# Patent

## Title
Genetic marker for prostate cancer

## Abstract

The invention provides a method for determining a genetic predisposition to prostate cancer in a male human subject. In one aspect, the method comprises determining whether the subject has the genotype GG of refSNP rs125555, where the genotype GG of refSNP rs125555 indicates that the subject is genetically predisposed to prostate cancer.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application Ser. No. 60/820,655, filed on Jul. 28, 2006, which is herein incorporated by reference in its entirety.


STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT
This invention was made with United States government support awarded by the following agency: National Cancer Institute, grant number 1R01 CA74103-01A2. The United States has certain rights in this invention.


BACKGROUND OF THE INVENTION
The prostate is the most common non-cutaneous cancer site in males in developed countries. It is the second most common cause of death from cancer in men aged 60 or older. Early detection methods, such as prostate specific antigen (PSA) testing and digital rectal exam (DRE), have been developed. PSA is a glycoprotein secreted by the prostate gland. However, the PSA test has limitations of sensitivity and selectivity. In general, PSA levels above 4 ng/mL are suggestive of cancer and levels above 10 ng/mL are highly suggestive. However, many individuals with elevated levels do not have prostate cancer, but exhibit benign prostatic hypertrophy. Conversely, many persons with prostate cancer have normal PSA levels at the time of diagnosis. In addition, while it is known that genetic factors are involved in the development of prostate cancer, few genes with a direct role in prostate cancer have been identified.
Considerable evidence demonstrates that inherited genetic variants or mutations predispose individuals to developing prostate cancer. Germline mutations are estimated to account for approximately 9% of all prostate cancers and 45% of cases in men under age 55. Numerous linkage-mapping studies have identified candidate regions throughout the genome that may contain genes that predispose to prostate cancer. However, the majority of sequence variants within these regions that cause disease have not yet been identified. Identification of genetic sequences that are linked to prostate cancer allows determination of those with a higher risk of developing prostate cancer. Such information can be used to monitor those individuals with higher prostate cancer risk more closely, thereby allowing opportunity to detect prostate cancer at an early stage, which increases the likelihood of survival.
Therefore, there is a continuing need for determining which patients are at risk of developing prostate cancer.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method for detecting a genetic predisposition to prostate cancer in a male human subject. The method generally comprises determining whether the subject has the genotype GG of refSNP rs125555 wherein the genotype GG indicates that the subject is genetically predisposed to prostate cancer. For example, individuals having the genotype GG of refSNP rs125555 are on average more likely to develop prostate cancer than individuals having the genotype GC or CC of refSNP rs125555. An individual identified to have the GG genotype can be subjected to one or more prostate cancer tests such as PSA test, DRE, and other known prostate cancer tests. If prostate cancer is not found, such an individual can be subjected to prostate cancer tests at a higher frequency in the future than the general population or individuals who have the genotype GC or the genotype CC. Whether an individual has the genotype GG of refSNP rs125555 can be determined directly by analyzing a DNA sample comprising refSNP rs125555. Whether an individual has the genotype GG of refSNP rs125555 can also be determined indirectly by analyzing at least a portion of a corresponding mRNA that comprises a transcribed product of refSNP rs125555 or by analyzing a protein product that comprises a translated product of refSNP rs125555. The scope of the invention comprises both direct and indirect methods of determining whether an individual has the genotype GG of refSNP rs125555.
One way to determine whether an individual has the genotype GG of refSNP rs125555 is to genotype refSNP rs125555. In this regard, any known methods can be used. There is a wide range of methods available, which differ in fundamental aspects of the genotyping process, such as assay chemistry (allele specific hybridization, polymerase extension, oligonucleotide ligation, enzymatic cleavage, flap endonuclease discrimination, among others), and detection methods (fluorescence, colorimetry, chemiluminiscence and mass spectrometry, to name some commonly used systems). For example, standard DNA sequencing technologies such as ABI dye terminator chemistry on an ABI sequencer can be used. In addition, standard SNP genotyping platforms such as TaqMan (ABI), mass spectroscopy (Sequenome), various single base pair extension assays, and chip based genotyping platforms (Affymetrix) or bead array platforms (Ilumina) can also be used. Various genotyping methods are described in Kowk P Y, Annual Review of Genomics and Human Genetics, 2:235-258, 2001, which is herein incorporated by reference in its entirety.
In some embodiments of the invention, the method of determining whether an individual has the genotype GG of refSNP rs125555 comprises obtaining a sample (e.g., a genomic DNA sample or an mRNA sample) comprising MBD1 encoding polynucleotides from the individual and analyzing the polynucleotides to determine the genotype of refSNP rs125555. Within these embodiments, in some instances the step of analyzing the polynucleotides comprises amplifying at least a fragment of the polynucleotides such as the MBD1 gene, where the fragment comprises a sequence which is a portion of the MBD1 gene that includes refSNP rs125555. The step of analyzing the polynucleotides may further comprises exposing the amplified fragment to an allele-specific probe (e.g., a polynucleotide probe) under hybridization conditions wherein a hybrid will form between the allele-specific probe and one but not the other of the G and C alleles of refSNP rs125555.
In other embodiments, whether an individual has the genotype GG of refSNP rs125555 is determined by analyzing position 1340 of the mRNA NM—015846. mRNA NM—015846 is a transcribed product of MBD1 encoding genomic DNA. The nucleotide at position 1340 of the mRNA NM—015846 corresponds to refSNP rs125555. Accordingly, these embodiments of the invention are directed to analyzing the nucleotide of the mRNA whose position corresponds to refSNP rs125555. As described in detailed below, there are four other transcript variants of the MBD1 gene that are generated by alternative splicing. Whether an individual has the genotype GG of refSNP rs125555 can be determined by similarly analyzing one or more of these four other MBD1 mRNAs.
Still in other embodiments, whether an individual has the genotype GG of refSNP rs125555 is determined by analyzing amino acid position 401 of the protein product of NP—056671. The amino acid at position 401 of the protein product of NP—056671 corresponds to the amino acid that is translated from the codon comprising refSNP rs125555. Accordingly, these embodiments of the invention are directed to analyzing the amino acid whose position corresponds to the translated product of refSNP rs125555. The presence of amino acid alanine at position 401 of the protein product of NP—056671 corresponds to a G at refSNP rs125555 and the presence of proline at position 401 of the protein product of NP—056671 corresponds to a C at refSNP rs125555. As described in detailed below, there are four other transcript variants of the MBD1 gene generated by alternative splicing that lead to four other MBD1 protein products. Whether an individual has the genotype GG of refSNP rs125555 can be determined by similarly analyzing one or more of these four other MBD1 protein products.
Another aspect of the invention provides a method for detecting a genetic predisposition to prostate cancer in a male human subject. The method comprises:

    
    
        obtaining a sample comprising MBD1 encoding polynucleotides from a male human subject; and
        determining whether the genotype of nucleotide 10,105 of SEQ ID NO:1 is GG, wherein the genotype GG of nucleotide 10,105 of SEQ ID NO:1 is an indication that the subject is genetically predisposed to prostate cancer.
    
    


SEQ ID NO:1 in the sequence listing provides a portion of human chromosome 18 containing refSNP rs125555 at nucleotide 10,105. SEQ ID NO:1 corresponds to the sequence from position 8281 to 28570 of genomic clone provided at NCBI GenBank Accession Number AC090246. Nucleotide 10,105 of SEQ ID NO: 1 corresponds to nucleotide 18,385 of AC090246, which in turn corresponds to nucleotide 46,054,177 of chromosome 18 (Build 36.2).
In some embodiments, the step of determining whether the genotype of nucleotide 10,105 of SEQ ID NO:1 is GG comprises hybridizing an allele-specific probe (e.g., a polynucleotide probe) to the polypeptides under hybridization conditions wherein a hybrid will form between the allele-specific probe and G or C allele of nucleotide 10,105 of SEQ ID NO:1 and will not form between the allele-specific probe and the other allele of nucleotide 10,105 of SEQ ID NO:1.
In other embodiments, the sample is digested with an enzyme prior to determining whether the genotype of nucleotide 10,105 of SEQ ID NO:1 is GG to produce a sample fragment comprising a portion of the MBD1 gene including nucleotide 10,105 of SEQ ID NO:1. In some instances within these embodiments, the method further comprises amplifying the sample fragment to produce an amplified product prior to whether the genotype of nucleotide 10,105 of SEQ ID NO: 1 is GG. Furthermore, in some cases the step of determining whether the genotype of nucleotide 10,105 of SEQ ID NO: 1 is GG comprises hybridizing the amplified product with a probe (e.g., a polynucleotide probe) under hybridization conditions wherein a hybrid will form between the probe and G or C allele of nucleotide 10,105 of SEQ ID NO:1, and the hybrid will not form between the probe and the other allele of nucleotide 10,105 of SEQ ID NO:1.
Yet another aspect of the invention provides a method for detecting a genetic predisposition to prostate cancer in a male human subject by determining the genotype of refSNP 125555 of MBD1 gene comprising:

    
    
        digesting a sample comprising MBD1 encoding target polynucleotides from the subject with a restriction endonuclease;
        separating the sample fragments obtained from said digestion and immobilizing the fragments on a membrane by gel-transfer hybridization;
        hybridizing the immobilized fragments to an allele-specific probe (e.g., a polynucleotide probe) under hybridization conditions wherein a hybrid will form between the allele specific probe and a fragment of the MBD1 encoding polynucleotide which comprises G or C allele of refSNP rs125555 and will not form a hybrid between the allele specific probe and a fragment of the MBD1 encoding polynucleotide which comprises the other allele of refSNP rs125555; and
        detecting any hybrids formed and correlating the presence of hybrids formed with a genetic predisposition to prostate cancer.
    
    


In some embodiments, the sample is a genomic DNA. In these embodiments, the gel-transfer hybridization comprises Southern blotting.
In other embodiments, the sample is an RNA. In these embodiments, the gel-transfer hybridization comprises Northern blotting.
BRIEF DESCRIPTION OF THE DRAWINGS
Not applicable.


DETAILED DESCRIPTION OF THE INVENTION
DNA methylation occurs throughout the eukaryotic genome and plays an essential role in gene regulation. The proteins MECP2, MBD1, MBD2, MBD3, and MBD4 are nuclear proteins, each containing a methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, binds specifically to methylated DNA and represses transcription from methylated gene promoters. Five transcript variants of the MBD1 are generated by alternative splicing resulting in different protein isoforms each containing at least one MBD domain. All five transcript variants repress transcription from methylated promoters. Variants with three CXXC domains also repress unmethylated promoter activity.
The present inventor has found that predisposition to prostate cancer can be correlated with a non-synonymous genetic variant in methyl-CpG binding domain (MBD)1, a gene that regulates transcription in association with DNA methylation. In one particular study, the frequency of the homozygous recessive mutant allele variant (rs125555) was about two times higher in cases than in controls in samples collected based on family history of prostate cancer (OR=2.63, CI=1.43 to 4.84) and also sporadic cancer samples (OR=1.9, P=0.002). The single nucleotide polymorphism (i.e., SNP) rs125555 is at nucleotide 18,385 of the human genomic clone provided at NCBI GenBank Accession Number AC090246 (10,105 of SEQ ID NO:1, which corresponds to AC090246 positions 8281 to 28570). Nucleotide 10,105 of SEQ ID NO:1 corresponds to nucleotide 46,054,177 of chromosome 18 (Build 36.2), which can be viewed using NCBI Map Viewer at the NCBI website. The SNP rs125555 corresponds to mRNA position 1340 of NM—015846 (SEQ ID NO:2) and to encoding of amino acid proline or alanine at amino acid position 401 of the protein product as described by NP—056671 (SEQ ID NO:3). The other four transcript variants of the MBD1 gene, including both the mRNA and the amino acid sequences, can be found at NCBI GenBank Accession numbers NM_015845 (SEQ ID NO:4 and SEQ ID NO:5), NM_015847 (SEQ ID NO:6 and SEQ ID NO:7), NM_002384 (SEQ ID NO:8 and SEQ ID NO:9), and NM_015844 (SEQ ID NO:10 and SEQ ID NO:11), respectively, all of which are herein incorporated by reference in their entirety. The SNP rs125555 corresponds to mRNA position 1271 and amino acid position 378 of the transcript and protein product found at NM_015845 (nucleotide position 1271 and amino acid position 378 of SEQ ID NO:4 and SEQ ID NO:5, respectively), mRNA position 1193 and amino acid position 352 of the transcript and protein product found at NM_015847 (nucleotide position 1193 and amino acid position 352 of SEQ ID NO:6 and SEQ ID NO:7, respectively), mRNA position 1172 and amino acid position 345 of the transcript and protein product found at NM_002384 (nucleotide position 1172 and amino acid position 345 of SEQ ID NO:8 and SEQ ID NO:9, respectively), and mRNA position 1172 and amino acid position 345 of the transcript and protein product found at NM_015844 (nucleotide position 1172 and amino acid position 345 of SEQ ID NO:10 and SEQ ID NO:11, respectively). Accordingly, unless explicitly stated, the term “determining whether an individual has the genotype GG of refSNP rs125555 (or rs125555)” includes indirect method of detecting determining whether an individual has the genotype GG of refSNP rs125555. For example, one skilled in the art can readily analyze mRNA position 1340 of NM—015846 (or the corresponding mRNA position of one of the other four MBD1 mRNAs) or amino acid position 401 of the protein product described as NP—056671 (or the corresponding amino acid position of one of the other four MBD1 protein products). In addition, when referring to genotype of the genomic DNA or mRNA, the genotype refers to the sense strand of genomic DNA or mRNA, respectively. Without being bound by any theory, the genotype GG of refSNP rs125555 is believed to disrupt function of an MBD1 protein suggesting a biochemical mechanism for predisposition to prostate cancer in individuals lacking a functional allele.
One aspect of the invention provides a method for detecting a genetic predisposition to prostate cancer in an individual (e.g., a male human subject). The method comprises determining whether the individual has the genotype GG of refSNP rs125555. The genotype GG of refSNP rs125555 is an indication that the individual is genetically predisposed to prostate cancer. As stated above, whether an individual has the genotype GG of refSNP rs125555 (i.e., rs125555) can be determined directly (i.e., by analyzing the genomic DNA) or it can be determined indirectly (i.e., by analyzing a corresponding mRNA or a corresponding protein product).
Genomic DNA and mRNA can be analyzed using a variety of methods known to one skilled in the art including amplification (e.g., using PCR) followed by analysis of the amplified product, e.g., by probe hybridization (such as fluorescence, Southern blotting, and Northern blotting), and radio-labeling, etc. Methods for analyzing the protein are also well known to one skilled in the art (e.g., ELISA or other antibody-antigen based analysis). It is well within the capability of a skilled artisan to generate an antibody that specifically binds to an MBD1 protein product having alanine at the amino acid position corresponding to refSNP rs125555 but not an MBD1 protein product having proline at the amino acid position corresponding to refSNP rs125555 or vise versa. As another example, an antibody that specifically binds MBD1 products but cannot distinguish those having alanine from those having proline at the amino acid position corresponding to refSNP rs125555 can be used to purify MBD1 proteins and the identity of the amino acid at the position corresponding to refSNP rs125555 can then be determined by known technologies.
In another aspect of the invention, polynucleotide probes and primers that specifically hybridize to a subsequence of MBD1 gene comprising rs125555 or its complement under stringent hybridization conditions are provided. The probes and primers of this invention are polynucleotides of at least 7 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides or at least 25 nucleotides. The resulting amplified product, which includes refSNP rs125555, is typically from at least about 20 nucleotides to about 1,000 nucleotides in length. In some embodiments, the nucleotide length of the amplified product is at least about 20 nucleotides to about 500 nucleotides, typically from about 30 nucleotides to about 300 nucleotides, and often from about 50 nucleotides to about 200 nucleotides. Regardless of the length of the amplified product described above, it should be appreciated that the amplified product is always at least as long as the total amount of the nucleotides in the primer pairs and more often is longer than the total amount of the nucleotides in the primer pairs that are used in amplification.
Various polynucleotide probes and primers are commercially available that can detect the genotype of rs125555 of MBD1 gene. One such commercially available polynucleotide probes and primer combination is TaqMan assay C—889712—10 available from Applied Biosystems (Foster City, Calif). However, it should be appreciated that any suitable region of the MBD1 gene comprising rs125555 can be chosen as a target for polynucleotide hybridization. One skilled in the art can readily design the appropriate polynucleotide probes and primers. Nucleotide substitutions, deletions, and additions can be incorporated into the polynucleotides as long as the characteristic ability to specifically hybridize to the target sequence or its complement is retained. Nucleotide sequence variation can result from sequence polymorphisms of various alleles, minor sequencing errors, and the like.
Various probes and primers can be used as probes in hybridization assays, such as fluorescence, and Southern and Northern blots, for identifying polynucleotides having a nucleotide sequence comprising rs125555 and as primers for amplification procedures. In general, any combination of probes and primers can be used for identifying allelic forms (i.e., genotype) of rs125555. Such probes and primers can be used to determine a subject's predisposition or risk factor for prostate cancer.
The probes also are useful in oligonucleotide arrays. Such arrays are used in hybridization assays to check the identity of bases in a target polynucleotide. In essence, when a target hybridizes perfectly to a probe on the array, the target contains the nucleotide sequence of the probe. When the target hybridizes less well, or does not hybridize at all, then the target and probe differ in sequence by one or more nucleotide. By proper selection of probes, one can check bases on a target molecule. See, e.g., Chee et al., WO 95/11995.
The polynucleotide can also comprise a label. A detectable moiety bound to either an oligonucleotide primer or a probe is subsequently used to detect hybridization of an oligonucleotide primer or probe to the genomic DNA, the corresponding RNA component, or an amplification product.
Although primers and probes can differ in sequence and length, the primary differentiating factor is one of function: primers serve as an initiation point for DNA synthesis of a target polynucleotide, as in reverse transcription and PCR reactions, while probes are typically used for hybridization to and detection of a target polynucleotide. Typical lengths of primers or probes can range from 7-50 nucleotides, preferably from 10-40 nucleotides, and most preferably from 15-35 nucleotides. A primer or probe can also be labeled with a detectable moiety for detection of hybridization of the primer or probe to the target polynucleotide.
As stated above, one skilled in the art readily recognizes and can readily synthesize the suitable polynucleotides for detecting rs125555. Such polynucleotides include both DNA and RNA molecules and naturally occurring modifications thereof, as well as synthetic, non-naturally occurring analogs of the same, and heteropolymers, of deoxyribonucleotides, ribonucleotides, and/or analogues of either. The particular composition of a polynucleotide or polynucleotide analog depends on the purpose for which the material is used and the environment in which the material will be placed. Modified or synthetic, non-naturally occurring nucleotides have been designed to serve a variety of purposes and to remain stable in a variety of environments, such as those in which nucleases are present.
While there are commercially available primers and probes for genotyping rs125555 as stated herein, other probes and/or primers can be readily synthesized for genotyping rs125555. Various suitable oligonucleotides can be readily synthesized, e.g., on an Applied BioSystems or other commercially available oligonucleotide synthesizer according to specifications provided by the manufacturer. Oligonucleotides can be prepared using any suitable method, such as the phosphotriester and phosphodiester methods, or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidates are used as starting materials and may be synthesized as described by Beaucage er al., Tetrahedron Letters, 1981, 22, 1859, and U.S. Pat. No. 4,459,066.
Polynucleotides, e.g., probes, also can be recombinantly produced through the use of plasmids or other vectors.
As stated above, various probes and primers can be used in genotyping rs125555 in a sample. In one embodiment, the method for determining the genotype of refSNP rs125555 in a sample involves amplifying a portion of the MBD1 gene (or a corresponding mRNA) comprising refSNP rs125555 and hybridizing a polynucleotide probe or primer to the amplified product, and (2) detecting the specific hybridization.
Typically, the polynucleotides used for specific hybridization is chosen to hybridize to a region of MBD1 gene (or a corresponding mRNA) that comprises refSNP rs125555 (or the corresponding portion of the mRNA). The polynucleotides can be a DNA or RNA molecule, as well as a synthetic, non-naturally occurring analog of the same. The polynucleotides in this step are polynucleotide primers and polynucleotide probes as discussed herein.
Any suitable method for detecting specific hybridization of a polynucleotide to refSNP rs125555 can be used. Such methods include, e.g., amplification by extension of a hybridized primer using reverse transcriptase; extension of a hybridized primer using RT (reverse transcription)-PCR or other methods of amplification; and in situ detection of a hybridized primer. In in situ hybridization, a sample of tissue or cells is fixed onto a glass slide and permeablized sufficiently for use with in situ hybridization techniques. Detectable moieties used in these methods include, e.g., labeled polynucleotide probes, direct incorporation of label in amplification or reverse transcription reactions, and labeled polynucleotide primers.
In another aspect, the present invention provides a kit that contains various primers, probes, or antibodies described above as well as various combinations of the primers, probes, and antibodies. For example, a kit of the present invention can contain a set of primers that can amplify a portion of the human genomic DNA or at least a portion of one of the MBD1 mRNAs that contains rs125555. The kit can further contain a probe that can hybridize to a portion of the human genomic DNA or one of the MBD1 mRNAs or complements thereof that contains rs125555. The probe is one that can distinguish whether the nucleotide at rs125555 is a G or a C. The kits may further contain one or more positive and negative control nucleic acids, MBD1 antibodies, and MBD1 proteins.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following example thereof, which is not intended to be limiting.
EXAMPLE
DNA from affected brothers with prostate cancer were collected and a whole-genome linkage scan was performed to identify regions that contain prostate cancer genes or prostate cancer aggressiveness genes. Regions of the genome that were identified to be associated with increased risk of prostate cancer include 2q, 12p, 15q, 16p, and 16q. Regions associated with family history of prostate cancer (1q), lack of family history of prostate cancer (18q), family history of breast cancer (1p35), and late age-at-diagnosis (4q) were also identified. Prostate cancer aggressiveness loci (5q31, 7q32 and 19q12) were identified using Gleason grade as an index of tumor aggressiveness.
Aberrant DNA methylation and misregulation of gene expression at methylated sites have been shown to contribute to carcinogenesis of many tissues, including prostate. The MBD1 gene is located within a small region on chromosome 18q21.1 that is associated with increased risk for prostate cancer. Within this region, the non-synonymous coding single nucleotide polymorphism (SNP) (rs125555) was tested in prostate cancer cases and controls. It should be noted that rs125555 (i.e., refSNP rs125555) corresponds to nucleotide 1340 of NM—015846 mRNA, which in turn corresponds to amino acid position 401 of the protein product NP—056671. The other four transcript variants of the MBD1 gene, including both the mRNA and the amino acid sequences, can be found at NCBI GenBank Accession numbers NM—015845, NM—015847, NM—002384, and NM—015844, respectively. The SNP rs125555 corresponds to mRNA position 1271 and amino acid position 378 of the transcript and protein product found at NM—015845, mRNA position 1193 and amino acid position 352 of the transcript and protein product found at NM—015847, mRNA position 1172 and amino acid position 345 of the transcript and protein product found at NM—002384, and mRNA position 1172 and amino acid position 345 of the transcript and protein product found at NM—015844. Accordingly, genotype of rs125555 can be determined by analyzing the genomic DNA, nucleotide 1340 of NM—015846 mRNA (or a corresponding nucleotide of one of the other four MBD1 mRNAs), and/or amino acid position 401 of the protein product NP—056671 (or a corresponding amino acid position of one of the other four MBD1 protein products).
It has been found that a common variant of MBD1, e.g., rs125555, is associated with prostate cancer. Accordingly, MBD1 gene, its mRNAs, and/or its protein products can be used to determine the risk of developing prostate cancer.
Two sets of prostate cancer cases and controls were used in this study. The family based cohort was collected from 275 multiplex prostate cancer sibships and 556 unrelated controls. Control subjects were followed for many years as part of a long-term prostate cancer screening study in which men were screened at 6 to 12 month intervals with PSA blood tests and DRE of the prostate.
The control subjects were required to meet the following four criteria: (1) be at least 65 years old, (2) never have registered a PSA level above 2.5 ng/mL, (3) always had DRE findings that were not suspicious for prostate cancer, and (4) have no known family history of prostate cancer. Family history of prostate cancer was assessed by inquiring about the subjects' brothers, fathers, grandfathers, and maternal and paternal uncles. As a result of the first criterion, the mean age of control subjects was greater than the mean age of case subjects. All the subjects were of European ancestry.
The sporadic prostate cancer cohort was collected. Incident prostate cancer cases were diagnosed from Jan. 1, 1999 through Dec. 31, 2000. The project over-sampled for younger men in order to reduce the number of controls with latent prostate cancers and to enhance investigations into genetic etiology. All cases diagnosed before age 60 and a 10% random sample of cases diagnosed between ages 60 and 79 were invited to participate. Overall case response rate was 68%. Case participants ranged in age from 38 to 80 years at the time of the study (mean=58 years). A comparable number of controls were identified through state driver's license. To reduce any bias, the small portion of prostate cancer cases without driver's licenses (<5%) were excluded from the study. All control participants were tested for PSA at the time of participation to exclude any controls with undiagnosed prostate cancer. Blood samples for DNA were provided by cases and controls. Information regarding the patient's prostate cancer including tumor stage and grade was collected by review of the patients' pathology reports and medical record.
Genotyping was done using the functionally tested TaqMan assay C—889712—10 purchased from Applied Biosystems (Foster City, Calif.).
To investigate the role of MBD1 in prostate cancer, the SNP rs125555 was tested in two independent case/control cohorts. Table 1 shows the allele distribution and frequency (%) obtained in each cohort. When the allele frequency of homozygous Ala/Ala was compared in cases and controls from the family cohort, there was an odds ratio of 2.63 (95% CI=1.43-4.84). For the sporadic cohort, the odds ratio for homozygous Ala/Ala was 1.865 (P=0.002).







TABLE 1







Distribution of MBD1 genotypes in two prostate cancer cohorts










Families
Sporadic














Pro/Pro (%)
Ala/Pro (%)
Ala/Ala (%)
Pro/Pro (%)
Ala/Pro (%)
Ala/Ala (%)

















Case
326 (60.3)
173 (32.0)
42 (7.8)
826 (61.2)
426 (31.6)
97 (7.2)


Control
311 (60.7)
186 (36.3)
15 (2.9)
826 (62.7)
439 (33.3)
53 (4.0)









MBD1 is located at chromosome 18q21.1 between markers AFM292wg1 and AFM312vc5 as provided at the Marshfield Clinic Mammalian Genotyping Service's website as well as NCBI's website. These markers map to 71.32 cM on the sex average map. Importantly, D18S363 at 71.3 cM has a Zlr score of 2.09 (P=0.018) and is the highest score over a 20 cM region on chromosome 18q in our whole genome scan of families affected with prostate cancer. The linkage at this region is in families without hereditary prostate cancer according to the Hopkins criteria. To be included in the hereditary prostate cancer group, a family must contain (1) two or more brothers with a diagnosis of prostate cancer at age <55, (2) at least three first-degree relatives with a diagnosis of prostate cancer, or (3) three consecutive generations with prostate cancer. Only six families met criterion 1 with five of them having an affected father. Therefore, virtually all hereditary prostate cancer families met criterion 2.
Without being bound by any theory, the rs125555 variant is believed to destroy protein activity of isoform 1 of MBD1 based on the PolyPhen (Polymorphism Phenotyping) computer calculation available at the website of the Genetics Division in the Department of Medicine at Brigham & Women's Hospital and Harvard Medical School. PolyPhen predicts the impact of an amino acid change on the structure and function of a protein using multiple sequence alignments and protein 3D-structures. This prediction that MBD1 activity is destroyed by the variant that associates with an increased risk of prostate cancer suggests that normal gene expression would be disrupted resulting in increased gene transcription at methylated sites that would normally be silenced.
The study showed that a non-synonymous coding variant rs125555 is associated with prostate cancer in two separate case/control cohorts of prostate cancer. The replication of results in two populations provides strong evidence that MBD1 is a prostate cancer gene, e.g., mysregulation of MBD1 protein expression is associated with prostate cancer. MBD1 expression was very high in benign prostatic hyperplasia and low-grade tissues with a decrease in MBD1 expression as tumor grade increased.
The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

## Claims

1. A method for detecting a genetic predisposition to prostate cancer in a male human subject, said method comprising:
detecting in said subject the presence of a G nucleotide in both alleles of the methyl-CpG binding domain protein 1 (MBD1) gene at the position corresponding to position 10,105 of SEQ ID NO: 1,
wherein the presence of a G nucleotide in both alleles of MBD1 gene at the position corresponding to position 10,105 of SEQ ID NO: 1 is indicative of a genetic predisposition to prostate cancer in said male human subject.

2. The method of claim 1, wherein whether the subject has a G nucleotide in both alleles of the MbD1 gene located at nucleotide position 10,105 of SEQ ID NO: 1 is determined by obtaining a sample comprising MBD1 encoding polynucleotides from the subject and analyzing the polynucleotides to determine whether the subject has a GG genotype at nucleotide position 10,105 of SEQ ID NO: 1.

3. The method of claim 2, wherein the sample is a genomic DNA sample or an mRNA sample.

4. The method of claim 2, wherein analyzing the polynucleotides comprises amplifying at least a fragment of the polynucleotides, wherein said fragment comprises nucleotide position 10,105 of SEQ ID NO: 1.

5. The method of claim 4, wherein analyzing the polynucleotides further comprises exposing the amplified fragment to an allele-specific probe under hybridization conditions wherein a hybrid will form between the allele-specific probe and one but not the other of the G and C alleles located at nucleotide position 10,105 of SEQ ID NO: 1.

6. The method of claim 1, wherein whether the subject has a G nucleotide in both alleles of the MBD1 gene located at nucleotide position 10,105 of SEQ ID NO: 1 is determined by analyzing an MBD1 mRNA.

7. The method of claim 1, wherein whether the subject has a G nucleotide in both alleles of MBD1 gene located at nucleotide position 10,105 of SEQ ID NO: 1 is determined by analyzing a MBD1 protein product.