WO2009097270A2 - Method of determining breast cancer risk - Google Patents

Abstract

The invention provides methods and reagents related to a chromosomal loci associated with altered risk for developing breast cancer. The method includes screening genomic sequence information for the presence or absence of one or more polymorphism alleles in chromosome 6q22.33 that indicates a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject. The invention also provides related machine readable media and methods of screening for a candidate therapeutic compound for breast cancer. The invention also provides an oligonucleotide or set of oligonucleotides that selectively anneal to genomic DNA comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 SNP alleles. The oligonucleotide or set of oligonucleotides can be provided in kits that also include reagents suitable for screening for the presence or absence of one or more polymorphism alleles in genomic sequence information.

Description

DHHS E-065-2008/0-PCT-02

METHOD OF DETERMINING BREAST CANCER RISK

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/023,936, filed January 28, 2008, the disclosure of which is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant Number NOl- CO- 12400 awarded by the Department of Health and Human Services The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Published reports estimated that the number of new invasive breast cancer cases will exceed 1.3 million and that the number of deaths caused by breast cancer will approach 470,000 for the year of 2007. Global Cancer Facts & Figures 2007, published by the American Cancer Society (Atlanta, GA 2007). In the United States, one in seven women is considered to be at risk of developing breast cancer, based on a life expectancy of 85 years. Breast Cancer Screening and Diagnosis Guidelines - V.I, 2007, published by the National Comprehensive Cancer Network (Ft. Washington, PA 2007). Thus, there is a need for reducing both the incidence and mortality associated with breast cancer. [0004] Risk factors associated with breast cancer include being a woman, inheritance of genetic markers such as BRCAl and BRCA2 mutations, personal and familial disease history, being overweight or obese after menopause, use of postmenopausal hormone therapy, physical inactivity, and consumption of one or more alcoholic beverages per day. Breast feeding, moderate or vigorous physical activity, and maintaining a healthy body weight are associated with lower risk of breast cancer. Global Cancer Facts & Figures 2007. It is widely recognized that early identification of individuals at increased risk for developing breast cancer coupled with appropriate screening and/or preventive treatment can be critical factors in reducing the incidence and mortality associated with breast cancer. [0005] With respect to genetic markers associated with a risk of developing breast cancer, cohort and twin studies have indicated that 5 to 15% of incident breast cancer cases result from autosomal dominant cancer susceptibility (Claus et al., Cancer 77: 2318-2324 (1996), Colditz et al., GA, JAMA 270: 338-343 (1993), Lichtenstein et al., N. Engl. J. Med., 343: 78- 85 (2000). Locatelli et al., Stat. Med., 26: 3722-3734 (2007), Slattery et al., JAMA 270: LVM 704227

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1563-1568 (1993)). However, only about 40% of the familial aggregation of breast cancers can be explained by mutations in BRCAl, BRC A2, or other identified cancer susceptibility genes (Ford et al., Am. J. Hum. Genet., 62: 676-689 (1998)). Attempts to utilize linkage strategies to localize other genes associated with an inherited predisposition to cancer have been somewhat hampered by genetic heterogeneity, decreased penetrance, and chance clustering (Kainu et al., Proc. Natl. Acad. Sci. USA, 97: 9603-9608 (2000), Kerangueven et al., Oncogene 10: 1023-1026 (1995), Rahman et al., Oncogene 19: 4170-4173 (2000), Risch et al., Science, 273: 1516-1517 (1996), Seitz et al., Oncogene 14: 741-743 (1997), Thompson et al. Proc. Natl. Acad. Sci. USA, 99: 827-831(2002). Accordingly, there is a desire for the identification of additional markers of genetic susceptibility for developing cancer, which could be used to identify individuals having a higher or lower risk of developing breast cancer.

BRIEF SUMMARY OF THE INVENTION

[0006] The invention provides reagents and methods of determining the risk of developing breast cancer in a subject. The method of determining the risk of developing breast cancer includes obtaining genetic sequence information from a subject and screening the sequence information for the presence or absence of one or more polymorphism alleles in chromosome 6q22.33. The presence or absence of one or more polymorphism alleles indicates a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject. The method can, for example, include screening for the presence or absence of one or more single nucleotide polymorphisms (SNP) alleles selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NOs: 1, 2, 3, and 4, respectively. Such alleles can, for example, be found in combinations that include the Hl and H2 haplotypes identified herein. [0007] The invention also provides a breast cancer model cell that includes a recombinant DNA that controls expression of a ECHDCl gene product, a RNF 146 gene product, or both gene products in the cell.

[0008] The invention provides a method of screening for candidate therapeutic compounds for breast cancer. The method includes contacting an breast cancer model cell with a test compound and assaying for increased or decreased expression of ECHDCl or RNFl 46 gene product in the cell. An increased or decreased expression of the gene product in the cell indicates that the test compound is a candidate therapeutic compound for breast cancer. LVM 704227

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[0009] The invention also provides a method of screening for a candidate therapeutic compound for estrogen receptor positive breast cancer. The method can be performed with a breast cancer model cell that includes an estrogen response element operatively linked to the coding sequence for a gene product. The RNF 146 gene product is overexpressed in the model cell. The cell is contacted with a test compound and assayed for inhibition of the gene product controlled by the estrogen response element relative to a control cell. The control is a breast cancer model cell that also includes an estrogen response element operatively linked to the coding sequence for a gene product and also overexpresseses the RNF 146 gene product. However, the control cell is not contacted by the test compound. The ability of the test compound to inhibit the gene product controlled by the estrogen response element indicates that the test compound is a candidate therapeutic compound for estrogen receptor positive breast cancer.

[0010] Additionally, the invention provides an oligonucleotide or set of oligonucleotides that selectively anneals to genomic DNA comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles corresponding to the sequence set forth in SEQ ID NOs:l, 2, 3, or 4, respectively. The oligonucleotide or set of oligonucleotides can be covalently linked to a reporter molecule, such as, for example, a fluorescent probe suitable for use in 5 '-nuclease ("Taqman") analysis, Invader or "flap-endonuclease" analysis, tetra- primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, or oligonucleotide ligase assay. [0011] The invention further provides kits that include the oligonucleotide or set of oligonucleotides according to the invention and reagents for determining the presence or absence of a SNP allele. Reagents can be suitable for use in, for example, 5'-nuclease ("Taqman") analysis, Invader or "flap-endonuclease" analysis, tetra-primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, or oligonucleotide ligase assay.

[0012] Furthermore, the invention provides machine-readable medium that comprises digitally encoded, processed genetic information indicating the presence or absence of one or more polymorphisms in chromosome 6q22.33 in a subject. Although such digitally encoded, processed information is not raw genetic sequence information from an oligonucleotide array, it can be information that results from the processing of such raw genetic sequence information to indicate the presence or absence of one or more polymorphisms in chromosome 6q22.33. LVM 7U4227

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BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures IA and IB are quantile-quantile plots. Figure IA shows the 391,467 SNPs that did not deviate from Hardy- Weinberg equilibrium and with a non-zero trend score in the initial survey of 249 familial cases and 299 controls. Figure IB shows the 150,071 SNPs with call rates of 99.6% or greater. Under the null hypothesis of no association at any locus, the points would be expected to follow the straight line.

[0014] Figure 2 is a plot showing the results of components cluster analysis of phase 1 cases (triangles), controls (circles) of Ashkenazi origin, and a reference set of northern Europeans (squares).

[0015] Figure 3 is a graph showing the genomic positions of multiple SNPs in the region at chromosome 6q22.33 associated with an increased or decreased risk of developing breast cancer. Figure 3 also summarizes the linkage disequilibrium (LD) for certain SNPs, which was found in the study of Example 3 below.

[0016] Figures 4A and 4B show plus (forward) strand genomic sequences on chromosome 6q22.33 that flank and include SNPs rs2180341, rs6569479, rs6569480, and rs7776136.

[0017] Figures 5A and 5B are graphs correlating 6q22.33 genotype with the results of ECHDCl and RNFl 46 expression analysis in peripheral blood lymphocytes (PBL). [0018] Figure 6 is a graph showing DLCl promoter-driven Luciferase activity in MCF7 cells contacted with estradiol (E2) relative to RNFl 46 gene product expression.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides methods and reagents based, at least in part, on the discovery that polymorphisms in chromosome 6q22.33 correlate with an altered risk for developing breast cancer in a subject. Thus, the invention provides a method of determining the risk for developing breast cancer in a subject that includes obtaining genetic sequence information from the subject and screening the sequence information for the presence or absence of one or more polymorphism alleles in chromosome 6q22.33. Examples of breast cancer include estrogen positive and estrogen negative tumors.

[0020] As used herein, the term "subject" refers to a human. The subject can be male or female. The subject can be one who has not been previously diagnosed with cancer or, alternatively, one that has been diagnosed with cancer.

[0021] In particular instances of the methods of the invention, the subject can be of Ashkenazi Jewish lineage. For example, the subject can have one, two, three, four, five, six, LV M 704227

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seven or eight, Ashkenazi Jewish great-grandparents. In another example, the subject can have one, two, three, or four Ashkenazi Jewish great-grandparents. In yet another example the subject can have one or two Ashkenazi Jewish parents. The methods disclosed herein can also be used to determine the risk of developing cancer in subjects that are not Ashkenazi Jewish. Further studes of those described in Examples 1-4 show that there is also a correlation between breast cancer and polymorphisms on chromosome 6q22.33 in subjects without an Ashkenazi Jewish lineage. Thus, for example, the subject can be of Caucasian or African American background.

[0022] In some (but not all) cases, a subject is free of a BRCA 1 or BRCA2 polymorphism associated with an altered risk for developing breast cancer. Inasmuch as the risk of developing breast cancer increases with age, the method of the invention can be advantageously used to screen for an altered risk of developing cancer in a subject that is at least 20, 25, 30, 35, 40, or 45 years old: Moreover, inasmuch as the risk of developing breast cancer also increases as women undergo the hormonal regulatory changes associated with menopause, the methods of the invention can be used to screen a female subject that is in perimenopause, menopause, or post-menopause, as these terms are generally used to refer to various characteristic symptoms (see, e.g., NIH State-of-the-Science Panel., Ann Intern Med., 42: 1003-13 (2005), the entire contents of which are specifically incorporated by reference herein).

[0023] For purposes herein, the term "genetic sequence information" can refer to genomic DNA, mRNA, cDNA, and fragments thereof that include sequence information evidencing the presence or absence of a polymorphism in a subject's genome. Additionally, "genetic sequence information" can refer to electronically generated or stored information that can be screened to determine the presence or absence of on one or more polymorphisms in the subject's genome. As used herein, the term "genomic DNA," unless otherwise clear from its context, can also refer to cloned genomic DNA and amplified genomic DNA. [0024] Methods of screening genetic sequence information for polymorphisms are known in the art (see, for example, Li et al., Nucl. Acids Res., 28: el (i-v) (2000); Liu et al., Biochem. Cell Bio. 80: 17-22 (2000); and Burczak et al., Polymorphism Detection and Analysis, Eaton Publishing, (2000)). Suitable methods include, for instance, DNA sequencing of genomic DNA and screening with oligonucleotide arrays ("gene chips") such as high density SNP arrays. Preferred methods include cloning for polymorphisms, nonradioactive PCR-single strand conformation polymorphism analysis, denaturing high pressure LVM 704227

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liquid chromatography (DHPLC), DNA hybridization, single-stranded conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), dynamic allele- specific hybridization (DASH) (Howell et al., Nat BiotechnoL, 17(l):87-8 (1999)), molecular beacon analysis (Abravaya et al., Clin. Chem. Lab. Med, 41 :468-474 (2003)), tetra-primer ARMS-PCR analysis (Ye et al., Nucl. Acids Res.,.29: e88 (2001)), Invader or "fϊap- endonuclease" analysis (Olivier M., Mutat Res., 573: 103-10 (2005)), primer extension analysis, such as, in combination with MALDI-TOF mass spectronomy or ELISA-like methods (Syvanen A.C., Nat. Rev. Genet., 2:930-42 (2001) and Rapley et al., (eds.), Molecular Analysis and Genome Discovery, published by John Wiley & Sons Ltd (Hoboken, NJ 2004)), oligonucleotide ligase assay, PCR amplification and high resolution melting point analysis of amplified DNA (Liew et al., CHn. Chem., 50:1156-1164 (2004)). A more preferred method of screening for a polymorphism includes the 5 '-nuclease ("Taqman") assay described, for example, in U.S. Patent Nos. 5,210,015 and 5,538,848. The entire contents of each of the foregoing references and patent documents disclosing methods for screening polymorphisms are specifically incorporated by reference herein. [0025] The method of determining risk for developing breast cancer of the invention requires specifically screening for one or more polymorphisms in chromosome 6q22.33. Thus, for example, when using an oligonucelotide (e.g., a genome wide) array to obtain raw genetic sequence information for various different genomic loci, the method of the invention requires further processing the raw genetic information to screen for and determine the presence or absence of one or more polymorphisms in chromosome 6q22.33 in a subject. Further processing of raw genetic information can be done on a machine running a software application that selectively extracts genetic information of interest (typically after refining the raw information obtained from the oligonucleotide array). In this regard, for example, the invention provides a machine-readable medium that includes digitally encoded, processed genetic information, which indicates the presence or absence of one or more of the polymorphisms in chromosome 6q22.33 in a subject. Machine-readable medium can include processed genetic information for fewer than 500,000 different genomic loci, e.g., fewer than 100,000, 50,000, 40,000, 30,000, 20,000, 10,000, 5,000, 1,000, 500, or 100 different genomic loci.

[0026] Preferred polymorphisms on chromosome 6q22.33 include those located within 100 kilobases of the genomic locus corresponding to ECHDCl; RNF 146. As used herein, "the genomic locus corresponding to ECHDCl; RNFl 46" is defined as the region beginning LVM 704227

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at nucleotide position 127,630 K (the approximate 5' end of the RNF146 gene, which is on the plus strand) and ending at nucleotide position 127,713 K (the approximate 5' end of the ECHDCl gene, which is on the minus strand) of chromosome 6q. More preferred polymorphisms include those located within 90, 80, 70, 60, 50, 40, 30, 20, or 10 kilobases (kb) flanking the genomic locus corresponding to ECHDCl; RNF 146. [0027] In a preferred embodiment, the method of determining the risk for developing breast cancer according to the invention includes screening for the presence or absence of an allele of one or more of the following single nucleotide polymorphisms (SNPs) on chromosome 6q22.33: rsl 134070, rs437186, rs6913350, rsl2199451, rs2180341, rs6569479, rs6569480, s7453272, rs7776136, and rs7761452. The SNP identifier numbers used herein, which include the prefix "rs", are the unique identifiers assigned to SNPs in the publicly available dbSNP database established by the National Center for Biotechnology Information in (NCBI) in collaboration with the Human Genome Research Institute. The chromosomal location as well as flanking genomic sequence for each SNP in the dbSNP database can be accessed via the NCBI website (see, e.g., Sherry et al., Nucl. Acids Res., 29: 308-311 (2001), which is specifically incorporated by reference herein in its entirety). [0028] In a more preferred embodiment, the method of determining the risk for developing breast cancer according to the invention includes screening for the presence or absence of an allele of one or more of the following SNPs: rs2180341, rs6569479, rs6569480, and rs7776136. Each of these SNPs is shown in the context of its flanking plus strand genomic sequence in Figure 4 and as set forth in SEQ ID NOs: 1, 2, 3, and 4, respectively.

[0029] Preferably, the method according to the invention provides a good level of confidence that the presence or absence of the screened-for polymorphism allele is associated with an altered risk for developing breast cancer. For example, the presence or absence of one or more screened for polymorphism alleles identified herein, indicates that the subject has a lower risk of developing cancer that is considered protective to about a 1 x 10^"4 or higher level of significance. Such higher levels of significance include, for example, a 5 x 10-⁴, 1 x 10^"5, 5 x 10^"5, 1 x 10-⁶, 5 x 10^'6, 1 x 10^"7, a 5 x 10^"7,l x 10^, 5 x 10^"8, 1 x 10^'9, 2 x 10^"9, or 5 x 10^"9 level of significance.

[0030] In another example, the presence or absence of one or more screened for polymorphism alleles identified herein, indicates that the subject has a higher risk of developing cancer at about a 1 x 10^"2 or higher level of significance. Such higher levels of LVM 704227

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significance include, for example, a 2 x 10^"2, 5 x 10^"2, a 1 x 10^"3, 5 x 10^'3, 1 x 10^"4, 5 X lO^"4, ! x 10^"5, 5 x 10^"5, 1 x 10^"6, 5 x 10^'6, 1 x 10^"7, or 5 x 10^'7 level of significance. The foregoing levels of significance can be calculated using the allele chi-square test. [0031] Thus, in one example, the method of determining the risk for developing breast cancer according to the invention includes screening for the presence or absence of the haplotype 1 (Hl) discussed more fully in Example 4 below. In the Hl haplotype, the rs2180341, rs6569479, rs6569480, and rs7776136 alleles correspond to SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively. The presence of the Hl haplotype is protective and indicates a lower risk of developing breast cancer in the subject.

[0032] In another example, the method of determining the risk for developing breast cancer according to the invention includes screening for the presence or absence of the haplotype 2 (H2) discussed more fully in Example 4 below. In the H2 haplotype, the rs2180341, rs6569479, rs6569480, and rs7776136 alleles correspond to SEQ ID NO:1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively. The presence of the H2 haplotype indicates a higher risk of developing breast cancer in the subject.

[0033] The method of the invention can further include screening for the presence or absence of one or more additional polymorphisms located at a position other than chromosome 6q22.33. Thus, in addition to screening for a polymorphism located on chromosome 6q22.33, the method can include screening for other polymorphisms associated with an altered risk of developing cancer. For example, gene studies carried out by the present inventors in multiplex kindreds affected by breast cancer have implicated polymorphisms associated with CHEK2, ATM, BRIPl and PALB2 in subsets of families lacking BRCAl or BRCA 2 mutations. Other studies have indicated that an increased risk of breast cancer is associated with polymorphisms in or near CASP8 D302H (rslO45485) and TGFBl LlOP (rsl 982073) (Cox et al., Nature Genet., 39: 352-358 (2007), which is specifically incorporated by reference herein in its entirety). Additionally, studies of selected kindreds and unselected individuals affected by breast cancer have implicated a locus near FGFR2 as associated with an approximately 1.2 fold increase risk of the disease, and identified a number of other SNPs as being associated with breast cancer risk (Easton et al., Nature, 447: 1087-1093 (2007); Hunter et al., Nat. Genet. 39: 870-874 (2007), both of which are specifically incorporated by reference herein in their entirety). Such additional SNPs LV M 704227

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include those located in or near the TNRC9 locus (e.g., rs8049226) on chromosome 16q, in or near the MAP3K1 locus (e.g., rs726501) on chromosome 5q , the LSPl region (e.g., rs7120258) and Hl 9 region (e.g., rs7120258 and rs7578974) on chromosome 1 Ip. Easton et al. reported significant association SNPs located on with 2p, 5p, 5q, and 8q. These reported additional SNPs are consistent with some of the results of the studies discussed more fully below in Examples 1-4, which have indicated a potential association between breast cancer and the following SNPs: (i) rs3803662 and rs3112625 located near TNRC9, (ii) rs3817198 and rs498337 located near LSPl, and (iii) rsl078806 near FGFR2, (iv) rs981782 located on chromosome 5 near the rs4866929 SNP identified by Easton et al. and which is in the HCNl gene locus.

[0034] Thus, the invention provides a method that can include screening for the presence or absence of one or more polymorphism alleles located on (i) chromosome 6q22.33 (such as, for example, the Hl or H2 haplotypes disclosed herein) and (ii) one or more genomic loci other than chromosome 6q22.33, wherein the presence or of both polymorphism alleles indicate a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject. For example, the method can include screening for the presence or absence of (i) one or more of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles that correspond to SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO: 3 when R is G, and SEQ ID NO:4 when W is T, respectively, and (ii) one or more of the following polymorphisms which have been associated with an increased risk of cancer: BRCAl, BRCA2, and the SNPs associated with CASP8 D302H (rsl 045485), TGFBl LlOP (rsl982073), or FGFR2. The presence of both the (i) and (ii) polymorphisms indicates that the subject has a higher risk of developing breast cancer.

[0035] In another example, the method can include screening for the presence or absence of (a) one or more of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively, and (b) one or more genomic loci, which are not on chromosome 6q22.33 and which have been associated with a decreased risk of cancer. The presence of both the (a) and (b) polymorphisms indicates that the subject has a lower risk of developing breast cancer.

[0036] Having determined whether a subject has an increased or decreased risk of developing breast cancer, in accordance with the method disclosed herein, the method of the invention can further include beginning or modifying a course of treatment for the subject. LV M 704227

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As used herein, the term "treatment" refers to both preventive and ameliorative treatment. It is expected that beginning or modifying a course of treatment that is appropriate to a subject's increased or decreased risk of developing breast cancer can contribute to an overall rational medical approach that diminishes the incidence and mortality associated breast cancer.

[0037] Thus, upon determining that a subject has an increased risk of developing breast cancer, the method of the invention can further include beginning or modifying a course of preventive treatment for the subject. A course of preventive treatment can include eliminating or reducing factors that contribute to the development of breast cancer. Such factors can include smoking, alcohol consumption, and being overweight, especially after menopause. Additional factors that can contribute to the development of breast cancer include female hormone regimens such as those in oral contraceptives ("the pill") and hormone replacement therapy (HRT). A course of preventive treatment can include promoting factors that are protective against breast cancer. Protective factors can include physical activity and breast-feeding of infant children as well as certain pharmaceuticals, nutraceuticals, or vitamin supplements. For example, aspirin and antiestrogen pharmaceuticals, such as tamoxifen, have been reported to be protective against the development of breast cancer in high risk subjects. Similarly, the selective estrogen receptor modulator (SERM) raloxifene HCl has been shown to be protective against the development of breast cancer in post-menopausal women. A somewhat controversial course of protective treatment in women with a very high risk of developing breast cancer is prophylactic mastectomy.

[0038] A course of preventive treatment can also include monitoring for the presence of breast cancer. Such monitoring can include mammography, clinical breast examination (CBE), and breast self-examination (BSE). Upon determining that a subject has an increased risk of developing breast cancer, the method of the invention can further include beginning a more aggressive form of monitoring for the presence of breast cancer. More aggressive monitoring can include, for example, magnetic resonance imaging (MRI), breast-specific gamma imaging (BSGI), positron emission tomography (PET), and/or ultrasound. Additionally, upon determining that a subject has an increased risk of developing breast cancer, the method of the invention can further include increasing the frequency of monitoring for the presence of breast cancer. LVM 704227

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[0039] Upon determining that a subject has a lower risk of developing breast cancer, the method of the invention can further include beginning an appropriate course of treatment or altering an existing course of treatment to a more appropriate one. An appropriate course of treatment for a person with a lower risk of developing cancer can include less frequent monitoring for the presence of breast cancer, as compared to the monitoring frequency for a person with an increased risk of developing cancer. In this regard, a person with a lower risk of developing breast cancer is less likely to benefit from more aggressive forms of monitoring for the breast cancer. It can be especially beneficial to reduce the frequency of a course of preventive treatment that has undesirably side effects, such as those associated with x-ray mammography or BSGI. Furthermore, by reducing the need for or frequency of certain courses of preventive treatment that consume health care resources, the method of the invention permits such resources to be more rationally allocated within a health care delivery system.

[0040] In another embodiment, the invention also provides a breast cancer model cell that includes a recombinant DNA for controlling the expression of a ECHDCl gene product and/or a RNFl 46 gene product, or both of the foregoing gene products in the cell. Breast cancer model cells are known in the art and include, for example, the MCF-7, BT20, MDA- MB-231. MDA-MB-435, MDA-MB-468, SK-BR3, T47D, ZR75.1 cell lines, which are available from the American Type Culture Collection (Rockville, MD). Breast cancer model cells also include primary breast tumor cells from a patient, which can, optionally, be transformed to establish a model breast cancer cell line. For purposes herein "gene product" refers to any molecule encoded by a gene. Gene products include, for example, proteins, mRNAs, primary RNA transcripts, alternatively spliced transcripts, allelic variants, and the like. Molecular biology techniques can be used to introduce into a host breast cancer model cell one or more recombinant DNA sequences to control the expression of ECHDCl and/or RNFl 46 gene product. Molecular biology techniques include those described in Ausubel et al., eds., Current Protocols in Molecular Biology, published by Wiley Interscience, (Hoboken NJ, 2004), the entire contents of which are specifically incorporated by reference herein. Preferred regulatory sequences include regulatory sequences that drive expression of a gene product in an inducible manner. In one example, the recombinant DNA includes a heterologous recombinant regulatory sequence, which is integrated into the genome of a host breast cancer model cell, such that the regulatory sequence is operably linked (i.e., can drive expression of) the host's endogenous genomic ECHDCl and/or RNF146 gene coding LVM 704227

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sequence, thereby producing a breast cancer model cell according to the invention. Techniques for integrating a heterologous regulatory sequence so that it is operably linked to the host's endogenous gene coding sequence have been described (for example, in U.S. Patent Nos. 5,981,214 and 6,544,748, the entire contents of which are specifically incorporated by reference herein in their entirety). In another example, recombinant DNA includes a heterologous regulatory sequence that is operably linked to an ECHDCl and/or a RNF 146 gene coding sequence that is not endogenous to the host cell. For example, the recombinant DNA can be a DNA vector that includes a regulatory sequence operably linked to cDNA encoding the ECHDCl and/or RNFl 46 gene product. Transfecting the vector into a breast cancer model cell line can thereby produce a breast cancer model cell in accordance with the invention. Molecular biology techniques for making and transfecting such vectors are known in the art and described in, for example, Ausubel et al, supra, which is specifically incoroporated by reference herein in its entirety.

[0041] The model cells of the invention can be used to screen for candidate therapeutic compounds for breast cancer. One candidate compound screening method includes providing a breast cancer model cell according to the invention, inducibly expressing the ECHDCl and/or RNFl 46 gene product in the model cell, contacting the cell with a test compound, and assaying for disruption of a breast cancer phenotype associated with the model cancer cell. Breast cancer phenotypes can include, for example, proliferation rate, cellular morphology, oncogene expression, and/or reduced tumor suppressor gene expression. Additionally, breast cancer phenotypes can include responses to molecular signaling events. For example, the MCF-7 cell line expresses estrogen receptor and responds to estrogen signaling by initiating a cascade of molecular events, such as for example, derepressing genes under the control of the estrogen response element. Some of the molecular events appear to promote proliferation of estrogen receptor positive breast cancer cells. Thus according to the invention, a method of screening for candidate compounds can include (i) providing a test MCF-7 cell that includes recombinant DNA for controlling the expression of ^'a ECHDCl and/or a RNFl 46 gene product, (ii) inducibly expressing the ECHDCl and/or a RNFl 46 gene product in the test cell, (iii) contacting the test cell with a test compound, (iv) contacting the test cell with estrogen, and (iv) assaying for inhibition of the test cell's response to estrogen signaling, relative to a control cell, which is the same as the test cell and has been contacted by estrogen but which has not been contacted by the test compound. Estrogen signaling in the test and control cell can be detected, for example, by including in the cells a heterologous estrogen response LVM 704227

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element operably linked to a reporter gene, such as, for example, a luciferase or green fluorescent protein (GFP) gene. Inhibition of estrogen signaling by the test compound indicates that the test compound is a candidate therapeutic compound for estrogen receptor positive breast cancer.

[0042] Another candidate compound screening method of the invention includes (i) providing a test cell that is a breast cancer model cell (which may or may not include recombinant DNA for controlling the expression of a ECHDCl gene product and/or a RNF 146 gene product), (ii) contacting the test cell with a test compound, and assaying for increased or decreased expression of ECHDCl and/or RNF 146 gene product, relative to a control cell, which is the same as the test cell but which has not been contacted by the test compound. Increased or decreased expression of ECHDCl and/or RNF 146 gene product indicates that the compound is a candidate therapeutic compound for breast cancer. [0043] The invention also provides reagents that can be used in the method of screening for a polymorphism on chromosome 6q22.33, in accordance with the invention. For example, the invention provides one or more oligonucleotide primers for amplifying a portion of chromosome 6q22.33 from a subject that comprises the rs2180341, rs6569479, rs6569480, or rs7776136 SNPs corresponding to the sequence set forth in SEQ ID NOs: 1, 2, 3, or 4, respectively. Such primers can be designed to anneal upstream and downstream of the polymorphic allele to thereby amplify the genomic sequence. The genomic sequence can then be screened, in accordance with the method of the invention, for determining the risk for developing breast cancer, for example, by DNA sequencing, RFLP, primer extension analysis, or any other appropriate method that can screen for the presence or absence of one or more particular rs2180341, rs6569479, rs6569480, or rs7776136 allele, which indicates a higher or lower risk for developing cancer.

[0044] The invention also provides one or more oligonucleotides that selectively anneal to genomic DNA (or amplified genomic DNA) comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles corresponding to the sequence set forth in SEQ ID NOs: 1, 2, 3, or 4, respectively. Such oligonucleotides can further include one or more covalently linked reporter molecules (such as, a radioactive molecule or a molecule capable of fluorescing) that can be used to indicate whether the genomic DNA (or amplified or cloned genomic DNA) includes one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles set forth in SEQ ID NOs: 1-4, respectively. Thus, for example, the primers of the invention can be designed for use in a method of the invention LVM 7U4227

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that includes using the 5 '-nuclease ("TAQMAN") analysis, INVADER or "flap- endonuclease" analysis, tetra-primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, or oligonucleotide ligase assay. [0045] The invention also provides kits with reagents suitable for use in the method of screening for a polymorphism on chromosome 6q22.33, in accordance with the invention. The kits can contain one or more primers of the invention, which are suitable for amplifying a portion of chromosome 6q22.33 from a subject that comprises the rs2180341, rs6569479, rs6569480, or rs7776136 SNPs corresponding to the sequence set forth in SEQ ID NOs: 1, 2, 3, or 4, respectively. The kits can further include reagents for analyzing the amplified DNA by one or more of the following methods: DNA sequencing, RFLP, DHPLC, DNA hybridization, or PCR amplification and high resolution melting point analysis of amplified DNA.

[0046] In preferred embodiments the kits include one or more one or more oligonucleotides that selectively anneal to genomic DNA comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles corresponding to the sequence set forth in SEQ ID NOs: 1, 2, 3, or 4, respectively. Such oligonucleotides can further include one or more reporter molecules that can be used to indicate whether the genomic DNA includes one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles, which correspond to the sequence set forth in SEQ ID NO:1, when R is T or A, SEQ ID NO:2, when Y is C or T, SEQ ID NO:3, when R is A or G, and SEQ ID NO:4, when W is A or T, respectively. Thus, the kits of the. invention can include one or more oligonucleotides of the invention and one or more additional reagents, such as buffers or control templates, that can be used in a method of the invention that includes using one or more of the following methods: 5 '-nuclease ("TAQMAN") analysis, INVADER or "flap-endonuclease" analysis, tetra-primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, oligonucleotide ligase assay, or DNA hybridization. [0047] The kits of the invention can, additionally, include one or more oligonucleotides that selectively anneals to genomic DNA (or amplified genomic DNA) comprising a SNP allele on a chromosomal locus other than 6q22.33.

[0048] It is generally preferred that the kits of the invention should include oligonucleotides that anneal, when used in conjunction with the reagents of the kit, to fewer than 5,000 different genomic loci, for example, fewer than 1 ,000 different genomic loci. Thus, the kits can include oligonucleotides designed to anneal to fewer than 900, 800, 700, LVM 7U4227

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600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 different genomic loci. This can reduce the number of signals generated by oligonucleotides of the invention, for example, when such oligonucleotides further include a covalently bonded reporter molecule, and thereby facilitate the detection of polymorphism alleles associated with an increased or decreased risk of cancer, in accordance with the methods of the invention.

[0049] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0050] This example demonstrates a first phase genome wide association study (GWAS) study with 249 familial breast cancer cases.

[0051] Genomic DNA samples were obtained from 249 Ashkenazi Jewish (AJ) kindreds with three or more cases of breast cancer but no identifiable mutations in BRCAl or BRCA2 and in 299 cancer-free AJ controls. Mean age of the patients affected by breast cancer was 55 years, median 55 (range 25-95). Control subjects included 300 healthy AJ women who either accompanied male urology patients identified through the Urology Clinic or who were participating in cancer screening at the Memorial Sloan-Kettering Cancer Center (MSKCC) and who were cancer free and did not have a family history of breast cancer. Control subjects also included 29 healthy AJ women enrolled at Sheba Medical Center, Tel-Hashomer, Israel. Remaining control subjects enrolled in the study were the first 299 control individuals from an ongoing study. Any woman who indicated a prior diagnosis of breast cancer, atypical hyperplasia, or lobular carcinoma in situ was not included as a control. To be eligible for enrollment in this study either as a case^' or control subject, individuals must have indicated that all four grandparents were Jewish and of Eastern European ancestry. [0052] Genotyping was performed on the Affymetrix EA 500K SNP platform. As an initial data quality control, SNPs were filtered out if they were out of Hardy- Weinberg equilibrium (HWE) in the controls. Quantile-quantile plot analysis showed that SNPs with Fisher exact test P- values < 0.02 were not in HWE, leaving 391,467 SNPs. Next, allele frequencies in cases were compared to those in controls. Chi-square and Cochran-Armitage tests produced closely comparable results, with the number of significant SNPs and level of significance far exceeding expectation, a finding similar to that reported by Easton, who used a genotyping platform similar to ours (Easton et al., Nature, 447: 1087-1093 (2007)). In view of reported discordances at large numbers of SNPs surveyed between genotype calls made with the BRLMM algorithm developed by Affymetrix and the fluorescence intensity LVM 7U4227

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values, genotypes were graphed versus relative fluorescence intensities for all SNPs. These analyses showed that miscalled genotypes were responsible for the exceedingly low P- values for most of these SNPs.

[0053] To ensure genotype accuracy, any SNP with more than two no calls was excluded, since a threshold of three or greater miscalls caused the BRLMM algorithm to fail, resulting in false positive calls at greater than the P=O.05 level of significance. Setting the miscall threshold at 2 or less effectively restricted the analysis to SNPs with >99.7% call rate. Although, this procedure reduced the effective size of the survey to 150,080 SNPs, the BRLMM genotypes correlated well with the relative fluorescence intensity values. It also furnished a more realistic estimate of the number of SNPs exceeding chi-square distribution expectations (Table 1 and Figure 1). Excess of smaller than expected chi-square P-values (points above the straight line in Figure 1) were attributed to significant associations with disease, rather than population stratification or genotyping miscalls. As can be seen in Table 1, the bin with the greatest credible P-value excess was that between 0.00001-0.0001 where there is a ratio of 1.75 in observed to expected P-values, which translates to 21 observed SNPs versus 12 expected in that category.

[0054] Table 1 shows the number of significant associations after initial AJ familial GWAS.

Table 1

* Observed includes only values with H-W Chi-Square in 299 Controls with P > 0.02; the number of finite P-values of this category were 150,080 of the 167,676 SNP with call rates greater than 99.7% surveyed.

[0055] To determine whether the case-control groups were sufficiently similar for study using SNP association analysis, the numerical methods developed by Price et al. were applied and implemented in the Eigenstrat package (Price et al., Nat Genet. 38: 904-909 (2006)) . This analysis (Figure 2) confirmed cluster overlap between the AJ cases and controls used in LVM 7U4227

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phase 1 of this study, and revealed distinct differences from a Northern European reference population. Filtering of outliers through the application of the PCA reduced the familial study to 299 research subject controls and 249 familial cases.

[0056] This example shows the sufficiency of the case-control groups for study using genome wide SNP association analysis.

EXAMPLE 2

[0057] This example demonstrates a second phase validation analysis of the implicated regions from the first phase of Example 1 in a second cohort using a custom SNP array. [0058] Genomic DNA samples were typed from a second cohort including (i) 950 additional breast cancer cases seen at MSKCC and unselected for family history of breast cancer and (ii) 979 controls from the New York Cancer Project (NYCP), which is a cohort study involving 8,000 healthy volunteers in the same geographical region as the cases employed in this study. MSKCC cases and NYCP controls were matched for age at diagnosis of breast cancer (cases) and age at genotyping (controls) to be within 2 years, and all were of AJ ancestry. Cases did not demonstrate any of the BRCA founder mutations. [0059] The top-ranking 123 chromosomal regions (each region spanning 200 kb) from the Phase 1 analysis of 249 BRCA wild-type breast cancer kindreds were selected. In order to achieve satisfactory density of SNPs in candidate regions for haplotype analysis, from 2 to 4 additional SNPs per region were added as well as an additional 18 SNPs that also showed strong association (p<0.001) and mapped within the distance of 200 kb from the top 123 loci. A total of 343 SNPs were selected for genotyping in a larger replication cohort that consisted of a fully independent set of 950 consecutive AJ breast cancer cases and 979 age-matched cancer-free AJ controls. This analysis was performed on the ILLUMINA GOLDENGATE platform. The 343 SNP allele frequencies were compared in the breast cancer cases and controls using both the chi square test, as well as the Cochran-Armitage trend test, which produced closely comparable results.

EXAMPLE 3

[0060] This example demonstrates a third phase validation analysis of the implicated regions from Example 1 in third cohort.

[0061] A third cohort included data from an additional and non-overlapping cohort of 243 AJ women who presented at the MSKCC clinic with 'sporadic' breast cancer, i.e., absent a first degree family history of breast cancer. As an addition to the control group we included 187 disease free AJ females obtained from the ongoing NYCP. The additional cases were LVM 704227

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genotyped on the EA Affymetrix 500K SNP array and the controls were genotyped on the Affymetrix Commercial Version 500K Genotyping Chips for a separate study; since both of these platforms included the key loci replicated in phase 2 of the current study, this cohort was included for separate analysis of these loci and these data were added to the aggregate analysis.

EXAMPLE 4

[0062] This example demonstrates the results of an aggregate analysis of SNP data from all three phases of the study described in Examples 1-3.

[0063] Chi-square P-values were generated from an aggregate analysis of the 1442 cases and 1465 controls from all three phases of the study. Table 2 lists 7 SNPs, which include those with the most significant P-values using the allele chi-square test in the aggregate statistics. Table 2 also lists the odds ratios (ORs) and 95% confidence intervals (CIs) for the top-ranked SNPs calculated using the chi square test based on a dominant or a recessive genetic model. Analysis using Cochran-Armitage trend tests produced comparable results. [0064] Table 2 (following page) shows regions of the genome that showed the strongest associations with Ashkenazi Jewish breast cancer after Phase 2 of the study.

LV M 704227 DHHS E-065-2008/0-PCT-02 Table 2

Chi-square P value of allele Chi-square P

Chrom.² Gene/ OR allele for OR dom.⁷ OR rec.⁸

SNP¹ MAFcs⁹ MAFcn¹⁰ Frequency (top rank#)⁴ value risk⁶ (95% CI) (95% CI)

Region³ aggregate⁵

Phase 1 Phase 2 Phase 3 (95% CI) rs6569479 6q22.33 ECHDCl; 6.0 x 1O^'J 9.8 x 10° 2.2 x 10^'2 1.2 x lO^'' 1.39 1.50 1.48 0.271 0.21 1 RNF146 (#1358) (#D (1.23- 1.57) (1.29-1.74) (1.07-2.04) rs7776136 6q22.33 ECHDCl; 2.7 x 10^"3 9.9 x lO-⁵ 2 x 10^"2 6.6 x lO^"8 1.39 1.51 1.42 0.278 0.217 RNF146 (#742) (#2) (1.23-1.57) (1.31-1.76) (1.05-1.90) rs2180341 6q22.33 ECHDCl; 8.9 X lO-⁴ 1.1 x lO-⁴ 1.8 x lO^"2 2.9 x lO^"8 1.41 1.53 1.51 0.273 0.211 RNF146 (#205) (#3) (1.25-1.59) (1.32-1.77) (1.10-2.08) rs6569480 6q22.33 ECHDCl; 2.2 x 10^"3 1.2 X lO^"4 2 x lO^"2 6.1 x 10^"8 1.40 1.51 1.51 0.272 0.211 RNF146 (#616) (#4) (1.24-1.58) (1.30-1.75) (1.10-2.08) rslO788O6 10q26.13 FGFR2 4.5-x 10^"2 8.6 x 10^"4 4 x 10^'2 1.5 x 10^"5 1.26 1.32 1.40 0.455 0.399 (#10062) (#5) (1.13-1.40) (1.13-1.54) (1.16-1.69) rs3012642 23ql3.1 PHKAl; 2.I x IO^'2 2.1 x 10^"3 7.7 x 10^"2 NS NS NS NS 0.034 0.039 HDAC8 (#4751) (#6) rs7203563 16pl3.3 A2BP1 2.5 x 10^'2 3.3 x 10-³ 0.57 1.8 x lO^'3 1.32 1.36 NS 0.110 0.086

(#5573) (#7) (1.11-1.57) (1.13-1.64)

1 - Included in this Table all SNPs that had P values < 0.01 based on the analysis of Phase 2 case-control data.

2 - Chromosome position by cytogenetic band

3 - Genes identified in the genome browser that are within 100 kb on either side of the SNP indicated. Dashes indicate that no gene was identified within 50 kb on either side of the SNP.

4 - Phase 1 consisted of 249 Ashkenazi Jewish (AJ) probands from multiplex families, in whom a mutation in BRCAl and BRCA2 was excluded, versus 299 cancer-free AJ controls; Phase 2 consisted of 950 consecutive AJ breast cancer cases versus 979 cancer-free AJ controls; Phase 3 consisted of additional 243 AJ breast cancer cases from MSKCC and independent 187 Ashkenazi Jewish cancer - free controls (rank # represents the top order of the SNP out of total SNPs analyzed in phases 1 and 2)

⁵ - Aggregate data consisted of the combined Phase 1, Phase 2, and Phase 3.

⁶ - Odds ratio calculated using aggregate data and based on the chi square test of the alleles inverted to express the risk allele when necessary; 95% confidence interval.

⁷ - Odds ratio calculated using the aggregate data and based on the chi square test of the dominant model.

⁸ - Odds ratio calculated using the aggregate data and based on the chi square test of the recessive model.

⁹ - MAFcs, minor allele frequency in cases

¹⁰ - MAFcn, minor allele frequency in controls NS - Not Significant

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[0065] Other groups have reported associations in the FGFR2 region in association with breast cancer (Easton et al, Nature, AAl: 1087-1093 (2007) and Hunter et al., Nat. Genet. 39: 870-874 (2007)). While a weak signal was observed for FGFR2 region in the present familial study at rsl078806 (P = 0.045), rslO788O6 and three other SNPs from this region (rs 1047111, rsl2776781, and rsl0886927) were also genotyped in the present phase 2 breast cancer cases and controls. Of these four SNPs, the most significant P- value in the allele frequency test was obtained at rsl078806 (P = 8.6 x 10^"4; OR = 1.24; 95% CI 1.09-1.41). The results indicated that rslO788O6 was in high linkage disequilibrium (D¹ =0.9778, r2 = 0.9354 ) to other SNPs used in the study by Hunter et al.: rs2981582, rsl219648, rs2420946, and rs2981579. As shown in Table 2, the allele frequency test for this SNP obtained a P-value of 1.5 x 10^"5 in the aggregate analysis. Along with rsl078806, rs 10886927 obtained a significant chi-square P-value in the comparison of the Phase 2 cases and controls (P = 0.036; OR = 1.15; 95% CI 1.01-1.31), though P-value in the aggregate data not as significant.

[0066] As shown in Table 2, the association with the RNFl 46; ECHDCl region at 6q22 was the strongest and most consistent in this study. The association with rs2180341, rs6569479, rs6569480 and rs7776136 was initially found in the familial study and confirmed in the subsequent supporting studies, including the aggregate analysis of all 1442 cases and 1465 controls in the study where the P-value for the allele frequency test was 2.9 xlO^"8 for rs2180341. The major haplotype (Hl) composed of the four SNPs was found to be protective at ~ 5.53 x 10^"5 level of significance (OR 0.564, 95% CI 0.422-0.752) (Table 3). This haplotype was confirmed in phase 2 at a significance threshold of 4 x 10^"5 with nearly identical ORs and 95% CIs as the familial study (data not shown). Because of the high linkage disequilibrium (LD) in the region (shown by longer vertical bars in Figure 3), the signal is consistent with one arising from the RNFl 46, the ECHDCl gene, or both. Table 3 (following page) shows the haplotype statistics using rs7776136, rs6569480, rs6569479, and rs2180341 in the ECHDC 1;RNF 146 locus; the genotype column of table 3 shows, from left to right, the SNP allele present on the plus strand of 6q22.33 for each of rs2180341, rs6569479, rs6569480, rs7776136, respectively. LV M 704227

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Table 3

Table 3 (cont.)

A re ate Stud

CI, confidence interval

[0067] Several additional loci were identified in phase 1 with associations and P-values in the range of 10^"3 to 10^"6, which were confirmed in phase 2 with P-values on the order of 10^"2, and aggregate P-values from 10^"4 to 10^"5. Some of these loci are shown in Table 2. Other than FGFR2, these additional loci are not among the associations previously reported in prior WGAS. These additional loci include 4q32.1, 3p21.31, 10q22.3, 17q25.3, and 5ql2.2, among which the most significant P-value found in the familial study was 5.7 x 10^~6 for SNP rs6449674 at 5ql2.2 (Table 2).

[0068] The foregoing example demonstrates that polymorphisms in chromosome 6q22.33 variously correlate with an increased risk of developing breast cancer or a decreased risk of developing breast cancer. Additionally, the foregoing example (a) confirms that polymorphisms near the FGFR2 gene locus and (b) shows that polymorphisms in chromosome 4q32.1, 3p21.31, 10q22.3, 17q25.3, and 5ql2.2 correlate with an altered risk of developing cancer.

EXAMPLE 5

[0069] This example extends the findings in Examples 1 -4 and demonstrates that polymorphisms in chromosome 6q22.33 correlate with an altered risk for breast cancer in non-Ashkenazi subjects.

[0070] Genetic sequence information was obtained from 751 non-AJ breast cancer patients and 840 healthy control individuals. The information was screened for the presence or absence of polymorphisms at the same chromosome 6 region associated with breast cancer LV M 704221

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risk in individuals of Ashkenazi ancestry. In this regard, all four SNPs alleles in the RNF 146; ECHDCl region that define the Hl or H2 haplotype are linked and have nearly identical effect. Accordingly, the genotype of just one SNP can be used to indicate the presence or absence of the Hl or H2 haplotype, with some statistical confidence. Stated differently, each of the four SNPs is a "tagging SNP" that can be used to screen for the presence or absence of the Hl or H2 haplotype.

[0071] As summarized by the data in Table 4, the study found a significant excess of GG homozygotes at the rs2180341 SNP locus (plus strand) in breast cancer cases compared to controls. This SNP allele indicates that the patients have the H2 haplotype. The P value of these results was 0.0093. Thus, the homozygous H2 haplotype correlates with an increased risk of developing breast cancer in non-AJ breast cancer patients.

Table 4 rs2180341 SNP genotype (on plus strand of both chromosomes)

G/G A/G A/A

Cases (751) 64 (8.5%) 279 408

Controls (840) 44 (5.2%) 326 470

P= 0.0093 (recessive model)

[0072] The foregoing example demonstrates that polymorphisms in chromosome 6q22.33 correlate with an altered risk of developing breast cancer in non-AJ subjects.

EXAMPLE 6

[0073] This example validates the association of 6q22 with increased BC risk and provides a replication analysis on independent cohorts of cases and controls for AJ subjects. [0074] The replication study used 487 breast cancer patients, ascertained by the Clinical Genetics Service at MSKCC. A substantial proportion of these breast cancer cases were described in the context of prior epidemiologic studies (Shaag, et al, Hum. MoI. Genet., 14:555-63(2005)). All breast cancer cases tested negative for AJ founder mutations in BRCAl and BRC A2 genes. Breast cancer cases were compared to 1,149 AJ controls from Examples 1-3. The replication study screened for rs2180341. [0075] Genomic DNA was prepared using Gentra Autopure system, according to manufacture's protocol (Qiagen, Valencia, CA). Other DNA extraction procedures were performed as previously described Kirchhoff, et al., CHn Cancer Res, 10:2918-21 (2004). Genotyping of rs2180341, rs65669479, rs65669480 and rs7776136 was performed by the TAQMAN allelic discrimination procedure using assays by design under standard conditions LVM 704227

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(Applied Biosystems, Foster City, CA). To verify the absence of bias by inclusion of data from samples previously genotyped in Examples 1-4 by other methods, Affymetrix 500K and Illumina GOLDENGATE assasy, all published cohorts were re-genotyped by conventional TAQMAN allelic discrimination. All genotypes showed 100% concordance. The clustering of genotype calls was performed using SDS 2.1 software (Applied Biosystems, Foster City, CA).

[0076] Deviations of genotype frequencies in the controls from those expected under Hardy- Weinberg equilibrium were evaluated by a chi-square test (1 degree of freedom). Breast cancer risk associated with rs2180341 was estimated as odds ratios (OR) for the genotype model, heterozygotes (dominant model), homozygotes (recessive model) and per- allele (each copy of rare allele) with the common homozygote as a reference category. Odds ratios were calculated using conditional logistic regression. All models were adjusted for exact age at diagnosis (cases) or at the time of inclusion in the study (controls) and ethnicity. [0077] Even with the smaller sample size of this replication case cohort (n=487), this analysis confirmed a significant increase of breast cancer risk under dominant model after stratification for age at diagnosis (OR=I.28, 95%CI 1.03-1.59, p=0.024). While the association was statistically marginal by per-allele test (OR= 1.18, 95% CI 0.99-1.41, P=O.066), the aggregate AJ analysis (total 1,565 cases and 1,149 controls including Examples 1-4) confirmed the strong association of 6q22.33 with breast cancer risk (per-allele OR= 1.32, 95% CI 1.15-1.50, P=0.00003). Results are summarized on Tables 5 and 6.

Table 5

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Table 6

[0078] The foregoing example confirms the association of polymorphisms in chromosome 6q22.33 with an altered risk of developing breast cancer in AJ subjects.

EXAMPLE 7

[0079] This example validates the association of 6q22 with increased breast cancer risk and provides a replication analysis on independent cohorts of breast cancer cases and controls in non-AJ subjects.

[0080] The replication analysis included a group of 171 cases ascertained from MSKCC based on the criteria of a strong family history of the disease with 3 individuals with breast cancer present in a single lineage. The replication analysis also included the 751 non-AJ sporadic (unselected for a family history of the disease) breast cancer cases from Example 5; this group included Caucasian (76%), African American (10%) and Hispanic (8.7%) subjects. The replication analysis further included a non-overlapping group of 544 non-AJ sporadic breast cancer cases collected from anonymized protocols at MSKCC; this group included Caucasian (74%), African American (8%) and Hispanic (12%) subjects. [0081] Non-AJ controls included an ascertainment of 837 females who were either participating in cancer screening and cancer free or were spouses of patients with prostate cancer and did not have a personal or family history of BC. The population structure of this control set was similar to the non-AJ sporadic breast cancer group: Caucasians (73%), African-Americans (11%), and Hispanics (7%). A second group of non-AJ controls included 630 cancer free females, all of Caucasian background, who participated in New York Cancer Project (NYCP), carried out by the AMDeC Foundation, Mitchell et al., J. Urban Health, 81 :301-10 (2004). Data from Example 5 was included in a final aggregate meta-analysis. Genomic DNA was prepared and genotyped as in Example 6.

[0082] Genotyping of rs2180341 in the 171 non-AJ cases enriched for family history of breast cancer are shown in Table 7. Although statistically insignificant (per-allele p=0.24), LV M 704227

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the genotype frequencies of G/G and A/G (5.3% and 41.5% in cases versus 4.8% and 37% in controls) showed a minor trend toward association that is also reflected in per allele OR (OR=1.17, 95%CI 0.9-1.52).

Table 7

[0083] When sample size was increased by adding the 1 ,295 non-AJ sporadic breast cancer cases and adjustments were made for age and ethnicity, the aggregated analysis of all three replication non-AJ cohorts (n= 1,466) showed a significant association for all modes of analysis with the strongest association signal under recessive model (OR= 1.57, 95% CI 1.15- 2.15, p=0.0039). See Table 8.

Table 8

[0084] Genotyping of rs2180341, rs65669479, rs65669480 and rs7776136 SNPs in a subgroup of 847 non-AJ controls confirmed a strong linkage disequilibrium (D'=0.98), comparable to observations in AJ.

[0085] Although non-AJ cohorts in this study were mostly from Caucasian background, there were significant differences in allele or genotype frequencies for rs2180341 in subjects with an African American (AA) background. Therefore, to correct for population stratification the statistical analyses of non-AJ cohorts were adjusted for age and ethnicity in multivariate models. Preliminary analysis of the data for AA subset showed a trend LVM 7U4227

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correlating 6q22.33 with breast cancer risk in AA. In particular, there was a trend towards higher minor allele (A/G, G/G) frequencies for both AA cases and controls as compared to Caucasians.

[0086] Because of the population substructure of the non-AJ cohorts used in this study, a separate analysis was performed on a subset of non-AJ cohorts limited to subjects of Caucasian background. The association of rs2180341 remained statistically significant (per- allele OR=I.19, 95%CI 1.04-1.35, p=0.01). See Table 9. Replication of the association of 6q22 with breast cancer risk was further demonstrated by limiting the analysis to replication cohorts only (excluding subjects from previous AJ association study). Significant associations were observed under all models of analysis with per-allele OR= 1.18 (95% CI 1.04-1.33, p=0.008). See Table 10.

Table 9

[0087] An aggregated meta-analysis (n=5,656) of all case and control populations described in the examples herein, genotyped for rs2180341 and adjusted for age and ethnicity, revealed a strong and consistent association with breast cancer (per-allele OR=I.24, 95% CI 1.13-1.36, p=3.85E-7). See Table 11. LVM 704227

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Table 1 1

[0088] The foregoing example confirms the association of polymorphisms in chromosome 6q22.33 with an altered risk of developing breast cancer in non-AJ subjects.

EXAMPLE 8

[0089] This example describes the results of sequencing and expression analysis of the transcribed regions within the 200 kb linkage disequilibrium region of 6q22.33 that includes rs2180341, rs65669479, rs65669480 and rs7776136 SNPs.

[0090] Candidate genes in 6q22.33 were sequenced in DNA from 40 matched pairs of normal and tumor tissues from biopsies of MSKCC BC patients. Coding regions of ECHDCl and RNFl 46 were sequenced by ABI3700 capillary sequencing. Primers were designed to cover entire transcribed regions of both genes and to capture ~50bp of sequence from both sides of each exon. Sequencing was performed from both directions and data were analyzed by both SEQUENCHER (Genes Codes, Ann Arbor, MI) and MUTATION SURVEYOR (Softgenetics, State College, PA) software.

[0091] For "constitutive" expression analysis, blood specimens were collected from 24 individuals who visited the Clinical Genetics Service at MSKCC for diagnostic purposes. The individuals represented three genotypes: 8 high-risk allele homozygotes (G/G), 8 heterozygotes (A/G) and 8 low-risk allele homozygotes (AJA). Total mRNA was extracted from blood using PAXgene extraction kit (Qiagen, Valencia, CA) according to manufacture's instructions. First strand c-DNA synthesis was performed with an ISCRIPT SELECT kit (Biorad, Hercules, CA) using oligo-dT primers according to standard method. Expression levels of ECHDCl and RNF 146 were determined using TAQMAN gene expression assays (Hs00929453_ml and Hs00258475_sl) and normalized to constitutive expression of beta- actin (Hs99999903_ml). Statistical differences in expression correlated to genotype were calculated using Wilcoxon Rank Test. ^• LVM 7U4227

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[0092] Besides several rare polymorphisms, for which no significant differences were observed after testing them independently in a subset of the case-control population, no obviously functional or potentially pathogenic mutation was found to be associated with the common susceptible haplotype tagged by rs2180341. These results leave open the possibility that further mapping may reveal that more distant variant (linked to the 200 kbp core region on 6q22.33) is involved in the distant transcriptional regulation of ECHDCl and/or RNF 146. [0093] Expression analysis revealed a weak trend (p=0.22) wherein rs2180341 minor alleles (G/G and A/G) correlated with increased expression level of the RNFl 46 gene product in peripheral blood lymphocytes (PBLs).

EXAMPLE 9

[0094] This example describes the association of 6q22.33 with estrogen receptor ("ER") positive tumors.

[0095] Genotyping for 1,658 breast cancer of the cases described in the foregoing examples indicated indicated the presence of (a) 248 ER negative and 983 ER positive tumors from AJ cases and (b) 100 ER negative and 333 ER positive tumors from non-AJ cases. Stratification of tumors by ER status, when compared to controls, indicated that rs2180341 in the 6q22.33 locus had a stronger association with ER positive (per-allele OR=I.35, 95% CI 1.20-1.51, p=0.000022) than ER negative tumors (per-allele OR=I.18, 95% CI 0.98-1.44, p=0.09). See Table 12. Despite the foregoing association of ER status and rs2180341, the latter was not associated with a higher age of onset.

Table 12

Table 12 cont.

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[0096] Per allele results within breast cancer cases-only, ER positive versus ER negative tumors, were not significant (per-allele P=O.19). See Table 13. This was likely the result of limited analytical power due to the small sample size for ER negative cases in this comparison. Breast cancer cases-only analysis did show a trend resulting from slightly elevated genotype frequency of G/G (7.1% ER positive versus 5.8% ER negative) and A/G (41% ER positive versus 39.1% ER negative) in ER positive tumors. The trend corresponds to an increased ORs (per-allele OR=I.14, 95% CI 0.94-1.38). See Table 13.

Table 13

[0097] The foregoing shows that, in a sufficiently large sample size, rs2180341 linkage disequilibrium was shown to correlate with ER positive tumor status.

EXAMPLE 9

[0098] This example demonstrates that RNFl 46 gene product down regulates estrogen response element.

[0099] MCF7 cells (American Type Culture Collection, Manassas, VA) were grown in normal DMEM supplemented with 10% (v/v) FCS, 0.01 mg/ml bovine insulin and antibiotics at 37⁰C in a humidified atmosphere of 95% air and 5% CO₂. MCF7 cells were grown in 35 mm dish to about 80% confluence and transfected with a mixture of 5 μl FUGENE HD transfection reagent (Roche Applied Science, Indianapolis, IN) and 2 μg pCMV6-XL5 (Origen Technologies, Burlingame , CA) or pCMV6-XL5/RNF146 in accordance with Roche's protocol. RNF 146 was also cloned into pCMV3Xflag (Sigma- Aldrich, St. Louis, MO) as 5' Flag fusion protein and transfected into MCF 7 cells. 48 or 72 hours after transfection, transfected cells were washed with IX PBS and lysed with 1 ml lysate buffer (Pierce Biotechnology, Rockford, IL) containing mammalian protease inhibitor mixture (Sigma- Aldrich, St. Louis, MO). Total protein quantity in lysates were measured using a kit (Bio-Rad, Hercules, CA). Equal protein amounts from each sample were subjected to immunoprecipitation and Western blot analysis with RNFl 46 gene product antibody (Novus LVM 7U4227

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Biologicals, Littleton, CO) as described by Chen et al., J. Biol. Chem., 281:7452-7(2006). Protein G/A-agarose beads were from Oncogene (Oncogene Science Biomarker Corp., Cambridge, MA). Horseradish peroxidase-conjugated secondary antibodies and an ECL chemiluminescent kit were purchased from Amersham Biosciences (GE Healthcare, Piscataway, NJ). Immunoblotting experiments showed that pCMV6-XL5/RNF146 and pCMV3Xflag/RNF146 constructs overexpressed the RNF 146 gene product in MCF7 cells. [00100] Luciferase assays were performed using a Dynein Light Chain (DLCl) promoter- Luciferase reporter construct. The DLCl promoter includes an estrogen response element half-sites; and DLCl promoter activity is upregulated in MCF7 cells contacted with estradiol (E2). Rayala et al., EMBO Rep., 6: 538-44 (2005). MCF7 cells were grown in 24 well plates and transfected with 3 μl FUGENE HD mixed with a combination of 200 ng DLCl promoter-Luciferase, 1 μg pCMV6-XL5, and/or pCMV6-XL5/RNF 146. After 24 hour transfection, estradiol (E2)(Sigma-Aldrich, St. Louis, MO) was added to 10 nM final concentration. Luciferase activity was measured using standard methods. [0100] Cotransfection of the DLCl reporter construct and the pCMV6-XL5/RNF 146 construct expressing the RNF 146 gene product showed that RNF 146 overexpression reduced the ability of E2 to drive DLCl promoter expression of luciferase. See Figure 6. [0101] The foregoing indicates that RNF146 downregulates estrogen response elements. [0102] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0103] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary LVM 704227

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language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0104] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

LVM 704227DHHS E-065-2008/0-PCT-0232CLAIMS:

1. A method of determining the risk for developing breast cancer in a subject, the method comprising: obtaining genetic sequence information from a subject; and screening the sequence information for the presence or absence of one or more polymorphism alleles in chromosome 6q22.33; wherein the presence or absence^' of the one or more polymorphism alleles is correlated with a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject.

2. A method of determining the risk for developing breast cancer in a subject, the method comprising: obtaining genetic sequence information from a subject; and screening the sequence information for the presence or absence of one or more polymorphism alleles in chromosome 6q22.33 and, optionally, the presence of the one or more additional polymorphism alleles located in a position other than chromosome 6q22.33; wherein the presence or absence of the one or more polymorphism alleles is correlated with a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject.

3. The method of claim 1 or 2, wherein the subject is free of a BRCAl or BRCA2 polymorphism associated with increased risk for developing breast cancer.

4. The method of any one of claims 1 to 3, wherein the one or more polymorphism alleles are within 100 kilobases of a genomic locus corresponding to ECHDCl; RNF 146.

5. The method of any one of claims 1 to 4, wherein the one or more polymorphism alleles are within 50 kilobases of a genomic locus corresponding to ECHDCl; RNF146. LVM 704227

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6. The method of any one of claims 1 to 5, wherein the one or more polymorphism alleles are within 20 kilo bases of a genomic locus corresponding to ECHDCl; RNFl 46.

7. The method of any one of claims 1 to 6, wherein the one or more polymorphism alleles are within 10 kilobases of a genomic locus corresponding to the ECHDCl or RNF146 gene.

8. The method of any one of claims 1 to 7, wherein the one or more polymorphism alleles comprise one or more single nucleotide polymorphisms (SNP) alleles selected from the group consisting of rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NOs: 1, 2, 3, and 4, respectively.

9. The method of claim 8, wherein the method comprises screening for the presence of one or more SNPs selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively, and wherein the presence of the one or more SNPs is correlated with a lower risk of developing breast cancer in the subject.

10. The method of claim 8 or 9, wherein the method comprises screening for the presence of haplotype 1 (Hl), which includes screening for the presence of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively, and wherein the presence of Hl is correlated with a lower risk of developing breast cancer in the subject.

11. The method of any one of claims 1-9, wherein the presence of the one or more polymorphisms in chromosome 6q22.33 is correlated with a lower risk of developing cancer that is protective to about 1 x 10^"4 or higher level of significance. LVM 704227

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12. The method of any one of claims 11 , wherein the presence of the one or more polymorphisms is correlated with a lower risk of developing cancer that is protective to about 1 x 10^~5 or higher level of significance.

13. The method of claim 11 or 12, wherein the presence of the one or more polymorphisms is correlated with a lower risk of developing cancer that is protective to about 1 x 10^"9 or higher level of significance.

14. The method of claim any one of claims 1 to 8, wherein the method comprises screening for the presence of one or more SNPs selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding SEQ ID NO:1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively, and wherein the presence of the one or more SNPs is correlated with a higher risk of developing breast cancer in the subject.

15. The method of claim 14, wherein the method comprises screening for the presence of haplotype 2 (H2), which includes screening for the presence of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO: 1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively, and wherein the presence of H2 is correlated with a higher risk of developing breast cancer in the subject.

16. The method of any one of claims 1 to 8, 14, and 15, wherein the breast cancer is estrogen receptor positive breast cancer.

17. The method of any one of claims 1 to 8, 14, and 15, wherein the presence of the one or more polymorphisms in chromosome 6q22.33 is correlated with that the subject has an increased risk of developing breast cancer at about a 1x10^'2 or higher level of significance .

18. The method of any one of claims 1 to 8, 14, and 15; wherein the presence of the one or more polymorphisms in chromosome 6q22.33 is correlated with that the subject LVM 704227

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has an increased risk of developing breast cancer at about a 1x10^"4 or higher level of significance.

19. The method of any one of claims 1 to 8, 14, and 15; wherein the presence of the one or more polymorphisms in chromosome 6q22.33 is correlated with that the subject has an increased risk of developing breast cancer at about a 1x10^'7 or higher level of significance.

20. The method of any one of claims 1 to 19, wherein the subject is of Ashkenazi Jewish lineage.

21. The method of any one of claims 1 to 20, wherein the subject has four Ashkenazi Jewish grandparents.

22. The method of any one of claims 1 to 19, wherein the subject is of African American lineage.

23. The method of any one of claims 1-22, wherein the subject is older than 35.

24. The method of any one of claims 1 to 23, wherein the subject is a female in pre-menopause, menopause, or post-menopause.

25. The method of any one of claims 1 to 24, wherein the method comprises screening for the presence or absence of one or more additional polymorphisms located in a position other than chromosome 6q22.33, and wherein the presence or absence of the one or more additional polymorphisms is correlated with a lower risk of developing breast cancer in the subject or an increased risk of developing breast cancer in the subject.

26. The method of claim 25, wherein the one or more additional polymorphisms are associated with a gene selected from the group consisting of TNRC9, MAP 3 Kl, LSPl, Hl 9, BARDl, MDM2, CHEK2, ATM, BRIPl PALB2, FGFR2, and CASP8. LVM 704227

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27. The method of claim 25, wherein the one or more additional polymorphisms are associated with BRCAl, BRCA2, or both.

28. The method of any one of claims 1 to 27, wherein the method further comprises beginning or changing a course of treatment for the subject based on the presence or absence of the polymorphism.

29. The method of claim 28, wherein the course of treatment is a preventive course of treatment.

30. The method of any one of claims 1 to 27, wherein the method further comprises beginning or changing a course of monitoring for breast cancer in the subject based on the presence or absence of the polymorphism.

31. The method of any one of claims 1 to 7 and 14 to 27, wherein the method comprises screening for the presence or absence of one or more additional polymorphisms located in a position other than chromosome 6q22.33, and wherein the presence or absence of the one or more additional polymorphisms is correlated with a higher risk of breast cancer in the subject,

32. The method of claim 30, wherein the method further comprises beginning an aggressive course of monitoring for breast cancer.

33. The method of claim 32, wherein the more aggressive course of monitoring for breast cancer includes more frequent monitoring for breast cancer, magnetic resonance imaging (MRI), breast-specific gamma imaging (BSGI), positron emission tomography (PET), or ultrasound breast examination.

34. A breast cancer model cell comprising a recombinant DNA that controls expression of a ECHDCl gene product, a RNF 146 gene product, or both of the gene products in the cell. LVM 704227

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35. The model cell of claim 34, wherein the one or more gene product is encoded by recombinant DNA.

36. The model cell of claim 34, wherein the gene product is encoded by endogenous chromosomal DNA.

37. A method of screening for a candidate therapeutic compound for estrogen receptor positive breast cancer, the method comprising providing a breast cancer model cell with an estrogen response element operatively linked to the coding sequence for a gene product; overexpressesing the RNFl 46 gene product in the cell; contacting the cell with a test compound; and assaying for inhibition of gene product controlled by the estrogen response element relative to a control cell, wherein the control cell includes an estrogen response element operatively linked to the coding sequence for a gene product, overexpresseses the RNFl 46 gene product, and is not contacted by the test compound; and wherein inhibition of gene product controlled by the estrogen response element indicates that the test compound is a candidate therapeutic compound for estrogen receptor positive breast cancer.

38. A method of screening for candidate therapeutic compounds for breast cancer, the method comprising contacting a breast cancer cell with a test compound; and assaying for increased or decreased expression of ECHDCl or RNF 146 gene product in the cell, wherein increased or decreased expression of the gene product in the cell indicates that the test compound is a candidate therapeutic compound for breast cancer.

39. An oligonucleotide or set of oligonucleotides that selectively anneals to genomic DNA comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles corresponding to the sequence set forth in SEQ ID NOs:l, 2, 3, or 4, LVM 704227

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respectively, wherein the one or more oligonucleotides are covalently linked to a reporter molecule.

40. An oligonucleotide or set of oligonucleotides according to claim 40, wherein the one or more oligonucleotides include a fluorescent probe suitable for use in an assay that indicates the presence of genomic DNA comprising one, but not both, of the rs2180341, rs6569479, rs6569480, or rs7776136 alleles corresponding to the sequence set forth in SEQ ID NOs: 1, 2, 3, or 4, respectively, wherein the assay is selected from the group consisting of 5 '-nuclease ("TAQMAN") analysis, Invader or "flap-endonuclease" analysis, tetra-primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, and oligonucleotide ligase assay.

41. An oligonucleotide or set of oligonucleotides according to claim 39 or 40, wherein the one or more oligonucleotides preferentially anneal to genomic DNA comprising one or more allele selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to the sequences set forth SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively.

42. An oligonucleotide or set of oligonucleotides according to claim 39 or 40, wherein the one or more oligonucleotides preferentially anneal to genomic DNA comprising one or more allele selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to the sequences set forth in SEQ ID NO:1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively.

43. A kit comprising the oligonucleotide or set of oligonucleotides according to any one of claims 39 to 42; reagents for determining the presence or absence of a SNP allele.

44. The kit of claim 43, wherein the reagents are suitable for use in a method of screening sequence information for the presence or absence of a SNP allele that includes using the 5 '-nuclease ("TAQMAN") analysis, Invader or "flap-endonuclease" analysis, tetra- LVM 704227

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primer ARMS-PCR analysis, molecular beacon analysis, dynamic allele-specific hybridization (DASH) analysis, or oligonucleotide ligase assay.

45. The kit of claim 43, wherein the kit includes oligonucleotides designed to anneal to fewer than 1,000 different genomic loci.

46. Machine-readable medium that comprises digitally encoded, processed genetic information indicating the presence or absence of one or more polymorphisms in chromosome 6q22.33 in a subject, wherein the digitally encoded information is not raw genetic sequence information from a nucleotide array.

47. The machine readable medium of claim 46, wherein the subject is free of a BRCAl or BRCA2 polymorphism associated with increased risk for developing breast cancer.

48. The machine readable medium of claim 46 or 47, wherein the one or more polymorphism alleles are within 100 kilobases of a genomic locus corresponding to ECHDCl; RNF146.

49. The machine readable medium of any one of claims 46 to 48, wherein the one or more polymorphism alleles are within 50 kilobases of a genomic locus corresponding to ECHDCl; RNF146.

50. The machine readable medium of any one of claims 46 to 49, wherein the one or more polymorphism alleles are within 20 kilobases of a genomic locus corresponding to ECHDCl; RNF146.

51. The machine readable medium of any one of claims 46 to 50, wherein the one or more polymorphism alleles are within 10 kilobases of a genomic locus corresponding to the ECHDCl or RNF 146 gene.

52. The machine readable medium of any one of claims 46 to 51 , wherein the one or more polymorphism alleles are one or more single nucleotide polymorphisms (SNP) LVM 704227

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alleles selected from the group consisting of rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NOs: 1, 2, 3, and 4, respectively.

53. The machine readable of medium any one of claims 46 to 52, wherein the one or more polymorphism alleles are one or more SNP alleles selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO: 1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively, and wherein the presence of the one or more SNP alleles indicates a lower risk of developing breast cancer in the subject.

54. The machine readable medium of any one of claims 46 to 53, wherein the processed genetic information indicates the presence or absence of haplotype 1 (Hl), which includes the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when SEQ ID NO:1 when R is A, SEQ ID NO:2 when Y is C, SEQ ID NO:3 when R is G, and SEQ ID NO:4 when W is T, respectively.

55. The machine readable medium of any one of claims 46 to 52, wherein the one or more polymorphism alleles are one or more SNP alleles selected from the group consisting of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively, and wherein the presence of the one or more SNP alleles indicates a higher risk of developing breast cancer in the subject.

56. The machine readable medium of any one of claims 46 to 52 and 55, wherein the processed genetic information indicates the presence or absence of haplotype 2 (H2), which includes screening for the presence of the rs2180341, rs6569479, rs6569480, and rs7776136 alleles corresponding to SEQ ID NO:1 when R is G, SEQ ID NO:2 when Y is T, SEQ ID NO:3 when R is A, and SEQ ID NO:4 when W is A, respectively, and wherein the presence of H2 indicates a higher risk of developing breast cancer in the subject.

57. The machine readable medium of any one of claims 46 to 56, wherein the medium includes processed genetic information for fewer than 5,000 different genomic loci.