer as it expressed ubiquitously unlike CHRNA5 which is not expressed at high levels in lung tissue.
  • a mechanism by which a nicotinic acid receptor could increase risk for lung cancer was given by Ho et al (2005), who showed that the CHRNA3 subunit binds NNK and subsequently upregulates nuclear factor Kappa B to induce cell proliferation.
  • PSMA4 is a component of the ATP/ubiquitin- dependent non- lysosomal pathway and although involved in the processing of class I MHC peptides, there is little evidence to date for a role in lung cancer.
  • Results show weak evidence that these SNPs influence smoking behavior; however, the effects only appeared consistently significant across studies in former but not in current smokers.
  • rs8034191 or rsl051730 directly contributes to lung cancer susceptibility.
  • Saccone et al. (2007) found an association of rsl6969968, a marker in strong LD with rsl051730, with Fagerstrom index in nondiseased individuals.
  • the present study shows a weak effect of rs8034191 or rsl051730 on smoking behaviors and an extremely significant effect on lung cancer risk, whether or not an adjustment for smoking behavior is made during the analysis.
  • Insertion/Deletion Variations involved in Lung Cancer Additional data set forth in Table 11 is a summary of insertion/deletion variations that are involved in lung cancer. The region where the insertion/deletions are located and the kind of mutation makes it likely these play a role in lung cancer. These insertions and deletions as important for lung cancer risk because they occur in the promoter regions of CHRNA3 and CHRNA5, areas known to be involved in regulation of genes, and because they show strong linkage disequilibrium with other SNPs in the region that show association with lung cancer risk.
  • Table 12A Primer Sequences for Variants 1 and 2 in Table 11

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Abstract

L'invention concerne des procédés pour identifier un sujet risquant de développer un cancer du poumon qui implique l'obtention d'un échantillon contenant de l'acide nucléique depuis le sujet, et l'analyse de l'acide nucléique en ce qui concerne un changement de séquence d'acide nucléique dans le chromosome 15. Par exemple, l'acide nucléique peut provenir d'une région de chromosome 15 s'étendant depuis 76593078 bp à 7671000 bp. Le changement de séquence d'acide nucléique peut être un SNP, comme rs1051730 ou rs8034191.
PCT/US2009/037694 2008-03-19 2009-03-19 Variations de séquence sur un risque de prédiction du chromosome 15 pour le cancer du poumon Ceased WO2009117595A2 (fr)

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