Aberrantly methylated genes as markers of breast malignancy

This is a continuation-in-part application of U.S. patent application Ser. No. 09/771,337, filed Jan. 26, 2001, co-pending, which is incorporated herein by reference in its entirety.

This invention was made with Government support under P50 CA88843-01, awarded by the National Institutes of Health; and U.S. Public health Service Grant CA48943. The government may have certain rights in the invention.

The present invention relates generally to a method of diagnosing a cell proliferative disorder of breast tissue by determining the DNA methylation status of nucleic acids obtained a subject.

Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorogenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R. D. eds. in DNA Methylation Biochemistry and Biological Significance , Springer-Verlag, New York, 1984). In eukaryotic cells, methylation of cytosine residues that are immediately 5′ to a guanosine, occurs predominantly in cytosine-guanine (CG) poor regions (Bird, Nature, 321:209, 1986). In contrast, CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation (Migeon, et al., supra) and parental specific imprinting (Li, et al., Nature, 366:362, 1993) where methylation of 5′ regulatory regions can lead to transcriptional repression. De novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas (Sakai, et al., Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700, 1994). Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated CpG island (Issa, et al., Nature Genet., 7:536, 1994; Herman, et al., supra; Merlo, et al., Nature Med., 1:686, 1995; Herman, et al., Cancer Res., 56:722, 1996; Graff, et al., Cancer Res., 55:5195, 1995; Herman, et al., Cancer Res., 55:4525, 1995).

Human cancer cells typically contain somatically altered nucleic acid, characterized by mutation, amplification, or deletion of critical genes. In addition, the nucleic acid from human cancer cells often displays somatic changes in DNA methylation (Fearon, et al., Cell, 61:759, 1990, Jones, et al., Cancer Res., 46:461, 1986; Holliday, Science, 238:163, 1987; De Bustros, et al., Proc. Natl. Acad. Sci., USA, 85:5693, 1988); Jones, et al., Adv. Cancer Res., 54:1, 1990; Baylin, et al., Cancer Cells, 3:383, 1991; Makos, et al., Proc. Natl. Acad. Sci., USA, 89:1929, 1992; Ohtani-Fujita, et al., Onco-gene, 8:1063, 1993). However, the precise role of abnormal DNA methylation in human tumorogenesis has not been established.

Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells, and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. This molecular defect has also been described in association with various cancers. CpG islands are short sequences rich in the CpG dinucleotide and can be found in the 5′ region of about half of all human genes. Methylation of cytosine within 5′ CGIs is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting. Aberrant methylation of CpG islands has been detected in genetic diseases such as the fragile-X syndrome, in aging cells and in neoplasia. About half of the tumor suppressor genes which have been shown to be mutated in the germline of patients with familial cancer syndromes have also been shown to be aberrantly methylated in some proportion of sporadic cancers, including Rb, VHL, p16,hMLH1, and BRCA1 (reviewed in Baylin, et al., Adv. Cancer Res. 72:141-196 1998). Methylation of tumor suppressor genes in cancer is usually associated with (1) lack of gene transcription and (2) absence of coding region mutation. Thus CpG island methylation can serve as an alternative mechanism of gene inactivation in cancer.

Breast cancer is by far the most common form of cancer in women, and is the second leading cause of cancer death in humans. Despite many recent advances in diagnosing and treating breast cancer, the prevalence of this disease has been steadily rising at a rate of about 1% per year since 1940. Today, the likelihood that a woman living in North America will develop breast cancer during her lifetime is one in eight.

Breast cancer is often discovered at a stage that is advanced enough to severely limit therapeutic options and survival rates. Accordingly, more sensitive and reliable methods are needed to detect small (less than 2 cm diameter), early stage, in situ carcinomas of the breast. In addition to the problem of early detection, there remain serious problems in distinguishing between malignant and benign breast disease, in staging known breast cancers, and in differentiating between different types of breast cancers (e.g., estrogen dependent versus non-estrogen dependent tumors). Recent efforts to develop improved methods for breast cancer detection have focused on cancer markers such as proteins that are uniquely expressed (e.g., as a cell surface or secreted protein) by cancerous cells, or are expressed at measurably increased or decreased levels by cancerous cells compared to normal cells. Accordingly, the use of the methylation status of certain genes as a marker of cancer or cancerous conditions provides an additional weapon in early detection and prognosis of breast cancers.

Identification of the earliest genetic changes in cells associated with breast cancer is a major focus in molecular cancer research. Diagnostic approaches based on identification of these changes in specific genes are likely to allow implementation of early detection strategies and novel therapeutic approaches. Targeting these early changes might lead to more effective cancer treatment.

The present invention is based on the finding that several genes are newly identified as being differentially methylated in breast cancers. This seminal discovery is useful for breast cancer screening, risk-assessment, prognosis, disease identification, disease staging and identification of therapeutic targets. The identification of new genes that are methylated in breast cancer allows accurate and effective early diagnostic assays, methylation profiling using multiple genes; and identification of new targets for therapeutic intervention.

In a first embodiment, the invention provides method of diagnosing a cellular proliferative disorder of breast tissue in a subject comprising determining the state of methylation of one or more nucleic acids isolated from the subject. The state of methylation of one or more nucleic acids compared with the state of methylation of one or more nucleic acids from a subject not having the cellular proliferative disorder of breast tissue is indicative of a cell proliferative disorder in the subject. In one aspect of this embodiment, the state of methylation is hypermethylation. The invention provides a method of diagnosing a cellular proliferative disorder of breast tissue in a subject by detecting the state of methylation of one or more of the following nucleic acids: Twist, cyclin D2, WT1, NES-1, HOXA5 and combinations thereof. Also methylated are RARβ2, 14.3.3 sigma, estrogen receptor, RASSF1A, HIN-1 and combinations thereof. In one aspect of the invention, nucleic acids are methylated in regulatory regions.

Invention methods can be used to diagnose disorders of the breast including breast cancers. In one aspect of the invention, disorders of the breast include ductal carcinoma in situ, lobular carcinoma, colloid carcinoma, tubular carcinoma, medullary carcinoma, metaplastic carcinoma, intraductal carcinoma in situ, lobular carcinoma in situ and papillary carcinoma in situ.

Another embodiment of the invention provides a method of determining a predisposition to a cellular proliferative disorder of breast tissue in a subject. The method includes determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the state of methylation of one or more nucleic acids compared with the state of methylation of the nucleic acid from a subject not having a predisposition to the cellular proliferative disorder of breast tissue is indicative of a cell proliferative disorder of breast tissue in the subject. The nucleic acids can be nucleic acids encoding Twist, cyclin D2, RARβ2, HOXA5, WT1, 14.3.3 sigma, estrogen receptor, NES-1, RASSF1A, HIN-1 and combinations thereof.

Still another embodiment of the invention provides a method for detecting a cellular proliferative disorder of breast tissue in a subject. The method includes contacting a specimen containing at least one nucleic acid from the subject with an agent that provides a determination of the methylation state of at least one nucleic acid. The method further includes identifying the methylation states of at least one region of at least one nucleic acid, wherein the methylation state of the nucleic acid is different from the methylation state of the same region of nucleic acid in a subject not having the cellular proliferative disorder of breast tissue.

Yet a further embodiment of the invention provides a kit useful for the detection of a cellular proliferative disorder in a subject comprising carrier means compartmentalized to receive a sample therein; and one or more containers comprising a first container containing a reagent that modifies unmethylated cytosine and a second container containing primers for amplification of a CpG-containing nucleic acid. The primers hybridize with target polynucleotide sequence having the sequence of certain nucleic acids described herein.

FIG. 1A shows the nucleotide sequence of the cyclin D2 promoter (SEQ ID NO:105). Regions highlighted indicate primer sequences. CG nucleotide pairs are shown capitalized and bolded. A highlighted box shows the location of an atg codon. FIG. 1B shows nucleotide sequences for forward (F) and reverse (R) primer external and internal pairs (SEQ ID NOS 21-24, 129 and 130) used to detect methylated (M) and unmethylated (U) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIGS. 2A and 2B show the nucleotide sequence of the TWIST promoter (SEQ ID NO:106). Regions highlighted indicate primer sequences. CG nucleotide pairs are shown capitalized and bolded. A highlighted box shows the location of an atg codon. FIG. 2C shows nucleotide sequences for forward (F) and reverse (R) external and internal primer pairs (SEQ ID NOS 109, 110, 107, 108, 131 and 132) used to detect methylated (M) and unmethylated (U) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIG. 3A shows the nucleotide sequence of the Retinoic Acid Receptor Beta (RARβ) promoter (SEQ ID NO:91). Regions highlighted indicate primer sequences. CG nucleotide pairs are shown capitalized and bolded. A highlighted box shows the location of an atg codon. FIG. 3B shows nucleotide sequences for forward (F) and reverse (R) external and internal primer pairs (SEQ ID NOS 92, 93, 135, 136, 133 and 134) used to detect methylated (M) and unmethylated (U) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIG. 4A shows the nucleotide sequence of Homo sapiens serine protease-like protease (NES-1) mRNA (SEQ ID NO:94). FIG. 4B shows the nucleotide sequence of the NES-1 region (exon 3) analyzed (SEQ ID NO:95). Regions highlighted indicate primer sequences. CG nucleotide pairs are shown capitalized and bolded. FIG. 4C shows nucleotide sequences for forward (F) and reverse (R) primer pairs (SEQ ID NOS 77-80) used to detect methylated (M) and unmethylated (U) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIG. 5A shows the nucleotide sequence of HOXA5 promoter (SEQ ID NO:96). CG nucleotide pairs are shown capitalized and bolded. A highlighted box shows the location of a cat codon. FIG. 5B shows the nucleotide sequence of the complementary region (5′ to 3″) analyzed (nucleotides -97 to -303) (SEQ ID NO:97). Regions highlighted indicate primer sequences. CG nucleotide pairs are shown capitalized and bolded. Highlighted box shows an atg codon. FIG. 5C shows nucleotide sequences for forward (F) and reverse (R) primer pairs (SEQ ID NOS 71, 98, 72, 69, 99 and 70) used to detect methylated (M) and unmethylated (U) nucleic acids. The base pair (BP) length of the primer pair product is also indicated. FIG. 5D shows forward and reverse (sense and antisense) primers (SEQ ID NOS 73, 100, 74, 75, 101, 76) used for sequencing and expression of HOXA5.

FIG. 6A to 6 F show the nucleotide sequence of Homo sapiens 14.3.3 sigma protein promoter and gene, complete cds.

FIG. 7A and 7B show the nucleotide sequence of Homo sapiens Wilms' tumor (WT1) gene promoter.

FIG. 8A and 8B show the nucleotide sequence of Homo sapiens (SEQ ID NO:104) estrogen receptor beta gene, promoter region and partial cds. CG nucleotide pairs are shown capitalized and bolded. FIG. 8C shows nucleotide sequences of forward (F) and reverse (R) primer (SEQ ID NOS 85, 86, 87, 88, 89 and 90) pairs used to detect methylated (M) and unmethylated (UM) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIG. 9A shows the nucleotide sequence of human HIN-1 (SEQ ID NO:120) cDNA Regions highlighted indicate primer sequences. FIG. 9B shows nucleotide sequences of forward and reverse external and internal primer pairs used to detect methylated and unmethylated nucleic acids. The base pair (bp) length of the primer pair product is also indicated.

FIG. 10A shows the nucleotide sequence of the RASSF1A promoter (SEQ ID NO:121). CG nucleotide pairs are shown capitalized and bolded. Regions highlighted indicate primer sequences. FIG. 10B shows nucleotide sequences of forward (F) and reverse (R) external and internal primer pairs (SEQ ID NOS 122, 123, 124, 126, 127 and 128) used to detect methylated (M) and unmethylated (UM) nucleic acids. The base pair (BP) length of the primer pair product is also indicated.

FIG. 11 is a schematic representation of the invention assay methods utilizing the technique of multiplex methylation-specific PCR,

The invention is based upon the discovery that the hypermethylation of certain genes can serve as markers for cellular proliferative disorders of breast tissue. This is the first time that promoter hypermethylation of certain genes such as Twist, cyclin D2, RARβ2, WT1, NES-1 and HOXA5 have been associated with breast cancer.

It has been determined that the methylation state of nucleic acids of certain genes, particularly regulatory sequences, is diagnostic for the presence or potential development of a cellular proliferative disorder of breast tissue in subjects. More particularly, the hypermethylation of certain nucleotides localized in CpG islands has been shown to affect the expression of genes associated with the CpG islands; typically such hypermethylated genes have reduced or abolished expression, primarily due to down-regulated transcription. Hypermethylation of, for example, Twist, cyclin D2, retinoic acid receptor β (RARβ), WT1, HOXA5, 14.3.3 sigma, estrogen receptor (ER) NES-1, the Ras association domain family 1A gene (RASSF1A), and the high in normal-1 gene (HIN-1) allows one to diagnose a cellular proliferative disorder of breast tissue. Using a recently developed PCR-based technique called methylated specific PCR (MSP), aberrantly methylated nucleic acids in breast cancer primary tumors and biological samples from individuals with breast cancer can be identified.

In a first embodiment, the invention provides a method of diagnosing a cellular proliferative disorder of breast tissue in a subject comprising determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the state of methylation of one or more nucleic acids as compared with the state of methylation of one or more nucleic acids from a subject not having the cellular proliferative disorder of breast tissue is indicative of a cellular proliferative disorder of breast tissue in the subject. A preferred nucleic acid is a CpG-containing nucleic acid, such as a CpG island.

A cell proliferative disorder as described herein may be a neoplasm. Such neoplasms are either benign or malignant. The term “neoplasm” refers to a new, abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal. A neoplasm creates an unstructured mass (a tumor), which can be either benign or malignant. The term “benign” refers to a tumor that is noncancerous, e.g. its cells do not invade surrounding tissues or metastasize to distant sites. The term “malignant” refers to a tumor that is metastastic, invades contiguous tissue or no longer under normal cellular growth control.

One type of cellular proliferative disorder is a cell proliferative disorder of breast tissue. Disorders of breast tissue or breast cancers can involve numerous cells and tissues resulting in various disorders of the breast including ductal carcinoma in situ, lobular carcinoma, colloid carcinoma, tubular carcinoma, medullary carcinoma, metaplastic carcinoma, intraductal carcinoma in situ, lobular carcinoma in situ, and papillary carcinoma in situ.

The invention method includes determining the state of methylation of one or more nucleic acids isolated from the subject. The phrases “nucleic acid” or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. As will be understood by those of skill in the art, when the nucleic acid is RNA, the deoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.

The nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a differentially methylated CpG island. The CpG island is a CpG rich region of a nucleic acid sequence. The nucleic acids includes, for example, a sequence encoding the following genes (GenBank Accession Numbers are shown, followed by the nucleotides corresponding to the region(s) examined for the presence or absence of methylation (numbers are relative to the first ATG codon unless otherwise indicated)): Twist (Accession No. AC003986; −51145 to 151750 (complement) (SEQ ID NO:106), cyclin D2 (Accession No. U47284; −1616 to −1394) (SEQ ID NO:105); RARβ2 (Accession No. AF; 157484; −196 to −357)(SEQ ID NO:91), WT1 (Accession No. AB034940) (SEQ ID NO:103); HOXA5 (Accession No. AC004080) (SEQ ID NO:96), 14.3.3 sigma (Accession No. AF029081) (SEQ ID NO:102); estrogen receptor (ER; Accession No. X62462) (SEQ ID NO:104); NES-1 (Accession No. AF024605) (SEQ ID NO:94), the nucleotide sequence of each of which is incorporated by reference herein.

WT1 encodes a transcriptional regulatory protein that binds DNA via four Cys 2 .His 2 zinc fingers. WT1 mRNA undergoes two independent splicing events leading to the expression of at least four predominant isoforms. These splices result in the inclusion or omission of exon 5 (51 base pairs) and the presence or absence of a nine base pair insert (encoding three amino acids, KTS) between the third and fourth zinc finger domains. Lack of expression has been observed in some Wilms'' tumors, leading to classification as a tumor suppressor gene. However, WT1 is overexpressed in 75% of cases of acute leukemia and is upregulated as chronic myeloid leukemia progresses into blast crisis. Thus, WT1 can apparently be either a tumor suppressor or an oncogene.

The cyclin D1, D2 and D3 proteins are involved in regulation of the cell cycle through phosphorylation and inactivation of the retinoblastoma protein and activation of cyclin E, leading to transition of the cells from G1 to DNA synthesis. In addition to their role in cell cycle regulation, the D-type cyclins have been implicated in differentiation and neoplastic transformation. Overexpression of cyclin D2 has been reported in gastric cancer, and was shown to correlate with disease progression and poor prognosis. Overexpression of cyclin D2 is also noted in ovarian granulosa cell tumors and testicular germ cell tumor cell lines.

14.3.3 σ is a member of a superfamily that is responsible for instituting the G2 cell cycle checkpoint in response to DNA damage in human cells (Hermeking, et at. (1997) Mol. Cell 1, 3-11; Chan, et al. (1999) Nature 401, 616-20). In addition to any growth advantage resulting from a loosening of this checkpoint control mechanism, loss of σ function is predicted to cause an increase in DNA damage in response to γ-irradiation. Loss of 14.3.3. σ in primary epithelial cells leads to immortalization (Dellambra et al. (2000) J. Cell Biol., 149:1117-1130), one of the earliest steps towards cancer.

Retinoic acid (RA) controls fundamental developmental processes, induces terminal differentiation of myeloid progenitors and suppresses cancer and cell growth. RA activity is mediated by nuclear receptors, the retinoic acid receptors, RARs, that act as RA-dependent transcriptional activators in their heterodimeric forms with retinoid X receptors, RXRs (Chambon, 1996). RARs induce local chromatin changes at level of target genes, containing responsive RA elements (RAREs) by recruiting multiprotein complexes with histone acetyltransferase (HAT) activity and histone deacetylase (HDAC) activity that dynamically pattern chromatin modification and regulate gene expression. RARs and RXRs, when disrupted, result in severe developmental defects and neoplastic transformation. In breast cancer cells, the expression of one member of the RARs family, RAR β is found consistently down regulated or lost. RAR β downregulation can be reversed by RA in estrogen receptor (ER)-positive, but not in ER-negative breast carcinoma cell lines, believed to represent more advanced forms of tumors. Loss of RA-induced RAR β expression is considered a crucial step in the development of RA-resistance in breast carcinogenesis. A complex regulatory region, with two promoters, regulates RAR β gene expression. Only one promoter, RAR β2, containing several RA-response elements, including a canonical and an auxiliary RA response element, βRARE, is active in human mammary epithelial cells (HMEC). The transcription of the RAR β2 promoter is mediated by multiple RARs including, RARα and RAR β itself able to recruit coactivator and corepressor protein complexes with HAT/HDAC activities, respectively.

The Twist gene product is a transcription factor with DNA binding and helix-loop-helix domains. Twist is a member of the bHLH transcription factor family and is involved in the development of mesodermally derived tissue including the skeleton. In humans, mutations in the Twist gene have been identified in patients with Saethre-chotzen syndrome, a relatively common craniosynostosis disorder with autosomal dominant inheritance. (see Gripp et al., (2000) Hum. Mutat. 15:479.) Twist also influences osteogenic gene expression and may act as a master switch in initiating bone cell differentiation by regulating the osteogenic cell lineage (Lee et al., (1999) J. Cell Biochem. 75:566-577).

NES1 (normal epithelial cell-specific 1) is a novel gene with a predicted polypeptide of about 30.14 kilodaltons and having a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor gamma, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator) (Liu et al., (1996) Cancer Res. 56:14 3371-9). All of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, indicating that it has protease-like activity. Immunolocalization studies with an antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detect a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive mammary epithelial cells (MECs), suggesting that NES1 is a secreted protein. The 1.4-kb NES1 mRNA is expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovaries. Although expression of the NES1 mRNA is observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines show a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorogenesis. Studies using fluorescence in situ hybridization localized the NES1 gene to chromosome 19q13.3, a region that contains genes for related proteases (Goyal et al., (1998) Cancer Res., 58:21 4782-6).

The HOX genes are expressed during embryonic development and have a role in specifying antero-posterior positional information. The genes are arranged in four clusters and a collinear relation exists between a gene's position in the cluster and its anterior boundary of expression. Genes with more anterior boundaries are also expressed earlier than genes with more posterior boundaries. Hox genes encode transcription factors; therefore, a model for the coordinate regulation of the genes within the HOX clusters is that Hox gene products regulate their own expression. The production of HOXA5 from an expression vector can activate a transient and simultaneous expression of other upstream and downstream genes of the same HOX cluster and genes from other clusters.

The estrogen receptor gene has been implicated in the initiation and/or progression of human breast cancer. Loss of expression of either gene has been associated with poorly differentiated tumors and poorer prognosis. Several studies have reported an association between estrogen receptor (ER) expression and breast tumors. A loss of ER expression has been associated with aberrant 5′ CpG island methylation in breast cancer cell lines and primary human breast tumors. Studies show that aberrant methylation of ER CpG islands begins before invasion of tumors into surrounding tissues and it increases with metastatic progression (Naas et al., (2000) Cancer Res., 60:4346-4348; incorporated by reference in its entirety).

Hypermethylation of the CpG island of Ras Association Domain Family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Hypermethylation of the RASSF1A promoter appears to be the main mechanism of inactivation. The high frequency of epigenetic inactivation of the RASF1A gene in breast cancer supports its role as a putative tumor suppressor gene (R. Dammann, et al., Cancer Research 61:3105-3109, 2001; K Dreijerink et al., PNAS 98(18):7504-7509, 2001; D. G. Burbee et al., J. National Cancer Institute 93(9):691-699, 2001, each of which is incorporated herein by reference in its entirety).

Expression of HIN-1 (high in normal-1) is significantly down regulated in 94% of human breast carcinoma and in 95% of preinvasive lesions, such as ductal and lobular carcinoma in situ. This decrease in HIN-1 expression is accompanied by hypermethylation of its promoter in the majority of breast cancer cell lines and primary tumors. This decrease in HIN-1 expression is accompanied by hypermethylation of its promoter in the majority of breast cancer cell lines (greater than 90%) and primary tumors (74%). HIN-1 is a putative cytokine with no significant homology to known proteins. Reintroduction of HIN-1 into breast cancel cells has been shown to inhibit cell growth, making HIN-1 a candidate tumor suppressor gene that is inactivated at high frequency in the earliest stages of breast tumorogenesis (I. E. Krop et al., PNAS 98(17):9796-9801, 2001, which is incorporated herein by reference in its entirety).

Any nucleic acid sample, in purified or nonpurified form, can be utilized in accordance with the present invention, provided it contains, or is suspected of containing, a nucleic acid sequence containing a target locus (e.g., CpG-containing nucleic acid). One nucleic acid region capable of being differentially methylated is a CpG island, a sequence of nucleic acid with an increased density relative to other nucleic acid regions of the dinucleotide CpG. The CpG doublet occurs in vertebrate DNA at only about 20% of the frequency that would be expected from the proportion of G*C base pairs. In certain regions, the density of CpG doublets reaches the predicted value; it is increased by ten fold relative to the rest of the genome. CpG islands have an average G*C content of about 60%, compared with the 40% average in bulk DNA. The islands take the form of stretches of DNA typically about one to two kilobases long. There are about 45,000 such islands in the human genome.

In many genes, the CpG islands begin just upstream of a promoter and extend downstream into the transcribed region. Methylation of a CpG island at a promoter usually prevents expression of the gene. The islands can also surround the 5′ region of the coding region of the gene as well as the 3′ region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns.

In general, the CpG-containing nucleic acid is DNA. However, invention methods may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample. A mixture of nucleic acids may also be employed. The specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA. The nucleic acid-containing sample used for determination of the state of methylation of nucleic acids contained in the sample or detection of methylated CpG islands may be extracted by a variety of techniques such as that described by Sambrook, et al. ( Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989; incorporated in its entirety herein by reference).

A nucleic acid can contain a regulatory region which is a region of DNA that encodes information that directs or controls transcription of the nucleic acid. Regulatory regions include at least one promoter. A “promoter” is a minimal sequence sufficient to direct transcription, to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents. Promoters may be located in the 5′ or 3′ regions of the gene. Promoter regions, in whole or in part, of a number of nucleic acids can be examined for sites of CG-island methylation.

Nucleic acids isolated from a subject are obtained in a biological specimen from the subject. The nucleic acid may be isolated from breast tissue, blood, plasma serum, lymph, duct cells, nipple aspiration fluid, ductal lavage fluid and bone marrow. Tissue, blood, lymph, lymph node, duct cells, nipple aspiration fluid, ductal lavage fluid and bone marrow are obtained by various medical procedures known to those of skill in the art. Duct cells can be obtained by nipple aspiration, ducal lavage, sentinel node biopsy, fine needle aspirate, routine operative breast endoscopy and core biopsy. Ductal lavage fluid can be obtained by using a DucWash procedure. In this procedure, a catheter is inserted into one or more of the four to eight ducts typically present in each human breast, lavage of the duct is performed, and the lavage fluid is collected. Alternatively, ductal lavage may be achieved through a microcatheter procedure known as ROBE (routine operative breast endoscopy), which allows visualization of a tumor at the same time as aspiration of fluid from the duct.

In one aspect of the invention, the state of methylation in nucleic acids of the sample obtained from a subject is hypermethylation compared with the same regions of the nucleic acid in a subject not having the cellular proliferative disorder of breast tissue. Hypermethylation, as used herein, is the presence of methylated alleles in one or more nucleic acids. Nucleic acids from a subject not having a cellular proliferative disorder of breast tissues contain no detectable methylated alleles when the same nucleic acids are examined.

A method for determining the methylation state of nucleic acids is described in U.S. Pat. No. 6,017,704 which is incorporated herein in its entirety and described briefly herein. Determining the methylation state of the nucleic acid includes amplifying the nucleic acid by means of oligonucleotide primers that distinguishes between methylated and unmethylated nucleic acids.

Two or more markers can also be screened simultaneously in a single amplification reaction to generate a low cost, reliable cancer-screening test for breast cancers. A combination of DNA markers for CpG-rich regions of nucleic acid may be amplified in a single amplification reaction. The markers are multiplexed in a single amplification reaction, for example, by combining primers for more than one locus. For example, DNA from a ductal lavage sample can be amplified with two or more different unlabeled or randomly labeled primer sets in the same amplification reaction. Especially useful are two or more markers selected from cyclin D2, RARβ2, Twist, NES-1, RASSF1A and HIN-1. The reaction products are separated on a denaturing polyacrylamide gel, for example, and then exposed to film or stained with ethidium bromide for visualization and analysis. By analyzing a panel of markers, there is a greater probability of producing a more useful methylation profile for a subject.

For example, a screening technique, referred to herein as “multiplex methylation-specific PCR” is a unique version of methylation-specific PCR. Methylation-specific PCR is described in U.S. Pat. Nos. 5,786,146, 6,200,756, 6,017,704 and 6,265,171, each of which is incorporated herein by reference in its entirety. Multiplex methylation-specific PCR utilizes MSP primers for a multiplicity of markers, for example up to five different breast cancer markers, in a two-stage nested PCR amplification reaction. The primers used in the first PCR reaction are selected to amplify a larger portion of the target sequence than the primers of the second PCR reaction. The primers used in the first PCR reaction are referred to herein as “external primers” or DNA primers” and the primers used in the second PCR reaction are referred to herein as “MSP primers.” Two sets of primers (i.e., methylated and unmethylated for each of the markers targeted in the reaction) are used as the MSP primers. In addition in multiplex methylation-specific PCR, as described herein, a small amount (i.e., 1 μl) of a 1:10 1 to about 10 6 dilution of the reaction product of the first “external” PCR reaction is used in the second “internal” MSP PCR reaction. The technique of multiplex methylation-specific PCR is illustrated schematically in FIG. 11 .

As shown in Table 1 below, multiplex methylation-specific PCR greatly enhances the accuracy of diagnosis obtainable from an amount of DNA available for analysis as compared with direct PCR analysis.

Multiplex methylation-specific PCR is also high specific. Tests conducted to compare the results of direct MSP with multiplex methylation-specific PCR in analysis of the methylation status of human primary breast tumor, and human breast cancer cell lines, are summarized, respectively, in Tables 5-7 below. The results shown in Tables 5-7 illustrate concordance in the results obtained by analysis of these various types of samples using direct MPC and multiplex methylation-specific PCR, as disclosed herein.

If the sample is impure (e.g., plasma, serum, lymph, ductal cells, nipple aspiration fluid, ductal lavage fluid, bone marrow, blood or breast tissue embedded in paraffin), it may be treated before amplification with a reagent effective for lysing the cells contained in the fluids, tissues, or animal cell membranes of the sample, and for exposing the nucleic acid(s) contained therein. Methods for purifying or partially purifying nucleic acid from a sample are well known in the art (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989, herein incorporated by reference).

Primers hybridize with target polynucleotide sequences. Nucleic acid sequences including exemplary primers are set forth in SEQ ID NO:1 to SEQ ID NO:128. Oligonucleotide primers specifically targeted to methylated and unmethylated genes including Twist, cyclinD2, RARβ2, WT1, HOXA5, 14.3.3 sigma, estrogen receptor, NES-1, RASSF1A, HIN-1, and their associated CpG islands include, respectively, SEQ ID NO:7-14, 21-24, 37-40, 49-64, 69-72, 77-80, 85-90, 107-110, 116-119, 124-128, 129-130, and 135-136. (See Table 4 below).

Detection of differential methylation can be accomplished by contacting a nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid. The sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG-sites under conditions and for a time to allow cleavage of methylated nucleic acid. Oligonucleotides are added to the nucleic acid sample under conditions and for a time to allow ligation of the oligonucleotides to nucleic acid cleaved by the restriction endonuclease, and the digested nucleic acid is amplified by conventional methods, such as PCR wherein primers complementary to the oligonucleotides are employed. Following identification, the methylated CpG-containing nucleic acid can be cloned, using methods well known to those of skill in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).

As used herein, a “methylation sensitive restriction endonuclease” is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated. Preferably, the methylation sensitive restriction endonuclease has inhibited activity when the C is methylated (e.g., Smal). Specific non-limiting examples of methylation sensitive restriction endonucleases include Sma I, BssHII, or HpaII, MspI, BSTUI, and NotI. Such enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleases will be known to those of skill in the art and include, but are not limited to SacII, and EagI, for example. An “isoschizomer” of a methylation sensitive restriction endonuclease is a restriction endonuclease that recognizes the same recognition site as a methylation sensitive restriction endonuclease but cleaves both methylated and unmethylated CGs. Those of skill in the art can readily determine appropriate conditions for a restriction endonuclease to cleave a nucleic acid (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).

A nucleic acid of interest is cleaved with a methylation sensitive endonuclease. Cleavage with the methylation sensitive endonuclease creates a sufficient overhang on the nucleic acid of interest, i.e., sufficient to allow specific hybridization of an oligonucleotide of interest. Following cleavage with the isoschizomer, the cleavage product can still have a sufficient overhang. An “overhang” refers to nucleic acid having two strands wherein the strands end in such a manner that a few bases of one strand are not base paired to the other strand. A “sufficient overhang” refers to an overhang of at least two bases in length or four or more bases in length. An overhang of a specific sequence on the nucleic acid of interest may be desired in order for an oligonucleotide of interest to hybridize. In this case, the isoschizomer can be used to create the overhang having the desired sequence on the nucleic acid of interest.

Cleavage with a methylation sensitive endonuclease results in a reaction product of the nucleic acid of interest that has a blunt end or an insufficient overhang. “Blunt end” refers to a flush ending of two stands, the sense stand and the antisense strand, of a nucleic acid. Once a sufficient overhang is created on the nucleic acid of interest, an oligonucleotide is ligated to the nucleic acid of interest, which has been cleaved by the methylation specific restriction endonuclease. “Ligation” is the attachment of two nucleic acid sequences by base pairing of substantially complementary sequences and/or by the formation of covalent bonds between two nucleic acid sequences.

An adaptor can be utilized to create DNA ends of desired sequence and overhang. An “adaptor” is a double-stranded nucleic acid sequence with one end that has a sufficient single-stranded overhang at one or both ends such that the adaptor cart be ligated by base-pairing to a sufficient overhang on a nucleic acid of interest that has been cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme. Adaptors can be obtained commercially. Alternatively, two oligonucleotides that are substantially complementary over their entire sequence except for the region(s) at the 5′ and/or 3′ ends that will form a single stranded overhang can be used to form an adaptor. The single stranded overhang on the adapter is selected to be complementary to an overhang on the nucleic acid cleaved by a methylation sensitive restriction enzyme or an isoschizomer of a methylation sensitive restriction enzyme, such that the overhang on the nucleic acid of interest will base pair with the 3′ or 5′ single stranded end of the adaptor under appropriate conditions. The conditions will vary depending on the sequence composition (GC vs AT), the length, and the type of nucleic acid (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1998).

Following the ligation of the oligonucleotide to the nucleic acid of interest, the nucleic acid of interest is amplified using a primer complementary to the oligonucleotide. Specifically, the term “primer” as used herein refers to a sequence comprising two or more deoxyribo-nucleotides or ribonucleotides, preferably more than three, and more preferably more than eight, wherein the sequence is capable of initiating synthesis of a primer extension product that is substantially complementary to a nucleic acid such as an adaptor or a ligated oligonucleotide. Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates, an agent for polymerization, such as DNA polymerase, and suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer can be an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact length of the primer will depend on many factors, including temperature, buffer composition (i.e., salt concentration), and nucleotide composition. The oligonucleotide primer typically contains 12-20 or more nucleotides, although it may contain fewer nucleotides.

Primers of the invention are designed to be “substantially” complementary to each strand of the oligonucleotide to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with a 5′ and 3′ oligonucleotide to hybridize therewith and permit amplification of CpG containing nucleic acid sequence.

Primers of the invention are employed in the amplification process, which is an enzymatic chain reaction that produces exponentially increasing quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction or PCR). Typically, one primer is complementary to the negative (−) strand of the locus (antisense primer) and the other is complementary to the positive (+) strand (sense primer). Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides, results in newly synthesized + and − strands containing the target locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target locus sequence) defined by the primer. The product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

The oligonucleotide primers used in invention methods may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphos-phoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. (Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.

Another method for detecting a methylated CpG-containing nucleic acid includes contacting a nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and non-methylated nucleic acid and detecting the methylated nucleic acid. The amplification step is optional and although desirable, is not essential. The method relies on the PCR reaction itself to distinguish between modified (e.g., chemically modified) methylated and unmethylated DNA.

The term “modifies” as used herein means the conversion of an unmethylated cytosine to another nucleotide that will facilitate methods to distinguish the unmethylated from the methylated cytosine. Preferably, the agent modifies unmethylated cytosine to uracil. Preferably, the agent used for modifying unmethylated cytosine is sodium bisulfite; however, other agents that similarly modify unmethylated cytosine, but not methylated cytosine, can also be used in the method. Sodium bisulfite (NaHSO 3 ) reacts readily with the 5,6-double bond of cytosine, but poorly with methylated cytosine. Cytosine reacts with the bisulfite ion to form a sulfonated cytosine reaction intermediate that is susceptible to deamination, giving rise to a sulfonated uracil. The sulfonate group can be removed under alkaline conditions, resulting in the formation of uracil. Uracil is recognized as a thymine by Taq polymerase. Therefore after PCR, the resultant product contains cytosine only at the position where 5-methylcytosine occurs in the starting template DNA.

The primers used in the invention for amplification of the CpG-containing nucleic acid in the specimen, after bisulfite modification, specifically distinguish between untreated or unmodified DNA, methylated, and non-methylated DNA. MSP primers for the non-methylated DNA preferably have a T in the 3′ CG pair to distinguish it from the C retained in methylated DNA, and the complement is designed for the antisense primer. MSP primers usually contain relatively few Cs or Gs in the sequence since the Cs will be absent in the sense primer and the Gs absent in the antisense primer (C becomes modified to U (uracil) which is amplified as T (thymidine) in the amplification product).

The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids in the polymorphic locus. Where the nucleic acid sequence of interest contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as a template for the amplification process. Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word “denaturing” includes all such means. One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105° C. for times ranging from about 1 to 10 minutes. Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA. The reaction conditions suitable for strand separation of nucleic acids with helicases are described by Kuhn Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).

When complementary strands of nucleic acids are separated, regardless of whether the nucleic acid was originally double or single stranded, the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands. This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, generally at a pH of about 7-9. Preferably, a molar excess (for genomic nucleic acid, usually about 108:1 primer:template) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. Large molar excess is preferred to improve the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to approximately room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein “agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40° C. Most conveniently the reaction occurs at room temperature.

The agent for polymerization may be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation such as Taq DNA polymerase, and the like). Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each locus nucleic acid strand. Generally, the synthesis will be initiated at the 3′ end of each primer and proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be agents for polymerization, however, which initiate synthesis at the 5′ end and proceed in the other direction, using the same process as described above.

Preferably, the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. However, alternative methods of amplification have been described and can also be employed. PCR techniques and many variations of PCR are known. Basic PCR techniques are described by Saiki et al. (1988 Science 239:487-491) and by U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference.

The conditions generally required for PCR include temperature, salt, cation, pH and related conditions needed for efficient copying of the master-cut fragment. PCR conditions include repeated cycles of heat denaturation (i.e. heating to at least about 95° C.) and incubation at a temperature permitting primer: adaptor hybridization and copying of the master-cut DNA fragment by the amplification enzyme. Heat stable amplification enzymes like the pwo, Thermus aquaticus or Thermococcus litoralis DNA polymerases which eliminate the need to add enzyme after each denaturation cycle, are commercially available. The salt, cation, pH and related factors needed for enzymatic amplification activity are available from commercial manufacturers of amplification enzymes.

As provided herein an amplification enzyme is any enzyme which can be used for in vitro nucleic acid amplification, e.g. by the above-described procedures. Such amplification enzymes include pwo, Escherichia coli DNA polymerase I, Klenow fragment of E. coli polymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney Murine Leukemia Virus reverse transcriptase, T4 DNA ligase, E. coli DNA ligase or Qβ replicase. Preferred amplification enzymes are the pwo and Taq polymerases. The pwo enzyme is especially preferred because of its fidelity in replicating DNA.

Once amplified, the nucleic acid can be attached to a solid support, such as a membrane, and can be hybridized with any probe of interest, to detect any nucleic acid sequence. Several membranes are known to one of skill in the art for the adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose (NITROPURE®) or other membranes used in for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GENESCREEN®, ZETAPROBE® (Biorad), and NYTRAN®. Methods for attaching nucleic acids to these membranes are well known to one of skill in the art. Alternatively, screening can be done in a liquid phase.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization of the probe of interest.

The probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.

Another embodiment of the invention provides a method of determining a predisposition to a cellular proliferative disorder of breast tissue in a subject comprising determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the nucleic acid is selected from the group consisting of Twist, cyclin D2, RARβ2, HOXA5, WT1, 14.3.3 sigma, estrogen receptor, NES-1, RASSF1A, HIN-1, and combinations thereof; and wherein the state of methylation of one or more nucleic acids as compared with the state of methylation of said nucleic acid from a subject not having a predisposition to the cellular proliferative disorder of breast tissue is indicative of a cell proliferative disorder of breast tissue in the subject.

As used herein, “predisposition” refers to an increased likely that an individual will have a disorder. Although a subject with a predisposition does not yet have the disorder, there exists an increased propensity to the disease.

Another embodiment of the invention provides a method for diagnosing a cellular proliferative disorder of breast tissue in a subject comprising contacting a nucleic acid-containing specimen from the subject with an agent that provides a determination of the methylation state of nucleic acids in the specimen, and identifying the methylation state of at least one region of least one nucleic acid, wherein the methylation state of at least one region of at least one nucleic acid that is different from the methylation state of the same region of the same nucleic acid in a subject not having the cellular proliferative disorder is indicative of a cellular proliferative disorder of breast tissue in the subject.

Invention methods are ideally suited for the preparation of a kit. Therefore, in accordance with another embodiment of the present invention, there is provided a kit it useful for the detection of a cellular proliferative disorder in a subject. Invention kits include a carrier means compartmentalized to receive a sample therein, one or more containers comprising a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of a CpG-containing nucleic acid, wherein the primers distinguish between modified methylated and nonmethylated nucleic acid. Primers contemplated for use in accordance with the invention include those set forth in SEQ ID NOs: 7-14, 21-24, 37-40, 49-64, 69-72, 77-80, 85-90, 116-119, 124-128, and combinations thereof.

Carrier means are suited for containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. In view of the description provided herein of invention methods, those of skill in the art can readily determine the apportionment of the necessary reagents among the container means. For example, one of the container means can comprise a container containing an oligonucleotide for ligation to nucleic acid cleaved by a methylation sensitive restriction endonuclease. One or more container means can also be included comprising a primer complementary to the oligonucleotide. In addition, one or more container means can also be included which comprise a methylation sensitive restriction endonuclease. One or more container means can also be included containing an isoschizomer of said methylation sensitive restriction enzyme.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

Although the invention has been described with reference to the presently preferred embodiment, is should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

RNA was treated with RNAse-free DNAse (Boehringer-Mannheim) (0.5-1 u/ul) for 30 min. at 37° C., followed by heat inactivation at 65° C. for 10 min. RT reactions contained 2 μg DNAse treated RNA, 0.25 μg/μl pdN6 random primers (Pharmacia), 1×first strand buffer (GibcoBRL), 1 mM dNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and were incubated for 1 h at 37° C. followed by heat inactivation at 75° C. for 5 min. PCR was performed using the primers 5′-CATGGAGCTGCTGTGCCACG-3′ (sense; SEQ ID NO:15) and 5′-CCGACCTACCTCCAGCATCC-3′ (antisense; SEQ ID NO:16) for cyclin D2 and primers 5′-AGCCATGGAACACCAGCTC-3′ (sense; SEQ ID NO:17) and 5′-GCACCTCCAGCATCCAGGT-3′ (antisense; SEQ ID NO:18) for cyclin D1. Co-amplified products of 36B4, a “housekeeping” ribosomal protein gene, was used as an internal control, using primers 5′-GATTGGCTAC CCAACTGTTGCA-3′ (sense; SEQ ID NO:19) and 5′-CAGGGGCAGCAGCCACAAAGGC-3′ antisense; SEQ ID NO:20). The 25 μl reactions contained 1×buffer (2× Reaction Mix, cat #10928-026, BRL) and 100 nM of each primer. The PCR conditions were: 1 cycle of 94° C. for 1 min “hot start” then addition of 1u of Taq polymerase (RedTaq), 1 cycle of 94° C. for 2 min, 35 cycles of: 94° C. for 15 sec, 55° C. for 30 sec, 72° C. for 45 sec, and finally 72° C. for 5 min. The PCR samples were resolved by electrophoresis on a 2% agarose gel in 1×TBE buffer.

Methylation-specific PCR (MSP)

One μg genomic DNA or the 50 ul extract of microdissected cells was treated with sodium bisulfite as described in Herman JG, (1996), and was analyzed by MSP using primer sets located within the CpG-rich island in the cyclin D2 promoter. Primers specific for unmethylated DNA were 5′-GTTATGTTATGTTmGTTGTATG-3′ (sense; SEQ ID NO:21) and 5′-GTTATGTTATGTTTGTTGTATG-3′ (antisense; SEQ ID NO:22) and yielded a 223 base-pairs PCR product. Primers specific for methylated DNA were 5′-TACGTGTTAGGGTCGATCG-3′ (sense; SEQ ID NO:23) and 5′-CGAAATATCTACGCTAAACG-3′ (antisense; SEQ ID NO:24) and yielded a 276 base-pair PCR product. The PCR conditions were as follows: 1 cycle of 95° C. for 5 min; 35 cycles of 95° C. for 30 s, 55° C. for 30 s and 72° C. for 45 s; and 1 cycle of 72° C. for 5 min. The PCR products were resolved by electrophoresis in a 2% agarose gel in 1×TBE buffer.

Treatment of Cells with 5′-aza-2′-deoxycytidine (5aza-dC) and Trichostatin A (TSA)

Cells were seeded at a density of 1×10 6 cells per 100-mm plate. 24 h later cells were treated with 0.75 μM 5-aza-dC (Sigma) or with 100 ng/ml of TSA (Sigma). Total cellular DNA and RNA were isolated at 0, 3 and 5 days after addition of 5-aza-dC and at 0, 24 and 48 hours after addition of TSA, as described above.

Western Blot Analysis

Proteins were extracted from cell pellets and from 8 micron sections of primary breast tumors in buffer containing 20 mM Tris pH 7.5, 150 nM NaCl and PMSF, and sonicated. Twenty μg of protein were fractionated on 12.5% SDS-PAGE and transferred by electrophoresis to a nylon membrane. The blot was incubated with anti-cyclin D2 antibody (Ab-4, “cocktail” mouse monoclonal antibodies, Neomarkers, San Diego, Calif.) diluted 1:200 in 5% skim milk, for 2h at room temperature. Horseradish peroxidase-conjugated antibody anti-mouse IgG (Amersham) was used at 1:1000, and binding was revealed using enhanced chemiluminescence (Amersham).

Cyclin D2 mRNA Expression in Breast Cancer

Serial analysis of gene expression (SAGE) and subsequent microarray analysis previously revealed that, compared with finite lifespan HMECs, cyclin D2 expression was significantly lower in a small panel of primary breast tumors (Nacht M, et al., Cancer Research 59:5464-5470 (1999). To confirm the validity of these findings, we investigated expression of cyclin D2 by RT-PCR in three finite life span and 6 immortal HMECs, 11 breast cancer cell lines and 24 primary breast carcinomas. A ribosomal protein RNA, 36B4, was co-amplified as an internal control. Abundant expression of cyclin D2 mRNA was noted in all three finite life span HMECs and in 4 of 6 immortalized HMECs. The two immortalized HMEC lines lacking cyclin D2 expression were HBL100 and MCF10A. In contrast, 10 of 11 breast cancer cell lines showed no detectable expression of cyclin D2. Only one breast cancer cell line, HS578T, expressed a low but detectable level of cyclin D2. Likewise, the results with primary tumors reflected the findings in cultured cells. Eighteen of 24 primary breast carcinomas expressed significantly lower levels of cyclin D2 mRNA as compared with finite lifespan HMEC 184 and five other HMECs. As an additional control for cyclin D2 expression, the expression of cyclin D1 was analyzed in the same panels of cell lines and tumors. Consistent with previous observations Cyclin D1 mRNA was detectable in all the cell lines and primary breast tumors tested. Thus, in both breast cancer cell lines and primary tumors specific loss of cyclin D2, but not cyclin D1, mRNA expression was observed.

Cyclin D2 mRNA Expression in Luminal and Myoepithelial Cells of the Breast

It has been reported that cyclin D2 is expressed in myoepithelial but not in luminal epithelial cells of the breast (Lukas J, (1995)). Therefore, lack of expression of cyclin D2 in breast cancers would be expected, since the vast majority of these tumors originate from luminal rather than myoepithelial cells. This conclusion was based, however, on the results from a single HMEC preparation. The present study used a larger panel of tissues. Luminal and myoepithelial cells isolated from four normal mammoplasty specimens from women aged 18 to 33 were used. Paired luminal and myoepithelial cells were obtained from the same breast of two women. Each cell type was purified using immunomagnetic beads. The human luminal and myoepithelial cells were separated by virtue of their exclusive expression of epithelial membrane antigen (EMA) and common acute lymphoblastic leukemia antigen (CALLA) respectively. The purity of the populations was checked by immunocytochemistry using cytokeratins 18 and 19 as markers for luminal cells and cytokeratin 14 as a marker for myoepithelial cells. These tests showed that the final population was 95-99% pure in each case. Cyclin D2 expression was assessed in the purified cell preparations by RT-PCR Cyclin D2 expression was observed in four of four purified luminal epithelial cells, as well as four of four myoepithelial cells. However, one luminal epithelial cell sample had a significantly lower expression of cyclin D2. Four HMECs of the 184 series, which stain for luminal cell markers-cytokeratins 8 and 18 and mucin, but not for myoepithelial cell marker-cytokeratin 14, also expressed cyclin D2 mRNA. Thus, cyclin D2 mRNA was expressed in all eight of eight luminal and four of four myoepithelial cell preparations from the normal breast.

Western Analysis Reveals Loss of Cyclin D2 Protein in Primary Tumors

For Western blot analysis specific anti-cyclin D2 antibodies that did not cross-react with cyclin D1 were used. While cyclin D2 protein was clearly detected in all seven HMECs tested (11-24, 1-26, 70N, 166372, 81N, 9F1403 and 184A1), it was undetectable in the majority (10/13) of primary breast tumors. Thus, HMECs that were derived from normal breast tissue and expressed high levels of cyclin D2 mRNA show clearly detectable levels of cyclin D2 protein as well. In contrast, primary breast tumors that exhibited low or absent cyclin D2 mRNA showed a corresponding loss of the cyclin D2 protein.

The Cyclin D2 Promoter is Hypermethylated in Breast Cancer Cell Lines and Primary Tumors

In Somatic Cells, about 80% of the CGs are methylated. Exceptions to this are the CpG islands in the promoter region of many genes. CpG islands are GC-rich regions of DNA, approximately 1 kb in length, present in the promoters of more than 60% of human genes. Normally CpG islands are unmethylated and the chromatin in those sites is enriched in hyperacetylated histone and deficient in histone H1, characteristic of active chromatin. Both unmethylated and methylated DNA are assembled into nucleosomes.

The cyclin D2 promoter contains a CpG-rich region at 1000 to 1600 base-pairs 5′ to the translation start site. To test whether aberrant methylation is associated with loss of cyclin D2 expression, primers for a Methylation Specific PCR (MSP) assay were designed to rapidly screen for cyclin D2 promoter methylation. Hypermethylation of the CpG rich region was detected in 11 of 11 breast cancer cell lines that also lacked expression of cyclin D2 protein. Aberrant methylation was also noted in 49 of 106 (46%) primary breast carcinomas.

Next, to determine whether cyclin D2 promoter-methylation is a tumor-specific phenomenon, DNA from histopathologically normal breast tissue adjacent to the surgically resected cancer was tested. All 11 samples of normal breast epithelial tissue adjacent to carcinoma were unmethylated at the CpG sites tested by MSP.

To further support the observation that cyclin D2 hypermethylation does not occur in normal HMECs and is associated with malignancy, normal breast epithelial cells prepared by a variety of techniques was examined. By MSP analysis, cyclin D2 promoter was found to be unmethylated in seven mammary organoid preparations from reduction mammoplasties, and in five finite life span HMECs cultured from non-malignant breasts. The only exception to this finding was in immortalized HMECs HBL100 and MCF10A, which contained hypermethylated cyclin D2. As expected, these HMECs were the only two that did not express cyclin D2 mRNA.

To rule out the contribution of inflammatory blood cells present in breast cancer specimens as the source of methylated cyclin D2, ten samples of peripheral blood cells (PBLs) from non-cancer patients were tested. All ten PBLs contained unmethylated cyclin D2 alleles.

Expression of cyclin D2 protein was undetectable in 10 of the 13 primary breast cancers tested. However, methylation of the cyclin D2 promoter was noted only in six of these ten primary tumors. This finding suggests that while methylation may cause silencing of cyclin D2 expression in many breast cancers, alternative pathways account for the loss of the protein in a proportion of these tumors.

Cyclin D2 Promoter Hypermethylation in Preneoplasla

Ductal carcinoma in situ (DCIS) is a preneoplastic lesion with a potential for progression to invasive cancer. To determine if hypermethylation of the cyclin D2 promoter occurs early in the evolution of breast cancer, MSP analysis was performed on DNA from carefully microdissected samples of DCIS. Hypermethylation was noted in 44% of DCIS samples. In the cases where adjacent invasive cancer was present as well, the methylation status of both lesions was concordant. This finding suggests that alteration of cyclin D2 expression may be an early event, and may precede transformation to the fully malignant stage of invasive carcinoma.

Re-expression of Cyclin D2 mRNA in Breast Cancer Cell Lines

Breast cancer cell lines MDAMB231 and MCF7 do not express cyclin D2 mRNA or protein. If silencing of expression is mediated by promoter methylation and/or altered chromatin conformation, then demethylation of the gene by exposure to 5-aza 2′-deoxycytidine (5aza-dC), or treatment with the histone deacetylase inhibitor, trichostatin A (TSA), should result in removal of the repressive mechanism and re-expression of the gene. Indeed, when MDAMB231 and MCF7 cells were exposed to 5-aza-dC in culture, the cyclin D2 promoter was partially demethylated (as analyzed by MSP), and cyclin D2 mRNA expression was restored (as analyzed by RT-PCR). Further, exposure to TSA also led to re-expression of the cyclin D2 mRNA. These results suggest that methylation at the promoter region plays a functional role in suppressing the expression of cyclin D2 in breast cancer.

Using RT-PCR, cyclin D2 expression was detected in four normal luminal epithelial cultures of the 184 series, in four of four purified luminal epithelial cell extracts, and in four of four myoepithelial cell extracts Using MSP, promoter hypermethylation was detected in 49/106 (46%) of the tumors. Hypermethylation of the gene correlated with lack of cyclin D2 mRNA and/or protein expression. Thus, in about 50% of breast cancers, cyclin D2 silencing may be attributed to tumor-specific methylation.

Hypermethylation and Loss of Expression of 14-3-3 Sigma

The extent of methylation of the cyclin 14-3-3 sigma-associated CpG islands in normal mammary epithelium, in breast cancer cell lines, and in primary mammary tumors, and expression of the 14.3,3 sigma mRNA and protein in the same cells and tissues was examined.

Cell Lines and Tissues

The breast cancer cell lines Hs578t, MDA-MB-231, MDA-MB-435 and MCF-7 and the human mammary epithelial cell lines, MCF-10A and HBL-100 were obtained and maintained according to instructions (ATCC). The two matched tumor cell lines, 21PT and 21MT were propagated as described (Band, et al. (1990) Cancer Res. 50:7351-7357). Cultured normal human breast epithelial cell (HMEC) strains, 161, 184, 172, and 48, and the conditionally and fully immortal cell lines, 184A1 (passage 15 and 99), and 185B5 were grown as described on the world-wide web at address lbl.gov/LBL-Programs/mrgs/review. Three additional short term cultures of HMECs,(#04372 and #16637) were grown according to specifications (Clonetics). Primary breast tumor tissues were obtained immediately after surgical resection at the Johns Hopkins University or Duke University, and stored frozen at −80° C. Microscopic examination of representative tissue sections from each tumor revealed that these samples contained greater than 50% tumor cells. Microdissection of primary tumor cryosections was performed by using a laser capture microscope (Schutze, et al. (1998) Nat Biotechnol 16:737-42) or by manually scraping the cells with a 20G needle under 40× magnification (Umbricht, et al. (1999) Oncogene 18:3407-14.).

Northern Blot Analysis

Total RNA was isolated from primary tumor tissues using Trizol Reagent (Life Technologies). Five micrograms were resolved on 1.5% agarose/formaldehyde gels, and transferred to a nylon filter using standard methods (Gene Screen, DuPont). A 375 bp σ-specific probe was generated using MCF-10A cDNA as a template and the primers 5′-ACAGGGGAACTATTGAGAGG-3′ (SEQ ID NO:25) and 5′-AAGGGCTCCGTGGAGAGGG-3′ (SEQ ID NO:26). Hybridizations were done in Quikhyb (Stratagene) according to the manufacturer's instructions. Filters were exposed to autoradiographic film for up to 5 days. To test for uniform loading of the samples, blots were stripped and reprobed with a 1.5 kb DNA fragment specific for 18S rRNA (ATCC, Clone #HHCSA65).

Loss of Heterozygosity (LOH) Studies

A TG repeat sequence in the 3′UTR of σ was amplified using: 5′-GAGGAGTGTCCCGCCTTGTGG-3′ (sense; SEQ ID NO:27) and 5′-GTCTCGGTCTTGCACTGGC-3′ (antisense; SEQ ID NO:28) primers, which yields a product of 117 bp. The 25 μl reactions contained 50 ng of template DNA (10), 17 mM NH 4 SO 4 , 67 mM TrisCl (pH 8.8), 6.7 mM MgCl 2 , 1% DMSO, 1.5 mM dNTP, 20 ng of each primer, 2 ng of γ- 32 P-labeled sense primer, and 0.5 μl Taq polymerase. PCR conditions were as follows: 1 cycle of 94° C. for 90 s; 35 cycles of 94° C. for 1 min, 57° C. for 30 s, 72° C. for 30 s; and 1 cycle of 72° C. for 5 min. PCR products were fractionated on a sequencing gel, which was exposed to autoradiographic film overnight (Evron, et al. (1997) Cancer Res, 57:2888-9).

Mutation Analysis

A 1.2 kb PCR product, encompassing the entire σ coding sequence, was generated using two primers, 5′-GTGTGTCCCCAGAGCCATGG-3′ (sense; SEQ ID NO:29) and 5′-GTCTCGGTCTTGCACTGGCG-3′ (antisense; SEQ ID NO:30). The PCR reaction contained 50 ng of DNA, 6.4% DMSO, 1.5 mM dNTPs, 100 ng of each primer and 0.5 μl Taq polymerase in a 50 μl reaction volume α- 33 P cycle sequencing was performed using the Amplicycle sequencing kit (Perkin Elmer). Four different α- 33 P-labeled primers were used to sequence the entire σ coding sequence:

5′-CACCTTCTCCCGGTACTCACG-3′  (antisense; SEQ ID NO:3 1),

5′-GAGCTCTCCTGCGAAGAG-3′  (sense; SEQ ID NO:32),

5′-GAGGAGGCCATCCTCTCTGGC-3′  (sense; SEQ ID NO:33)

and

5′-TCCACAGTGTCAGGTTGTCTCG-3′  (antisense; SEQ ID NO:34).

Transfection of Human Breast Cancer Cell Lines

1.5×10 5 of MCF-7, MDA-MB-231, and Hs578t, or 2.5×10 5 cells of MDA-MB435 breast cancer cells were seeded in six-well plates. The following day, transfections were performed using Trans IT-LT1 (Mirus Corp.) as per manufacturer's instructions. Plasmids used in the transient transfections include: KKH luciferase, containing 4 kb of the σ-promoter linked to the luciferase gene in the pGL3-Basic vector (Promega); pCMV-β-gal (Clontech), which was used to correct for the efficiency of transfection; and pGL3-Basic (Promega), which was used as a negative vector control against which KKH luciferase activities were compared. Two μg of luciferase reporter plasmid or the pGL3-Basic vector control and 0.5 μg of CMV-β-gal reporter plasmid were used for each transfection.

Luciferase and β-galactosidase Assays

Cell lysates were made approximately 48 hr post-transfection as per manufacturer's instructions (Promega, Luciferase Assay System). Luciferase and β-galactosidase activities were quantitated using the luciferase assay system (Promega) and the Aurora GAL-XE® reporter gene assay (ICN Pharmaceuticals, Inc), respectively. Experiments were done in triplicate. Luciferase activity was first normalized for efficiency of transfection by using the ratio of luciferase to β-galactosidase activity. For each transfected cell line, the results were compared with the mean of pGL3 vector control levels and expressed as fold elevated expression above pGL3. The means and standard deviations of the results of all experiments were calculated.

Sodium Bisulfite DNA Sequencing

Genomic DNA was subjected to sodium bisulfite modification as described in Herman et al. ((1996) Proc. Nail. Acad. Sci. USA, 93:9821-9826). Bisulfite-converted DNA was amplified, as described above, using primers that encompass the first exon of the σ gene: 5′-GAGAGAGTTAGTTTGATTTAGAAG-3′ (sense primer with start at nt 8641; SEQ ID NO:35) and 5′-CTT ACTAATATCCATAACCTCC-3′ (antisense primer with start at nt 9114; SEQ ID NO:36) which generated a 474 bp PCR product. Conditions for PCR were as follows: 1 cycle at 95° C. for 5 min; 35 cycles at 95° C. for 45 s, 55° C. for 45 s and 72° C. for 60 s; and 1 cycle at 72° C. for 4 min. The product was purified using a Qiagen PCR purification kit (Qiagen Corp) and sequenced using the sense primer with an ABI automated fluorescent sequencer according to the manufacturer's instructions.

Methylation-specific PCR (MSP) One μg genomic DNA was treated with sodium bisulfite as described in (Herman, et al., (1996) Proc. Natl. Acad. Sci. USA 93, 9821-9826), and was analyzed by MSP using a primer set that covered CG dinucleotide numbers 3, 4, 8 and 9. Primers specific for methylated DNA: 5′-TGGTAGTTTTATGAAAGGCGTC-3′ (sense; SEQ ID NO:37) and 5′-CCTCTAACCGCCCACCACG-3′ (antisense; SEQ ID NO:38), and primers specific for unmethylated DNA: 5′-ATGGTAGTTTTTATGAAAGGTGTT-3′ (sense; SEQ ID NO:39) and 5′-CCCTCTAACCACCCACCACA-3′ (antisense; SEQ ID NO:40) yielded a 105-107 bp PCR product. The PCR conditions were as follows: 1 cycle of 95° C. for 5 min; 31 cycles of 95° C. for 45 s, 56° C. for 30 s and 72° C. for 30 s; and 1 cycle of 72° C. for 4 min.

Treatment of Cells with 5′-aza-2′-deoxycytidine (5-aza-dC)

Cells were seeded at a density of 2×10 6 cells per 100-mm plate. 24 h later cells were treated with 0.75 μM 5-aza-dC (Sigma) (Ferguson, et al. (1995) Cancer Res 55:2279-2283). Total cellular RNA and genomic DNA were isolated from the cells at 0 and 3 days after addition of 5-aza-dC as described herein.

RNA was treated with RNase-free DNAse (Boehringer-Mannheim) (1 μg/μl) for 2 h at 37° C., followed by heat inactivation at 65° C. for 10 min. RT reactions contained 1 μg DNAse treated RNA, 0.25 μg/μl pdN6 random primers (Pharmacia), 1×first strand buffer (GibcoBRL), 0.5 mM DNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and were incubated for 1 h at 37° C. PCR was performed using the a-specific primers 5′-GTGTGTCCCCAGAGCCATGG-3′ (SEQ ID NO:41) and 5′-ACCTTCTCCCGGTACTCACG-3′ (SEQ ID NO:42) using buffer conditions described herein. The PCR conditions were: 1 cycle of 95° C. for 5 min; 30 cycles of 60° C. for 45 s, 72° C. for 45 s and 95° C. for 45 s. PCR samples were resolved by electrophoresis in a 2% agarose gel.

Assay for G1 and G2 Checkpoint and Chromosomal Aberrations

The G1 cell cycle checkpoint and chromosomal aberrations in mitosis were assessed as described previously (Pandita, et al. (1996) Oncogene 13:1423-1430). Specifically, cells in plateau phase were irradiated with 3 Gy, sub-cultured after 24h, and metaphases were collected. G1 type aberrations were examined at metaphase. All categories of asymmetric chromosome aberrations were scored: dicentrics, centric rings, interstitial deletions/acentric rings, and terminal deletions.

The efficiency of G2 checkpoint control was evaluated by measuring the proportion of cells in metaphase after irradiation. Chromosomal aberrations at mitosis were assessed by counting chromatid breaks and gaps per metaphase as described elsewhere (Morgan, et al. (1997) Mol Cell Biol 17:2020-2029). Specifically, cells in exponential growth phase were irradiated with 1 Gy. Metaphases were harvested 45 and 90 minutes following irradiation and examined for chromatid type breaks and gaps. Fifty metaphases each were scored for G1 and G2 types of chromosomal aberrations.

Introduction of σ into the σ-negative Breast Cancer Cell Line MDA-MB435 by Adenoviral Infection

Cells were seeded and grown to 50% confluence. Adenovirus encoding either σ or β-galactosidase (Hermeking, et al. (1997) Mol. Cell 1:3-11) was added to the culture at a multiplicity of infection of 5000:1 and infection was allowed to take place overnight. The cells were harvested, fixed and stained with Hoechst dye and subjected to FACS analysis.

σ Expression in Normal, Immortalized and Tumorigenic Breast Epithelial Cells

By SAGE analysis, σ was found to be expressed at an average of 7-fold lower levels in three human breast cancer cell lines, 21PT, 21MT and MDA-MB468 than in two populations of normal human mammary epithelial cells (HMEC). Northern blot analysis was performed to confirm this finding in other breast cancer cell lines and in primary breast tumors. No expression of σ was detected in 45 of 48 (94%) primary tumors. In contrast, σ was expressed at easily detectable levels in all 6 cultured HMEC populations and 5 immortalized but nontumorigenic cell lines. These results indicate that loss of σ gene expression is a frequent event in human breast cancer.

Genetic Alterations within the σ Gene

Possible causes for loss of σ gene expression in breast tumors include deletion of the chromosomal region containing the gene or intragenic mutations that lead to decreased mRNA stability. σ localizes to chromosome 1p35, an arm that has been extensively studied for LOH in breast cancer (Hermeking, et al. (1997) Mol. Cell 1, 3-Bieche, et al. (1995) Genes Chrom. Cancer 14:227-251). LOH has been observed for the 1p32-36 region at a frequency of 15-25%. However, it is not known whether the region lost in these tumors includes σ (Genuardi, et al. (1989) Am. J. Hum. Genet. 45:73-82; Trent, et al. (1993) Genes Chrom Cancer 7:194-203; Nagai, et al. (1995) Cancer Res. 55:1752-1757: Tsukamoto, et al. (1998) Cancer 82:317-322). Therefore, the loss of σ by utilizing a TG repeat sequence within the 3′ UTR of the σ gene itself was examined. Using primers that span the TG repeats, the locus in 45 sets of normal and tumor DNA pairs was studied. Twenty of 45 (44%) of the patients were found to be heterozygous with respect to the length of the PCR-product. Only one of the 20 tumor specimens exhibited LOH (Table 2). Eleven of these 20 samples were tested by Northern blot analysis, and no σ transcripts were detectable. These results prompted an examination whether there were smaller genetic changes within the coding region of σ. The entire 1190 bp coding region from σ-nonexpresssing (σ-negative) breast cancer cell lines, MDA-MB435 and Hs578t and 7 primary tumor tissues was amplified with PCR and sequenced. No mutations were found. In addition, 25 primary tumor DNA samples were analyzed by single stranded conformation polymorphism, and no abnormalities were detected. These results suggest that genetic alterations within a are not a primary mechanism for loss of gene expression.

Epigenetic Alterations of the σ Gene

Next tested was whether the lack of σ mRNA was due to deficiencies in factors required for σ transcription. The two breast cancer cell lines, MDA-MB435 and Hs578t, served as model systems for σ-negative primary tumors that harbored wild type σ alleles, while the two breast cancer cell lines, MCF-7 and MDA-MB-231, served as σ-positive controls since they both express detectable levels of σ. The plasmid KKH-luciferase contains 4 kb of sequence upstream of the transcriptional start site of σ linked to the luciferase reporter gene; this upstream region contains the sequences necessary for p53 and γ-irradiation-inducible transcription of σ (5). Following transient transfection of the four cell lines with the reporter plasmid, high levels of expression was observed (70- to 300-fold above the promoterless parental vector) in both σ-negative and σ-positive breast cancer cell lines. These results indicate that the σ-negative breast cancer cells, like the σ-positive cells, are able to support transcription from the σ promoter equally well, and contained factors required for transcription.

σ has a CpG rich region (CpG island) within its first and only exon that begins near the transcription initiation site and ends approximately 800 bp downstream. To explore a role of hypermethylation in silencing a gene expression, the nucleotide sequence of this region was determined after treating the DNA with sodium bisulfite (Frommer, et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). PCR primers were designed to amplify a region spanning 27 CpG dinucleotides within the CpG island. No significant methylation was observed using DNAs from four σ-positive cell lines including 2 HMECs (184, MCF-10A) and two tumorigenic breast cancer cell lines (MCF-7 and MDA-MB-231). In contrast, DNAs from two σ-negative breast cancer cell lines, HS578t and MDA-MB435, were fully methylated at all of the CpG sites. Since there was a strong correlation between σ-methylation status and mRNA expression in all the cell lines examined, 10 σ-negative primary breast tumors were also examined. All of the tumor DNAs exhibited partial or complete methylation of the 27 CpG dinucleotides.

Next, an MSP assay was utilized to detect methylation of the CpG island, using primers spanning the region between CpG dinucleotides 3 and 9 within the σ gene. Primers were designed that take advantage of the nucleotide sequence differences between methylated and unmethylated DNA as a result of bisulfite modification. By this method, 5/5 σ-positive HMEC strains were completely unmethylated. In addition, DNAs from the σ-positive immortalized breast epithelial cells (MCF-10A, HBL-100) and breast cancer cell lines (MCF-7 and MDA-MB-231), were also unmethylated at the sites examined. In contrast, DNAs from the σ-negative breast cancer cell lines, Hs578t and MDA-MB435, were fully methylated. Similarly, 43 of 50 samples from primary breast tumors were partially or completely methylated. Of these 43 tumors, 26 were examined by Northern blot analysis, and all 26 lacked detectable σ gene expression. Three of the seven unmethylated breast tumor samples also lacked a transcripts; the expression pattern for the remainder was not tested. These results demonstrate that aberrant methylation of σ is a frequent event in breast cancer, but that other mechanisms are responsible for silencing the gene in a small fraction of breast tumors.

Previous reports indicate that σ gene expression is restricted to differentiated epithelial cells. In order to clearly ascertain the cellular origin of methylated DNA, normal and tumor tissues were microdissected and analyzed for σ methylation by MSP. All six DNA samples of microdissected mammary epithelial cells obtained from reduction mammoplasty specimens were unmethylated. In contrast, all 32 samples of DNA from microdissected breast carcinomas were methylated within the σ CpG island. These results indicate that hypermethylation of the σ gene is associated with loss of gene expression in the majority of primary breast tumors. The data from gene expression, genetic and epigenetic studies are summarized in Table2.

In order to determine the effect of methylation on a gene expression, two fully methylated, σ-negative cell lines, Hs578t and MDA-MB-435, were treated with the DNA methyltransferase inhibitor, 5-aza-dC. Treatment of cells with 0.75 μM 5-aza-dC for 3 days led to demethylation of the CpG rich region encompassed by the MSP primers. Moreover, 5-aza-dC treatment resulted in reactivation of gene expression, as demonstrated by RT-PCR. These results demonstrate that methylation is at least partially responsible for loss of σ transcription in breast cancer cells.

Functional Consequences of Loss of σ in Breast Cancer Cells

The function of human σ has been analyzed in human colon carcinoma cells, These studies demonstrated that following ionizing irradiation, a sequesters cdc2-cyclin B1 complexes in the cytoplasm, thus arresting the cells in G2. These actions prevent the cell from initiating mitosis before repair of its damaged DNA. Colon carcinoma cells lacking a can still initiate, but do not maintain, G2 arrest, leading to mitotic catastrophe and cell death.

In an attempt to determine the effects of loss of σ gene expression on cell cycle regulation in breast cancer cells, the effects of γ-irradiation on the σ-negative breast cancer cell lines, MDA-MB-435, 21NT, and 21MT, and the σ-positive breast cancer cell line, MCF-7 were tested. First, G1 type chromosomal aberrations were examined 24 h after cells were exposed to 3 Gy of γ-irradiation. All categories of G1-type chromosomal aberrations were scored at metaphase; their frequency was identical in the two cell types. These results indicate that the examined cell lines have similar G1 cell cycle checkpoint control responses to ionizing radiation.

Next, the G2 checkpoint function in the four cell lines was evaluated. Cells in exponential growth phase were γ-irradiated with 1 Gy and metaphases were examined for chromatid type breaks and gaps. Defective G2 arrest will increase these values. The results show a striking difference in the ability of σ-negative cells and σ-positive cells to repair their damaged DNA. Forty-five minutes post irradiation, σ-negative cells exhibited up to twice as many G2 type chromosomal aberrations as MCF-7 cells. This number increases to three-fold by 90 minutes. Moreover, while repair of DNA damage was evident in the MCF-7 cells, as evidenced by a decrease in the number of G2 type aberrations between 45 and 90 minutes, no decrease was seen in σ-negative cells.

Finally, in order to further demonstrate the role of σ in G2 checkpoint function in breast cells, a cloned copy of the gene was overexpressed in the σ-negative cell line MDA-MB-435 as well as in normal breast epithelial cells using the adenovirus expression system used to express σ in colon cancer cells (5). Overexpression of σ in these breast epithelial cells led to a rapid and permanent G2 arrest, whereas the control virus-infected cells showed no effect. These results indicate that although the σ-negative cell lines have a functional G1 cell cycle checkpoint, they accumulate more genetic damage following irradiation, which is consistent with its failure to arrest in G2 in response to DNA damage.

In summary, these results show that in striking contrast to normal breast tissue, greater than 90% of breast cancers lack detectable expression of σ. Hypermethylation of the σ gene occurs in a CpG-rich region that extends from the transcriptional initiation site to the middle of the coding region. Bisulfite genomic sequencing of this 500 bp region showed that is consistently and densely methylated in σ-negative cell lines and primary breast tumors. Several studies have clearly documented that gene activity correlates inversely with the density of gene-specific CpG island methylation, but is less dependent on the position and distance of the methylated DNA sequences from the transcriptional initiation site. With respect to σ, dense methylation just downstream of its transcriptional start site is strongly associated with gene silencing. Furthermore, in σ-negative cell lines, 5-aza-dC-induced demethylation of the CpG island leads to reactivation of gene expression, indicating that hypermethylation plays a causal role in σ gene inactivity.

Hypermethylation and Loss of Expression of RAR β2

The extent of methylation of the RAR β2-associated CpG islands in normal mammary epithelium, in breast cancer cell lines, and in primary mammary tumors, and expression of the RAR β2 mRNA and protein in the same cells and tissues was examined.

Cell Cultures

Human epithelial mammary cells (HEMC) from reduction mammoplasty including three mortal strains, 184, 48R and 172R, and two immortal strains, 184A1 and 184B5, were obtained and cultured according to the protocols designed by Dr. Martha Stampfer (see the HMEC Homepage, http://www.lbl.gov/*mrgs/index.htlm) using Clonetics (Walkersville, Md., USA) reagents. Human breast cancer cell lines were maintained in Dulbecco's modified Eagle's medium (GIBCO) (Hs578t,MCF-7, MDA-MB-231 and T47D) or IMEM medium (Biofluids) (MDA-MB-435, MDA-MB-468, ZR751) with 5% fetal calf serum (FCS). For drug treatments, exponentially growing cells were seeded in 10 cm 2 plates at a density of 36105 cells/plate or in 6-well plates at 16105 cells/well. Cells were allowed to attach overnight before the addition of the appropriate concentration of 5-Aza-2′ deoxycytidine (5-Aza-CdR) (Sigma), Trichostatin A (TSA) (Sigma) or RA(Sigma). When reduction of retinoids was required, cells were treated in either medium with 0.5% FCS or charcoal-dextran stripped FCS (Hyclone). At the indicated time points, both attached and detached cells were harvested, counted with Trypan Blue (Life Technologies) and processed for DNA or RNA extraction. 5-Aza-CdR was dissolved in 0.45% NaCl containing 10 mM sodium phosphate (pH 6.8). Trichostatin A and all-trans-retinoic acid (RA) (Sigma) were reconstituted in absolute ethanol (solvent). The growth inhibition (%) was calculated as: (1-NT/NC)6100, where NT is the number of treated cells and NC is the number of control cells.

Tissue Samples

Normal and tumor tissues were collected from existing tumor banks (Instituto per lo Studio e la Cura dei Tumori, Milan; the Cancer Center, Rotterdam, the Johns Hopkins Breast Cancer Program, Baltimore, Md., USA). All tumor samples were obtained from excess clinical specimens and institutional guidelines for the acquisition and maintenance of such specimens were followed. DNA and RNA extraction: Extraction of DNA and RNA from breast cancer cell lines was performed by using DNAzol and Trizol respectively (LifeTechnologies) according to the manufacturer's instructions. Genomic DNA was further treated with 500 mg/ml proteinase K at 55° C., extracted with phenol-chloroform-isoamylic alcohol (24:24:1) (CIA) and ethanol precipitated. Extraction of DNA from paraffinated breast cancer and lymph node tissues was essentially performed as previously described (Formantici et al., 1999). One to three consecutive sections estimated to contain at least 90% tumor cells were incubated at 58° C. overnight in 200 ml of extraction buffer (50 mM KCl, 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 0.1 mg/ml gelatin,0.45% NP-40, 0.45% Tween 20, and the solution was heated at 95° C. for 15 min to inactivate the proteinase K and then centrifuged at 6000 r.p.m. The DNA in the supernatant was used for analysis.

Southern Blotting

Genomic DNA (7 mg) was digested overnight with 15 U/mg of XbaI, HpaII and MspI enzymes, electrophoresis on a 0.8% agarose gel and transferred to Hybond-N filter. A 227 bp probe was amplified using the sense 5′-AGA GTT TGA TGG AGTTGG GTG GAG-3′ (SEQ ID NO:43) and antisense 5--CAT TCG GTT TGGGTC AAT CCA CTG-3′ (SEQ ID NO:44) primers, gel purified and labeled with 32 P-dCTP using the Megaprime DNA labeling system (Amershan). After hybridization the filters were washed and exposed to X-ray film at −80° C. for autoradiography.

Methylation Specific PCR

(MSP) Bisulfite modification of genomic DNA was essentially performed as described by Herman et al. (1996) and described herein. Modified DNA was used immediately or stored in aliquots at −20° C. The PCR mixture contained 1×PCR buffer (16.6 mM ammonium sulfate, 67 mM Tris (pH 8.7), 1.5 mM MgCl 2 ), dNTPs (each at 1.25 mM), primers (300 ng each per reaction),and bisulfite-modified DNA (50 ng) or unmodified DNA(50 ng). Reactions were hot started at 95° C. before the addition of 2.5 U of Taq polymerase (Qiagen). Amplification was carried out in a Thermal Cycler 480 Perkin Elmer for 30 cycles (1 min at 94° C., 1 min at the annealing temperature (at) selected for each primer pair, 1 min at 72° C.), followed by 4 min at 72° C. Twelve μl of the PCR reaction were electrophoresed onto 1.5% agarose gels, stained with ethidium bromide and visualized under UV. Two primer pairs, W3 sense 5′-CAGCCCGGGTAGGGTTCACC-3′ (SEQ ID NO:45), W3 antisense 5′-CCGGATCCTACCCCGACGG-3′ (SEQ ID NO:46), and W4sense 5′-CCGAGAACGCGAGCGATCC-3′ (SEQ ID NO:47) and W4 anti-sense 5′-GGCCAATCCAGCCGGGGCG-3′ (SEQ ID NO:48), were designed on the human RAR β2 sequence (Shen et al., 1991) and used to control the Na bisulfite modification. The primer pairs selected to detect the unmethylated DNA were as follows: U1sense 5′-GTG GGT GTA GGT GGA ATA TT-3′ (SEQ ID NO:49) and U1antisense 5′-AAC AAA CAC ACA AAC CAA CA-3′ (SEQ ID NO:50) (at 55° C.); U2 sense 5′-TGT GAG TTA GGA GTA GTG TTTT-3′ (SEQ ID NO:51) and U2 antisense 5′-TTC AAT AAA CCC TAC CCA-3′ (SEQ ID NO:52) (at 49° C.); U3 sense 5′-TTA GTA GTT TGG GTA GGGTTT ATT-3′ (SEQ ID NO:53) and U3 antisense 5′-CCA AAT CCT ACC CCAACA-3′ (SEQ ID NO: 54) (at 55° C.); U4 sense 5′-GAT GTT GAG AAT GTGAGT GAT TT-3′ (SEQ ID NO:55) and U4 antisense 5′-AAC CAA TCC AACCAA AAC A-3′ (SEQ ID NO:56) (at 55° C.); The sequences of the primers to detect the methylated DNA were: M1 sense 5′-AGC GGGCGT AGG CGG AAT ATC-3′ (SEQ ID NO:57) and M1 antisense 5′-CAACGA ACG CAC AAA CCG ACG-3′ (SEQ ID NO:58) (at 63° C.); M2 sense 5′-CGT GAG TTA GGA GTA GCG TTT C-3′ (SEQ ID NO:59) and M2 antisense 5′-CTT TCG ATA AAC CCT ACC CG-3′ (SEQ ID NO:60) (at 57° C.); M3 sense 5′-GGT TAG TAG TTC GGG TAG GGTTTA TC-3′ (SEQ ID NO:61) and M3 antisense 5′-CCG AAT CCT ACC CCGACG-3′ (SEQ ID NO:62) (at 64° C.); M4 sense 5′-GTC GAG AAC GCG AGCGAT TC-3′ (SEQ ID NO:63) and M4 antisense 5′-CGA CCA ATC CAA CCGAAA CG-3′ (SEQ ID NO:64) (at 64° C.).

M and U primers were designed in the same regions, with one or two nucleotide differences to meet annealing requirements. Fragment M3 (position 773±1007) contains the βRARE (792±808) and the transcription start site (position 844);fragment M4 (position 949±1096) contains an Sp1 element (position 1074±1081).

The exon 5 (sense primer 5′-GAC TGT ATG GAT GTTCTG TCA G-3′; SEQ ID NO:65) and exon 6 (antisense primer 5′-ATT TGTCCT GGC AGA CGA AGC A-3′; SEQ ID NO:66) were designed on the basis of published RAR β2 transcript (de The' et al., 1990;van der Leede et al., 1992) and used to amplify 50 ng of DNase treated total RNA using the Superscript One-StepRT±PCR System (Life Technologies). RT±PCR with actin primers (sense primer 5′-ACC ATG GAT GAT GAT ATCG-3′; SEQ ID NO:67 and antisense primer 5′-ACA TGG CTG GGG TGTTGA AG-3′; SEQ ID NO:68) was used as an internal RNA control.

The RAR β2 Promoter Is Methylated in Breast Cancer Cell Lines Independently of Their ER Status and RA-inducibility

RAR transcription was first tested in a panel of breast cancer cell lines grown in the absence of exogenous RA, by reverse transcriptase-PCR (RT±PCR), using primers encompassing exons 5 and 6 (de The' et al.,1990; van der Leede et al., 1992; Toulouse et al., 1997). Under these conditions, only one cell line, Hs578t,produced a detectable 256 bp RT±PCR product. Thus, previous reports were confirmed that RAR β gene expression is down regulated/lost in breast cancer cell lines. Growing cells in the presence of RA can assess the distinction between down regulation and loss. As previously reported (Swisshelm et al., 1994; Liu et al., 1997; Shang et al., 1999), we observed induction of RAR β expression and growth inhibition inT47D, MDA-MB-435, MCF7 and ZR75-1 cell lines treated for 48 h with 1 μM RA, but not in the MDA-MB-231 and MDA-MB-468 cell lines.

To see whether the RAR β2 methylation status correlated with the ER status, the methylation status was examined at RAR β2 in a panel of ER-positive (MCF7,T47D, ZR75-1) and ER-negative (Hs578t, MDA-MB-231, MDA-MB-435, MDA-MB-468) cell lines.

By Southern blotting, the CpG island of the RAR β2 promoter within a 7.5 kb XbaI DNA fragment encompassing the TATA box, the βRARE, the transcriptional start site (TS) and the 5′ untranslated region of exon 5 was examined. In this region nine HpaII sites can be identified (Shen et al., 1991; Baust et al.,1996). The DNA methylation status was analyzed by using the methylation-sensitive enzyme, HpaII. MspI, the isoschizomer of HpaII, insensitive to methylation, was used as a positive control. The PCR probe spans the βRARE and the TATA box regions. The same 7.5 kb region was previously analyzed in a colon carcinoma cell line, and the size of all the possible fragments relative to the most 3′ HpaII site a were reported (Cote' and Momparler, 1997). Genomic DNA from the ER-positive, RA-inducible cell line T47D is digested to completion, indicating that it is not methylated at any of the HpaII sites. In contrast, DNA from the ER-positive, RA-inducible ZR75-1 cell line and DNA from the ER-negative, RA-resistant MDA-MB-231 cell line showed to be differentially methylated at the methylation-sensitive sites. Using methylation-specific PCR (MSP), we further analyzed a 616 bp long RAR β2 region from nucleotide 481 to nucleotide 1096 (Shen et al., 1991) in all the cell lines. MSP entails the modification of genomic DNA by sodium bisulfite that converts all unmethylated, but not methylated, cytosine to uracil (Herman et al., 1996). The genomic DNAs from four breast cancer cell lines ZR751,MCF7, MDA-MB-231, MDA-MB-468 showed partial to complete methylation of the promoter region. The human mammary epithelial cell (HMEC) strain48R, expressing RAR β and three breast cancer cell lines, the RAR β-positive Hs578t and the RA-inducible MDA-MB-435 and T47D, revealed only the (U) unmethylated PCR products.

These results indicate that hypermethylation of the RAR β2 promoter occurs in breast cancer cell lines irrespective of the ER status, and can be detected in both RA-inducible, and RA-resistant breast cancer cells.

RAR β2 Is Unmethylated in Both Mortal and Immortalized HMEC, but Is Methylated in Primary Breast Tumors

The next question examined was whether hypermethylation of RAR β2 promoter in cell lines has correlates in clinical breast cancer. As a normal control, the HMEC mortal strains (48R, 172R), that are the closest representation of normal mammary epithelial cells available were examined. Also analyzed were two immortal mammary epithelial strains (184A1 and 184B5). The DNA of these strains was found to be unmethylated. Consequently, methylation of RAR β2 may be an event in the progression of breast cancer, following immortalization. Genomic DNAs from three paraffinated samples of breast tumors, two ER-positive (T1, T2)and one ER-negative (T3), estimated to contain more than 90% tumor cells, were analyzed with all MSP primer pairs, and shown to be partially methylated. Both microdissected breast stroma, and microdissected normal epithelial cells were found unmethylated at RAR β2, making it very likely that the U products in the tumor samples were amplified either from residual normal epithelial cells, or stromal cells mixed to tumor cells. DNAs from matching histologically tumor free lymph node samples (N1±N3), were similarly analyzed and produced only the unmethylated PCR products. The DNA of additional 21 tumors was performed using two sets of primer pairs (U3/M3 andU4/M4). Fifteen (7 ER-positive and 8 ER-negative) of the 24 tumors presented methylation at the RAR β2promoter. With the same primer sets hypermethylation at RAR β2 was detected in the DNA of ten out of 39 primary breast tumors collected, and analyzed independently, at the Johns Hopkins University. The overall data indicate that hypermethylation at RAR β2 promoter occurs in approximately one third of primary breast tumors, and that the RAR β2 methylation state is independent of the ER status of the tumor.

5-Aza-CdR Induces Partial Demethylation at the RAR β2CpG Island and Reactivation of RAR β Gene Expression

In order to determine whether DNA methylation is affecting, at least in part, RAR β gene expression, all cell lines showing methylation at the RAR β2 promoter were treated with the DNA methyl transferase inhibitor, 5-Aza-CdR. Treatment of cells with either0.4 or 0.8 mM 5-Aza-CdR for 3 days, led to partial demethylation of the CpG rich RAR β2 region. This was evident both by Southern analysis in the MDA-MB-231cell line, and by MSP in all cell lines. Moreover, 5-Aza-CdR treatment resulted in reactivation of gene expression both in RA-inducible MCF7 and ZR75-1, and RA-resistant MDA-MB-231 and MDA-MB-468 cells. Subsequent studies examined whether reactivation of RAR β expression by 5-Aza-CdRA-resistant cells could be enhanced by RA. Using non-quantitative RT±PCR, a difference could not appreciated in the level of RAR β transcription in MDA-MB-231 cells treated with 0.4 mM5-Aza-CdR alone, or in combination, with 1 μM RA. In this experiment, 5-Aza-CdR alone, or in combination with RA, produced 63 and 96% growth inhibition respectively. In the same experiment, treatment with 1 μM RA alone produced a negligible effect on growth inhibition (52%). A synergistic effect of the two drugs on cancer cells was previously reported (Cote' and Momparler, 1997; Bovenzi et al., 1999).

These data indicate that DNA methylation is, at least, one factor influencing the down regulation/loss of RAR β transcription in breast cancer cell lines with a methylated RAR β2 promoter. Cells treated with 5-Aza-CdR alone, or in combination with RA, showed re-expression of RAR b, which may have contributed, along with the toxic5-Aza-CdR, to the observed growth inhibition.

The HDAC Inhibitor TSA Can Reactivate RAR β Expression in RA-resistant Cells; Demethylation of the RAR β2 Promoter Is Not an Absolute Requirement for RAR β Reactivation

The chromatin status at a given locus can be dynamically influenced by the degree of acetylation/deacetylation due to HAT/HDAC activities. Absence of RAR β regulatory factors, like RAR a, as well as DNA-methylation, can contribute to pattern chromatin modifications at RAR β promoter in RA-resistant cell lines. One of these cell lines, MDA-MB-231, lacks RA-inducible RARα activity (Shao et al., 1994) and displays a RAR β2 methylated promoter. A subsequent study was designed to probe indirectly whether the level of HDAC at RAR β2 can influence RAR β expression, by testing the effect of TSA, a HDAC inhibitor on MDA-MB-231cells (Yoshida et al., 1995). Cells were treated for 2 days, in the presence or absence of 100 ng/ml TSA alone, or in combination, with 1 μm RA. By using RT±PCR, it was clear that, unlike cells treated with RA alone, cells treated with a combination of RA and TSA re-expressed RAR β mRNA. Under the same experimental conditions, 100 ng/ml TSA alone, or in combination with 1 mm RA, produced 77and 92% growth inhibition, respectively. Treatment with 1 μm RA alone did not affect significantly growth inhibition (52%). By MSP analysis, it was assessed that RAR β expression was restored in the presence of a methylated RAR β2 promoter. This finding indirectly shows that global alterations of HDAC activity, generated by TSA in MDA-MB-231 cells, involved RAR β2 resulting in RA-induced RAR β expression. Further, demethylation at RAR β2 did not seem to be an absolute requirement for RAR β gene expression inMDA-MB-231 cells. Noteworthy, persistence of methylation at RAR (2 was observed also in MCF7 cells where RAR β transcription could be restored in the presence of RA. Growth inhibition was observed in cells treated with TSA alone, or in combination, with RA. Very likely, RAR β along with TSA, a drug known to induce growth inhibition(Yoshida et al., 1995), contributed to the massive growth inhibitory effect that was observed.

These results show that RAR β2 promoter is methylated in breast cancer. This study presents evidence that, in breast cancer cells, RAR β2 promoter undergoes DNA hypermethylation, an epigenetic change known to induce chromatin modifications and influence gene expression. Methylation of the RAR β2 promoter region was detected, both in breast carcinoma cell lines, and a significant proportion of primary breast tumors. RAR β2 methylation status did not correlate with the ER status of breast cancer cells and was observed both in in situ lesions and invasive tumors. It is not clear when epigenetic changes occur during breast cancer progression. However, methylation of the promoter was not detected in both mortal, and immortal human mammary epithelial cell (HMEC) strains, as well as in normal microdissected breast epithelial cells. These results suggest that aberrant methylation of the RAR β2CpG island may be a later event following immortalization. Treatment of breast cancer cells presenting with a methylated RAR β2, with the demethylating agent 5-Aza-CdR, induced partial DNA demethylation and restored RAR β gene expression. This evidence clearly indicates that DNA methylation is at least a component contributing to RAR β downregulation/loss.

RNA was treated with RNAse-free DNAse (Boehringer-Mannheim) (0.5 1 u/ul) for 30 min. at 37° C., followed by heat inactivation at 65° C. for 10 min. RT reactions contained 2 μg DNAse treated RNA, 0.25 μg/μl pdN6 random primers (Pharmacia), 1×first strand buffer (GibcoBRL), 1 mM of each dNTP (Pharmacia), and 200 U MMLV-RT (GibcoBRL), and were incubated for 1 h at 37° C. followed by heat inactivation at 75° C. for 5 min. PCR was performed using the primers 5′-ACCAGAGTTGGGTGCTGAC-3′ (sense; SEQ ID NO:81) and 5′-ACCTGGCACTGGTCTCCG-3′ (antisense; SEQ ID NO:82) for Nes1. A “housekeeping” ribosomal protein gene 36B4 was co-amplified as an internal control, using primers 5′-GATTGGCTACCCAACTGTTGCA-3′ (sense; SEQ ID NO:83) and 5′-CAGGGGCAGCAGCCACAAAGGC-3′ (antisense; SEQ ID NO:84). The 25 μl reactions contained 1×buffer (1:10 of 10×PCR buffer BRL#, 1.2 mM MgSO4, 0.2 mM of each dNTP) and 100 nM of each primer. The PCR conditions were: 1 cycle of 94° C. for 1 min “hot start” then addition of 1 u of Taq polymerase (RedTaq); 1 cycle of 94° C. for 2 min; 35 cycles of: 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 45 sec and finally 72° C. for 5 min. The PCR samples were resolved by electrophoresis on a 2% agarose gel in 1×TBE buffer.

NES-1 expression was observed in mammary organoids and HMEC's from mammoplasty specimens of normal and benign disease breast. In finite life span HMEC primary breast carcinomas analyzed by RT-PCR, NES-1 expression was observed in seven of eleven samples. MSP analysis for a CpG-rich island at NES-1 third exon in the same samples showed methylated sequences in samples that showed NES-1 expression and unmethylated sequences in samples without NES-1 expression. Methylated NES-1 is absent in normal tissue.

In earlier examples use of methylation-specific polymerase chain reaction (PCR) technology (MSP) for detection of the promoter methylation status of human cyclin D2, retinoic acid receptor beta (RARβ), and Twist genes (called “direct MSP”) is described. These genes are essentially unmethylated in normal tissue, but high levels of methylation were found in carcinoma. The present example illustrates a broad study of ductal and lobular carcinoma employing two additional markers, RASSF1A, and Hin-1 genes, in order to achieve the goal of detection of 100% of breast carcinomas. Results of this study show that 100% of invasive ductal carcinoma patients can be detected using the combination of Cyclin D2, RARβ, Twist, and RASSF1A markers (N=27 patient). In addition 100% of invasive lobular carcinoma patients can be detected using the combination of Cyclin D2, RARβ, Twist, and Hin-1 markers (N=19 patients). In the study of 129 patients, the incidence (%) of patients detected with methylation of each of these genes in breast carcinoma is as indicated in Table 3 below.

Thus, the direct MSP technology provides a mechanism for detection of most human breast cancer by molecular methods.

Potential problems limiting such analyses are mainly the small amount of DNA that is available under certain circumstances (e.g. in ductal lavage, where fluid and cells are obtained from the breast duct) and the need to enhance detection of trace amounts of methylated tumor (e.g. in analyses of blood for circulating tumor DNA, diluted by the presence of a vast excess of unmethylated DNA from blood cells). These problems have now been overcome by development a new technology called multiplex MSP. The procedure for multiplex MSP is basically the following three steps:

1. DNA is isolated and treated with sodium bisulfite, as in direct MSP.

2. PCR reaction #1 is performed using 2 μl DNA (□0.1 μg) in the presence of 5 pairs of primers that will specifically amplify Cyclin D2, RARβ, Twist, RASSF1A, and Hin-1 in the same tube. These primers bind DNA whether or not it is methylated and they bind outside the region that is amplified in PCR reaction #2.

3. PCR reaction #2 is performed using 1 μl diluted PCR-derived DNA from the first PCR reaction. As in direct MSP, one pair of primers is used per tube that will amplify one gene (either Cyclin D2, RARβ, Twist, RASSF1A, or Hin-1) and the primers are methylation status-specific. Thus two tubes are run per test (patient sample) in PCR reaction #2, each for detection of either unmethylated or methylated DNA respectively. In this reaction PCR-derived DNA is diluted between 10 1 and 10 7 fold (See FIG. 11 ).

In more detailed terms, multiplex methylation-specific PCR was accomplished by performing two sequential PCR reactions. The first PCR reaction used 5 pairs of gene-specific external primers to co-amplify Cyclin D2, RARβ, Twist, RASSF1A, and Hin-1. The external primer pairs hybridized to sequences outside the region covered by the second PCR reaction. External primers do not contain CpG sequences, thus DNA amplification was independent of methylation status of the genome. The second PCR reaction used 1 pair of gene-specific internal primers to amplify DNA. Unlike the first PCR reaction, for the second PCR reaction primers were methylation status-specific. All primers recognized only sodium bisulfite treated DNA (data not shown). The primer sequences utilized are shown in Table 4 for each gene.

For the first PCR reaction 2 μl sodium bisulfite-treated DNA was added to a reaction mixture containing 166 mM (NH 4 ) 2 SO 4 , 670 mM Tris, pH 8.8, 67 mM MgCl 2 , 100 mM β-mercaptoethanol, 1% DMSO and 4 μg/ml of each external primer, in a final volume of 25 μl. The reaction was overlaid with 2 drops oil in a 500 μl eppendorf tube. Samples were incubated at 95° C. for 5 min, and then 35 cycles of 95° C. for 30 sec, 56° C. for 30 sec, and 72° C. for 45 sec. The final extension was performed at 72° C. for 5 min. For the second PCR reaction, 1 μl of the first PCR reaction (diluted 1:10 2 -1:10 6 ) was added to the PCR reaction mix, as described above, which in addition contained 4 μg/ml of each of two internal primers (forward and reverse). External primers were not added. Reactions to detect methylated and unmethylated genome were carried out in separate reaction tubes, in 8-well strip tubes covered with 2 drops of oil/well. PCR reaction conditions were identical to the first reaction.

Using this technique, it was determined that multiplex MSP greatly enhances the amount of DNA available for analyses of markers of tumor methylation. The test capacity for direct MSP if ˜1 μg starting DNA is used enables evaluation of 5 genes in duplicate. By comparison, if ˜0.1 μg of starting DNA is used in multiplex MSP, a panel of 5 genes can be evaluated in 25 replicate tests, and there is the potential that 10 panels of 5 genes in replicates of 25 tests could be evaluated from ˜1 μg starting DNA. This would be true if the PCR reaction DNA was conservatively diluted only 10 1 fold, and we have observed that it may be possible to dilute it much higher (i.e. 10 5 -10 6 fold) to further enhance the availability of sample DNA.

Multiplex MSP was found to be highly specific, demonstrating concordance with direct MSP analyses of samples obtained from normal human white blood cells (WBC), breast cancer cell lines, and primary breast tumors. Samples found unmethylated by direct MSP were unmethylated by multiplex MSP as well. Furthermore, higher sensitivity for detection of methylated DNA was observed with multiplex MSP, as traces of methylated DNA were detectable by multiplex MSP that were not detectable by direct MSP in some samples.

In these studies, Cyclin D2, ASSF1A and/or Twist were found to be methylated (at least one marker) in 100% of invasive ductal carcinomas in a sample of 27 cell lines tested. Also gene promotor methylation was found in invasive lobular carcinoma cells as follows: RASSF1A=85% (n=20); HIN-1=79% (n=19); Twist=20% (n=20); RARβ=20% (n=20); and CyclinD2=35% (n=20). Gene promotor methylation was found in invasive ductal carcinoma cells as follows: RASSF1A=66% (n=20); HIN-1=59% (n=19); Twist=47% (n=20); RARβ=30% (n=20); and CyclinD2=54% (n=20). The incidence of various combinations of cyclin D2, RARβ, Twist, TASSF1A and HIN-1 in invasive ductal carcinoma in a study of breast cancer cell lines (n=27) was also determined using Multiplex methylation-specific PCR. The combination of cyclin D2, RARβ and Twist occurred in 89% of the samples; the combination of cyclin D2, RARβ, Twist and RASSF1A occurred in 100% of the samples; and the combination of Cyclin D2, RARβ, Twist, and Hin-1 occurred in 93% of the samples tested. The combination of RASSF1A and HIN-1 detected invasive lobular carcinoma with 95% accuracy. These studies show that RASSF1A and HIN-1 are preferred markers for evaluating a subject having or suspected of having early stage tumorogenesis of breast tissue and that a Multiplex methylation-specific PCR assay utilizing the five markers RASSF1A, Twist and Cyclin D2 will provide an accuracy of 100% detection of invasive ductal carcinoma.

In conclusion, the multiplex MSP technology can greatly enhance the detection of trace amounts of methylated DNA from patient samples, in a manner which is highly specific. Multiplex MSP can also greatly increase the amount of DNA available for analyses of a wider number of markers of tumor methylation than can presently be analyzed by direct PCR. This technology could allow for analyses of up to 50 genes (10 panels of 5 genes) from the same amount of starting material that can maximally be used to analyze 5 genes using direct MSP.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

1

gcggcgcagt tccccaacca 20

2

22

Artificial Sequence

PCR primer (Antisense)

2

atggtttctc accagtgtgc tt 22

3

20

Artificial Sequence

PCR primer (Sense)

3

gcatctgaaa ccagtgagaa 20

4

18

Artificial Sequence

PCR primer (Antisense)

4

tttctctgat gcatgttg 18

5

22

Artificial Sequence

PCR primer (Sense)

5

gattggctac ccaactgttg ca 22

6

22

Artificial Sequence

PCR primer (Antisense)

6

caggggcagc agccacaaag gc 22

7

24

Artificial Sequence

PCR primer (Sense)

7

tttgggttaa gttaggcgtc gtcg 24

8

23

Artificial Sequence

PCR primer (Antisense)

8

acactactcc tcgtacgact ccg 23

9

24

Artificial Sequence

PCR primer (Sense)

9

tttgggttaa gttaggtgtt gttg 24

10

23

Artificial Sequence

PCR sequence (Antisense)

10

acactactcc tcatacaact cca 23

11

21

Artificial Sequence

PCR primer (Sense)

11

cgtcgggtga aggcgggtaa t 21

12

21

Artificial Sequence

PCR primer (Antisense)

12

cgaacccgaa cctacgaaac c 21

13

21

Artificial Sequence

PCR primer (Sense)

13

tgttgggtga aggtgggtaa t 21

14

21

Artificial Sequence

PCR primer (Antisense)

14

caaacccaaa cctacaaaac c 21

15

20

Artificial Sequence

PCR primer(Sense)

15

catggagctg ctgtgccacg 20

16

20

Artificial Sequence

PCR primer (Antisense)

16

ccgacctacc tccagcatcc 20

17

19

Artificial Sequence

PCR rimer (Sense)

17

agccatggaa caccagctc 19

18

19

Artificial Sequence

PCR primer (Antisense)

18

gcacctccag catccaggt 19

19

22

Artificial Sequence

PCR primer (Sense)

19

gattggctac ccaactgttg ca 22

20

22

Artificial Sequence

PCR primer (Antisense)

20

caggggcagc agccacaaag gc 22

21

22

Artificial Sequence

PCR primer (Sense)

21

gttatgttat gtttgttgta tg 22

22

21

Artificial Sequence

PCR primer (Antisense)

22

taaaatccac caacacaatc a 21

23

19

Artificial Sequence

PCR primer (Sense)

23

tacgtgttag ggtcgatcg 19

24

20

Artificial Sequence

PCR primer (Antisense)

24

cgaaatatct acgctaaacg 20

25

22

Artificial Sequence

Sense primer

25

acaggggaac tttattgaga gg 22

26

19

Artificial Sequence

Antisense primer

26

aagggctccg tggagaggg 19

27

21

Artificial Sequence

PCR primer (Sense)

27

gaggagtgtc ccgccttgtg g 21

28

19

Artificial Sequence

PCR primer (Antisense)

28

gtctcggtct tgcactggc 19

29

20

Artificial Sequence

PCR primer (Sense)

29

gtgtgtcccc agagccatgg 20

30

20

Artificial Sequence

PCR primer (Antisense)

30

gtctcggtct tgcactggcg 20

31

21

Artificial Sequence

Alpha -33P-labeled primer (Antisense)

31

caccttctcc cggtactcac g 21

32

18

Artificial Sequence

Alpha -33P-labeled primer (Sense)

32

gagctctcct gcgaagag 18

33

21

Artificial Sequence

Alpha -33P-labeled primer (Sense)

33

gaggaggcca tcctctctgg c 21

34

22

Artificial Sequence

Alpha -33P-labeled primer (Antisense)

34

tccacagtgt caggttgtct cg 22

35

24

Artificial Sequence

PCR sense primer

35

gagagagtta gtttgattta gaag 24

36

22

Artificial Sequence

PCR antisense primer

36

cttactaata tccataacct cc 22

37

23

Artificial Sequence

PCR sense primer

37

tggtagtttt tatgaaaggc gtc 23

38

19

Artificial Sequence

PCR antisense primer

38

cctctaaccg cccaccacg 19

39

24

Artificial Sequence

PCR sense primer

39

atggtagttt ttatgaaagg tgtt 24

40

20

Artificial Sequence

PCR antisense primer

40

ccctctaacc acccaccaca 20

41

20

Artificial Sequence

PCR primer

41

gtgtgtcccc agagccatgg 20

42

20

Artificial Sequence

PCR primer

42

accttctccc ggtactcacg 20

43

24

Artificial Sequence

PCR sense primer

43

agagtttgat ggagttgggt ggag 24

44

24

Artificial Sequence

PCR antisense primer

44

cattcggttt gggtcaatcc actg 24

45

20

Artificial Sequence

PCR W3 sense primer

45

cagcccgggt agggttcacc 20

46

19

Artificial Sequence

PCR W3 antisense primer

46

ccggatccta ccccgacgg 19

47

19

Artificial Sequence

PCR W4 sense primer

47

ccgagaacgc gagcgatcc 19

48

19

Artificial Sequence

PCR W4 antisense primer

48

ggccaatcca gccggggcg 19

49

20

Artificial Sequence

U1 sense primer

49

gtgggtgtag gtggaatatt 20

50

20

Artificial Sequence

U1 antisense primer

50

aacaaacaca caaaccaaca 20

51

22

Artificial Sequence

U2 sense primer

51

tgtgagttag gagtagtgtt tt 22

52

18

Artificial Sequence

U2 antisense primer

52

ttcaataaac cctaccca 18

53

24

Artificial Sequence

U3 sense primer

53

ttagtagttt gggtagggtt tatt 24

54

18

Artificial Sequence

U3 antisense primer

54

ccaaatccta ccccaaca 18

55

23

Artificial Sequence

U4 sense primer

55

gatgttgaga atgtgagtga ttt 23

56

19

Artificial Sequence

U4 antisense primer

56

aaccaatcca accaaaaca 19

57

21

Artificial Sequence

M1 sense primer

57

agcgggcgta ggcggaatat c 21

58

21

Artificial Sequence

M1 antisense primer

58

caacgaacgc acaaaccgac g 21

59

22

Artificial Sequence

M2 sense primer

59

cgtgagttag gagtagcgtt tc 22

60

20

Artificial Sequence

M2 antisense primer

60

ctttcgataa accctacccg 20

61

26

Artificial Sequence

M3 sense primer

61

ggttagtagt tcgggtaggg tttatc 26

62

18

Artificial Sequence

M3 antisense primer

62

ccgaatccta ccccgacg 18

63

20

Artificial Sequence

M4 sense primer

63

gtcgagaacg cgagcgattc 20

64

20

Artificial Sequence

M4 antisense primer

64

cgaccaatcc aaccgaaacg 20

65

22

Artificial Sequence

PCR sense primer

65

gactgtatgg atgttctgtc ag 22

66

22

Artificial Sequence

PCR antisense primer

66

atttgtcctg gcagacgaag ca 22

67

19

Artificial Sequence

PCR sense primer

67

accatggatg atgatatcg 19

68

20

Artificial Sequence

PCR antisense primer

68

acatggctgg ggtgttgaag 20

69

18

Artificial Sequence

PCR sense primer

69

tttagcgggt ggcgttcg 18

70

20

Artificial Sequence

PCR antisense primer

70

atacgacttc gaatcacgta 20

71

18

Artificial Sequence

PCR sense primer

71

ttggttggaa gttgggtg 18

72

22

Artificial Sequence

PCR antisense primer

72

aatacaactt caaatcacat ac 22

73

23

Artificial Sequence

PCR sense primer

73

attttgttat aatgggttgt aat 23

74

21

Artificial Sequence

PCR antisense primer

74

aacatatact taattccctc c 21

75

20

Artificial Sequence

PCR sense primer

75

tcattttgcg gtcgctatcc 20

76

18

Artificial Sequence

PCR antisense primer

76

gccggctggc tgtacctg 18

77

20

Artificial Sequence

PCR sense primer

77

ttgtagaggt ggtgttgttt 20

78

22

Artificial Sequence

PCR antisense primer

78

cacacaataa aacaaaaaac ca 22

79

20

Artificial Sequence

PCR sense primer

79

ttcgaagttt atggcgtttc 20

80

20

Artificial Sequence

PCR antisense primer

80

ttatttccgc aatacgcgac 20

81

19

Artificial Sequence

PCR sense primer

81

accagagttg ggtgctgac 19

82

18

Artificial Sequence

PCR antisense primer

82

acctggcact ggtctccg 18

83

22

Artificial Sequence

PCR sense primer

83

gattggctac ccaactgttg ca 22

84

22

Artificial Sequence

PCR antisense primer

84

caggggcagc agccacaaag gc 22

85

21

Artificial Sequence

Forward primer

85

gggtgttttt gagattgttg g 21

86

20

Artificial Sequence

Complement to reverse primer

86

tgagttgtga tgggttttgg 20

87

20

Artificial Sequence

Reverse primer

87

ccaaaaccca tcacaactca 20

88

18

Artificial Sequence

Forward primer

88

agagtaggcg gcgagcgt 18

89

20

Artificial Sequence

Complement to reverse primer

89

cgggaaaagt acgtgttcgt 20

90

20

Artificial Sequence

Reverse primer

90

acgaacacgt acttttcccg 20

91

1462

Homo sapiens

91

gtgacagaag tagtaggaag tgagctgttc agaggcagga gggtctattc tttgccaaag 60

gggggaccag aattccccat gcgagctgtt tgaggactgg gatgccgaga acgcgagcga 120

tccgagcagg gtttgtctgg gcaccgtcgg ggtaggatcc ggaacgcatt cggaaggctt 180

tttgcaagca tttacttgga aggagaactt gggatctttc tgggaacccc ccgccccggc 240

tggattggcc gagcaagcct ggaaaatgca attgaaacac agagcaccag ctctgaggaa 300

ctcgtcccaa gccccccatc tccacttcct ccccctcgag tgtacaaacc ctgcttcgtc 360

tgccaggaca aatcatcagg gtaccactat ggggtcagcg cctgtgaggg atgtaagggc 420

tttttccgca gaagtattca gaagaatatg atttacactt gtcaccgaga taagaactgt 480

gttattaata aagtcaccag gaatcgatgc caatactgtc gactccagaa gtgctttgaa 540

gtgggaatgt ccaaagaatc tgtcaggaat gacaggaaca agaaaaagaa ggagacttcg 600

aagcaagaat gcacagagag ctatgaaatg acagctgagt tggacgatct cacagagaag 660

atccgaaaag ctcaccagga aactttccct tcactctgcc agctgggtaa atacaccacg 720

aattccagtg ctgaccatcg agtccgactg gacctgggcc tctgggacaa attcagtgaa 780

ctggccacca agtgcattat taagatcgtg gagtttgcta aacgtctgcc tggtttcact 840

ggcttgacca tcgcagacca aattaccctg ctgaaggccg cctgcctgga catcctgatt 900

cttagaattt gcaccaggta taccccagaa caagacacca tgactttctc agacggcctt 960

accctaaatc gaactcagat gcacaatgct ggatttggtc ctctgactga ccttgtgttc 1020

acctttgcca accagctcct gcctttggaa atggatgaca cagaaacagg ccttctcagt 1080

gccatctgct taatctgtgg agaccgccag gaccttgagg aaccgacaaa agtagataag 1140

ctacaagaac cattgctgga agcactaaaa atttatatca gaaaaagacg acccagcaag 1200

cctcacatgt ttccaaagat cttaatgaaa atcacagatc tccgtagcat cagtgctaaa 1260

ggtgcagagc gtgtaattac cttgaaaatg gaaattcctg gatcaatgcc acctctcatt 1320

caagaaatgc tggagaattc tgaaggacat gaacccttga ccccaagttc aagtgggaac 1380

acagcagagc acagtcctag catctcaccc agctcagtgg aaaacagtgg ggtcagtcag 1440

tcaccactcg tgcaataaga ca 1462

92

21

Artificial Sequence

Forward primer

92

ggattgggat gttgagaatg t 21

93

21

Artificial Sequence

Reverse primer

93

caaccaatcc aaccaaaaca a 21

94

1454

Homo sapiens

94

accagcggca gaccacaggc agggcagagg cacgtctggg tcccctccct ccttcctatc 60

ggcgactccc agatcctggc catgagagct ccgcacctcc acctctccgc cgcctctggc 120

gcccgggctc tggcgaagct gctgccgctg ctgatggcgc aactctgggc cgcagaggcg 180

gcgctgctcc cccaaaacga cacgcgcttg gaccccgaag cctatggcgc cccgtgcgcg 240

cgcggctcgc agccctggca ggtctcgctc ttcaacggcc tctcgttcca ctgcgcgggt 300

gtcctggtgg accagagttg ggtgctgacg gccgcgcact gcggaaacaa gccactgtgg 360

gctcgagtag gggatgatca cctgctgctt cttcagggcg agcagctccg ccggacgact 420

cgctctgttg tccatcccaa gtaccaccag ggctcaggcc ccatcctgcc aaggcgaacg 480

gatgagcacg atctcatgtt gctaaagctg gccaggcccg tagtgccggg gccccgcgtc 540

cgggccctgc agcttcccta ccgctgtgct cagcccggag accagtgcca ggttgctggc 600

tggggcacca cggccgcccg gagagtgaag tacaacaagg gcctgacctg ctccagcatc 660

actatcctga gccctaaaga gtgtgaggtc ttctaccctg gcgtggtcac caacaacatg 720

atatgtgctg gactggaccg gggccaggac ccttgccaga gtgactctgg aggccccctg 780

gtctgtgacg agaccctcca aggcatcctc tcgtggggtg tttacccctg tggctctgcc 840

cagcatccag ctgtctacac ccagatctgc aaatacatgt cctggatcaa taaagtcata 900

cgctccaact gatccagatg ctacgctcca gctgatccag atgttatgct cctgctgatc 960

cagatgccca gaggctccat cgtccatcct cttcctcccc agtcggctga actctcccct 1020

tgtctgcact gttcaaacct ctgccgccct ccacacctct aaacatctcc cctctcacct 1080

cattccccca cctatcccca ttctctgcct gtactgaagc tgaaatgcag gaagtggtgg 1140

caaaggttta ttccagagaa gccaggaagc cggtcatcac ccagcctctg agagcagtta 1200

ctggggtcac ccaacctgac ttcctctgcc actccccgct gtgtgacttt gggcaagcca 1260

agtgccctct ctgaacctca gtttcctcat ctgcaaaatg ggaacaatga cgtgcctacc 1320

tcttagacat gttgtgagga gactatgata taacatgtgt atgtaaatct tcatgtgatt 1380

gtcatgtaag gcttaacaca gtgggtggtg agttctgact aaaggttacc tgttgtcgtg 1440

aaaaaaaaaa aaaa 1454

95

181

Homo sapiens

95

ccgcagaggc ggcgctgctc ccccaaaacg acacgcgctt ggaccccgaa gcctatggcg 60

ccccgtgcgc gcgcggctcg cagccctggc aggtctcgct cttcaacggc ctctcgttcc 120

actgcgcggg tgtcctggtg gaccagagtt gggtgctgac ggccgcgcac tgcggaaaca 180

a 181

96

1150

Homo sapiens

96

accaagagag actgggagag ggcggcagag aagagagggg ggaccgagag ccgcgtcccc 60

gcggtcgcgt ggatttagaa aaaggctggc tttaccatga cttatgtgca gcttgcgcat 120

ccaggggtag atctggggtt gggcgggcgg cgccgggctc ggctcgctct gcgcactcgc 180

ctgctcgctg ctggcagggg cgtcctcctc ggctccggac gccgtgccaa ccccctctct 240

gctgctgatg tgggtgctgc cggcgtcggc cgaggcgccg ctggagttgc ttagggagtt 300

tttcccgccg tggtggctgt cgctgccggg cgagggggcc acggcggagc agggcagcgg 360

atcgggctga ggagagtgcg tggacgtggc cggctggctg tacctgggct cggcgggcgc 420

cgcgctggcg ctggcagcgt agctgcgggc gcgctctccg gagccaaagt ggccggagcc 480

cgagcggccg acgctgagat ccatgccatt gtagccgtag ccgtacctgc cggagtgcat 540

gctcgccgag tccctgaatt gctcgctcac ggaactatga tctccataat tatgcaactg 600

gtagtccggg ccatttggat agcgaccgca aaatgagttt acaaaataag agctcatttg 660

ttttttgata tgtgtgcttg atttgtggct cgcggtcgtt tgtgcgtcta tagcaccctt 720

gcacaattta tgatgaatta tggaaatgac tgggacatgt acttggttcc ctcctacgta 780

ggcacccaaa tatggggtac gacttcgaat cacgtgcttt tgttgtccag tcgtaaatcc 840

tgcctgatga cctctagagg taaactcgtg cactaatagg ggagttgggt ggaggcgagg 900

ggggtggcgc gcgcgccccg ggcgcgtgcc cgccgccagt tgccgccgtt cagccggact 960

cgagcgccac ccgctggagg cagggctcat cgcccagctt ccgaccgggg gctgcaaggg 1020

ccggggtcga attgaggtta cagcccatta tggcaaaatt attgcatttc cctcgcagtt 1080

ccattaggat gtaccaattg ttaggccgtc agctgccgat cgcgcgcccg gcgaggatgc 1140

agaggattgg 1150

97

790

Homo sapiens

97

ccaatcctct gcatcctcgc cgggcgcgcg atcggcagct gacggcctaa caattggtac 60

atcctaatgg aactgcgagg gaaatgcaat aattttgcca taatgggctg taacctcaat 120

tcgaccccgg cccttgcagc ccccggtcgg aagctgggcg atgagccctg cctccagcgg 180

gtggcgctcg agtccggctg aacggcggca actggcggcg ggcacgcgcc cggggcgcgc 240

gcgccacccc cctcgcctcc acccaactcc cctattagtg cacgagttta cctctagagg 300

tcatcaggca ggatttacga ctggacaaca aaagcacgtg attcgaagtc gtaccccata 360

tttgggtgcc tacgtaggag ggaaccaagt acatgtccca gtcatttcca taattcatca 420

taaattgtgc aagggtgcta tagacgcaca aacgaccgcg agccacaaat caagcacaca 480

tatcaaaaaa caaatgagct cttattttgt aaactcattt tgcggtcgct atccaaatgg 540

cccggactac cagttgcata attatggaga tcatagttcc gtgatcgagc aattcaggga 600

ctcggcgagc atgcactccg gcaggtacgg ctacggctac aatggcatgg atctcagcgt 660

cggccgctcg ggctccggcc actttggtct cggagagcgc gcccgcagct acgctgccag 720

cgccagcgcg gcgcccgccg agcccaggta cagccagccg gccacgtcca cgcactctcc 780

tcagcccgat 790

98

22

Artificial Sequence

Complement to reverse primer

98

gtatgtgatt tgaagttgta tt 22

99

20

Artificial Sequence

Complement to reverse primer

99

tacgtgattc gaagtcgtat 20

100

21

Artificial Sequence

Complement to reverse primer

100

ggagggaatt aagtatatgt t 21

101

19

Artificial Sequence

Complement to reverse primer

101

ccaggtacag ccagccggc 19

102

10034

Homo sapiens

102

ggatcccagc ctgcccctcc acttctctcc caagccaggt cccggcatgg gtgggttatg 60

ctcatgctgg caatacttga aacgggttta ttaatgctgg gtattttgca caattttata 120

gacctctttt ctacatagtc ttttttaaat ggaaggagaa aatgtcagcc acattactgt 180

ctgtgtagtg ccaggtgaag ggttatcaga aggctggttg gttttaataa gtttattcca 240

agagaccttc tggctggaat gagtgagagt gtgtgtgcat gtgtgtgtgt gttcatgtgt 300

gccctgtatg aatgtggctg gctcccagat cccctgggct gccccctgcc ccatcccctt 360

tgagtatcag aagcactctg agccaagggg acagggggca cgtgcactgg tcacgagaaa 420

accctgggct cccactgggg ctcagcccag cctcctatct ttccttcttc tatggacttc 480

agacagccag tgtctgggga ctctgccact ctacccccag ccctacccac cagcccccag 540

gtgaggcttc cagctgggac ctgcccagac aggctgagcc tgggcgtggt gggtggggtg 600

atggctctgg ggagcggctg ccatcctaca agccacaccc cctcctctga gctctgaata 660

tgggacccag tgccaggagc tggaagacaa ggtgtttctg ccaaacggga cctccatcca 720

gagaaaagga agaaggtgca gggtgggcca agaggcaagt gaaggttggc ctgagtctgg 780

gccggaaact cagaggatgt ttctcctctg ctgggagctg tagtttctta tcaaaataga 840

tattgttcca ccatccccct ccttggccct tcaagtgggc tgaagccttg gaaagtgaca 900

taggaagtcc ccagatcttg cccttctcac tccagaggct agtggtcaca gacagctggg 960

aatggcagcc acagagggtc cctctggaga aacagcttca ccccagcctc agggccctgg 1020

gcatcactgc agtggccctg ggaggtgagg aagaagctgg ctagaggagg gggctcccac 1080

ctacctttta tttaagccag tattctttgt tcctgcttgt aataaaactt cagtttataa 1140

gagttgcttt gctttggttt ggtttttgtt tgcttttcct ttgctgaggc cccaactggg 1200

agccctctgt tctttcagac aaatttggtt ctttcctggg gagactgtga gaaggcaggc 1260

agcccagtga tctggctaca ttttccctca cctggctgga gctctgtccg ctggaggaag 1320

agcagagagg gctgcggctg agcccccatg ggcacgtgaa aagaggccat cctgtcccct 1380

ctttgtcccc tccaccttcc cctgcctcag gggcttggag accccaaatt cttcttccct 1440

actgcctttc cactccgatc cccaatgagt gcccagctaa gaaaatgttt gagacagtag 1500

attccagttt gagagccgga gcttccctgg ctaccacctc caacctgggc accagggccc 1560

agccagacaa ctcataacac tggcccacct ctctggtatc tccctcagga ggacacctgt 1620

caggattttg ccatctcctg cacagcctga ggggagctaa caggcctctt tgcagagggt 1680

tagctggtaa gaccgtttct tccctgtcgg ccagcactgc ccgctcccct ccacacacca 1740

tctcatcctc atcgcatgcc tcgccaaccc catggagccc gtccatctgt ctggtgtgtg 1800

gtgcggtgtg tgtgctggtg gtggtagggt ctccagggac tccccgctaa gcagaaggat 1860

cgggatatag ggcaaggcta aaagcccagc cccattgtgg actgaggaag tacgttcgcg 1920

cagagcagct ctccagctgg aagaggaggt ggagggtgag gctggggaga ggatggcgaa 1980

cctgccctga ggtgcttggg tctgtgctgg tggggtcctg gtatgcaggg gccaccggtc 2040

actaacactc ttatgtcctg gctttctgtc cccgctgagc tttctctcac ccgcccgttt 2100

tctctcctgc ttcattgcct gctgcctaag ccttggccct tctctcgggc agaggcaggt 2160

gctgtggcag cacctctccc caccaccggg cccctgcagg ccgcctccct cctcccaggc 2220

ctgctaaccc tctctcttct ccttctttgc tgtcctgccg gggatctcca gtgtgtgcgg 2280

gggcttaagg acctcctgag gaccgctgct ctctgcctct ccaggaatgg cctgggggga 2340

gccaggcacc cggcacctcc acctgcctaa cctgtggccc atctgccacc atctgtgcct 2400

acagggtctg ccccccagcc tgcccggcct gtgtgctctc taggacccca tagggggcag 2460

gggctggcct ctttgcccca ttcccgctcc atgccggcca gagtgtagaa agccataacg 2520

cacgcagcca tcagcacaat aatgtgactc tacgctgata tgctccctct ctcctccact 2580

gacttcccct tcccggattt gtgaggtgtc aagactagga atctggcctt agagcctgcc 2640

cctccacccc ctcagatcag gcatagccat agtcaagccc agcaggtttc ctcaggagct 2700

gtctggggtg ttgatggtgg atgacgctgc tgaacaagtt tggtgactgt tctaagcaca 2760

actggcttga tactgttccc acggcctgtc cacctcccac ccccaaccct ccaccagagt 2820

aggtaggatg tagggagggt gcgtgccgcc tttgctctag gcactgaggg accaagctag 2880

ccgtgcacag ccccatacac ttcaggggcg taaaggaaag agctgagcca aggaaaatca 2940

gctgagccca gggctggggg ctgcttgtct gctatcctgt accttttttt tttttaacca 3000

aaataaagat tcccctcttc ttgccatacc attggctgtc tggtggcgcc tttactttgg 3060

ggcccaggga tgggacctgc agtgggcgtg tggaacatat ggctccccct cgctcccagc 3120

tttcttccag ctggccagtg ctgctctgga gatttacaag cacaacgaag ccaggaggga 3180

cacaggaaaa gtggctgaca tccttttcac tctgcccctc cagaactctt ggtctcaatt 3240

ccagacacca cccagcctta gctgacctct ggattctgat aggtcccagt gcaggctgag 3300

acagagggtt taactccagt ttgggactgc catacccatg aactgagccc agcccagggt 3360

aacgatctca tggaaacttc tctctcccca gttgctgcac tacatcaaga tacacacatg 3420

tgcatacact gtactatggg ctaaaaaaat acgtaccgct accgttcagc aagggcttgc 3480

cgagtcccgg gcccattttc tcatcttaac ctgtgaggag gatgatgtca gcctttttac 3540

agatgaggga actgagactc aaggaagaaa caggagctgc ccaaggtcac ccagctggca 3600

aagcagcaaa tcccagatcg gaacctgatc tctgccccga gctctgagcc atctgcacta 3660

cccaaggaat gaatacagcg gtgggaggat gagatcttgg agaaacccta aaattagaga 3720

atgtcatagc cagtagaggg cttagagttg atctgggcca gcctccttgt tttactgatg 3780

gagaaattga agcccagagg caggaaggga cctgcccaag gccttataac agagctggga 3840

tgcagtccca cactctgacc tcattccatt ctctctccat aaattctgca ctgtctctag 3900

actggactgg tttagatgtg ggatactcta aacagcagtg ccttcaagag aaaaagaatc 3960

agaactacga atcacttaaa agtaatgtaa gctactctgg gcacactgcc tatggggtcg 4020

ccctgctcca caaggagcca caaaaataat taaaataatt taatatccct tcccaaaggt 4080

aaccagtaaa gtaagctctt ggctaggtaa ctggactctt gttcacaact agccagtggg 4140

aaaaggtgct agagcttcct ctggccacct gtttaatttg atcattccaa gacagaaaca 4200

tttcttagga agttctttct agaatctacc tggtgtccct cccactgcta tcagagccct 4260

gtcctctgtc ctcagtggag gtagagagca aatggttgct gctttcttca tcacaaccct 4320

tcaaagccta ttattaccag ctaagaagga ttggttgact atgggccaga gcccctgagc 4380

ctgctggtag aatggatgct gtacaggagg gtggggaggt agcaggcaga atgaggaaag 4440

cccctttgag ctgcaacccc agctcctgtc ctgctgactc agacagctga ctgtggagct 4500

ccatgccctg ccagggcctg ctgcctcctg cccgtctgag ctcctgaact tgggaaatgg 4560

aggcccagag gcaaagggag gtacctgaga caggaactga gtcaggatca acaggccaga 4620

gcgggcagga ggtatcaggc agcctggctc ccagatgcac ccctgagctc cagcagggga 4680

ggagtaggaa tgaaggggct tccttgccct tgctcatggc tatgcggagg gcgtgaacca 4740

ccaccaggtc ctctggctta agtggcggga agcaaatggt ccctccctgg actcaggctc 4800

caaagttcct gggcctgcct tccaggttcc cagtgtcctg ggatctccag ctttccccag 4860

gacttgggga agccccggct ggatgactag tacaaatgaa ggcccctgag gttccaggac 4920

ctgctgaggt cacaggaata tcctagatca agcttgtcca acccacggcc cacaggctgc 4980

atgtggccca gaatggcttt gaatgcagcc caacacaaat tagtaaactt tcttaaaaca 5040

ttatgagatt tttttgcaaa tttttttttt ttttttagct catcagttat tggtagtgtt 5100

ggtatatttt atgtgtggcc caagacaatt cttccaatgt ggcccaggga agccaaaaga 5160

ttggacacgc ctgtcctaga tggagaggaa ggaggcagtg ctgagcacat ctggccattc 5220

atccatctgg agagagaagg ctatgggcaa actgcttcct ctcccctgta gacacccagc 5280

tgggaaggtc tggcctttgg taagtcctgg cttggggtcc ttcctcattt cacagaacct 5340

aactctatgt tagtgctttg tgagtatatg ttgatcataa taaagttgac gggatttttt 5400

cacatgataa taatagttgt catctggccg ggcatggtgg cttatgccta taatttcagc 5460

actttggaag gctgaggcag gtggatcact tgaggtcagc tgttcgagac cagcctggcc 5520

aacatggtga aaccacatct ctacttaaaa aaaaaaaaaa tacaaaaatt agctgggtgt 5580

ggtggtgcac ccttgtaatc ccagctactc gggaggctga ggcaggagaa tcacttgaac 5640

ccaggaggtg gaggttgcag tgagctgaga ttgtgccact acactccagc ctgggtgaca 5700

agagcgaaac tccgtctcaa aaaaaaagaa aataataata ataatagttg ccatccattc 5760

tactgtgctt tccattaact cgtgtaatcc tcacaagtcc cattttatag ttacaggaac 5820

tgaggctcac agagcttaaa tcacttggcc aaggccacaa acagctataa gaattacatt 5880

taggcagtct gattccaaag atactagtct attctgtatc tcatagacaa acaatacata 5940

ttcacttttt tgttgttgtt ttgttttgag acggagtctt gctctgtcac ccaggctgga 6000

gtgcagtggc gccatctcgg ctcactgcaa cgtccgcctc ccgggttcaa gcgattctcc 6060

tgcctcagcc tcccgagtag ctgggactac aggcatgtgc caccatgccc ggctaatttt 6120

ttgtattttt agtagagaca gggttttcct gggttagcca gaatggtctc gatctcctga 6180

ccttgtgatc cacccacctc agcctcccaa agtgctgaga tgacaggcgt gagccaccgc 6240

gtccgaccta tattcactat ttataaattg gagagaataa gaaaatcaaa agggccaggt 6300

gtagtgactc acacctgtaa tcccagcact ttgggaagcc aaggcaggag gattgcttga 6360

acccagaagt tcgagaccag cctgggcaac atggtgagac cctgtctcta caaaaaatac 6420

aaaaattagc tgggcgttgt ggtgagcacc ttattcttag gaagctgagg caggaggatc 6480

acctgaggcc aaggaggttg agactgcagt gagctgtgat cataccactg tacttcagcc 6540

tggacatcag agtaagaccc tatctctaaa aaggaaattg agaagaaaga aaatcaaagg 6600

gaagcaaaat cactcactct cactacctca agataccctc tagaagttgg tattttagtg 6660

tggttcctat tgttttctgt gtcagttctc tgatttgagc aaaatctttg ggacgtcaaa 6720

cttaaaatcc cctttacttc cttggaaacc ctgtagcatt agcccagaca tgtccctact 6780

cctccttgtg gcaaagagaa ggatctcgtc tttggtcccc agagttctgg cctaagcctc 6840

cctccaggag ggaagatgag tgttcagaca ctcagagtag ctgggggaga cacaggcctg 6900

tgaaattatc ctggctcaac tattaggtcg gcagaatccc agtgaaggga gccctacctc 6960

tgagccccat ctaagctttg gctatgggtg gggcagataa gcaggaatcc atccctatag 7020

gctcaatgcc aacaccctta ggtgaaactc ttgatgaaac ttgaggccag ggctccggca 7080

agcagggaaa gaacgttggc aacagaggtc tccatctctg aggactctgc caggggtcag 7140

agatggggca atggtcaaaa ggaaggaaca ggccaggcac agtggctcat gcccataatc 7200

ccagcacttt gggaggctga ggcaggagga tcgcttgagc ccaggagttt gagacctgcc 7260

tgggcaatgt agtgagatct gctctctatt taaaaaaaaa aaaaaggaaa gaacaagtaa 7320

acttctgaga aacaggctgg gggaggcatc acgtagctgg aattgctgcc ccataaaaca 7380

gaatggtatg tgtcactgcc acctcccttt ctcagtcctc tctctcccca ggttgctagc 7440

gtccccctgg gggatcaaac tggactgctt cccagcctca gacagagagc agtctgagtc 7500

aggcaggaaa gtgggacagc cggggagctg gaccccaccc tctgtgagcc ccgctggtac 7560

ctgatggcat gtggcttgga gagggcaggt gacctggcgt ggagggccag agggtaaatc 7620

ctcaaacaag tggcaacagg ccaccaactt gaaagggaaa attgtgtagt gatgggaaat 7680

gtgtccaaca aacctactgg gtgactaatt acaaaggctg ggctggagct tcagaggctg 7740

cttgttaaac acttcattaa gcggcactct gaaagctgcc acctgcgcat tctgggagct 7800

cagaggggac cctgaggggg aatgaggcct ggaggatgga accatcttca ggtagactga 7860

gaaggagcct ggatctcact tccaaacaca gtctggagct cataggtcag aggcctcaat 7920

gggagaaaag ctaaaggaag agggtgcaga aaggagtttc agggaattgg tggctatgtg 7980

actttgagca aatctcaccc ctctctgaga cttagtgttc ccatctctat ggtcctgtgt 8040

gtgtcacaga gacatggtgg ggattaaatt cgatcgtgat atgaaagtgc ttgggaaact 8100

ccatggccct acctaaacat gagttatcct cacctgaacc aaggggggaa gttacctggc 8160

aggattagga accccatcct cctgaacctt tatgggctct gtcgaggctg aagcagccag 8220

gggctaaagc cagtccttag cccctggaag ggcactgtga aagtggatct gatttgagaa 8280

gccgtttcct gatgtgggca gccatgtgat gccagccccg aacaagaggg ggcagcctgg 8340

agcctggaaa ggtgccagtg caggtggggc ccacgcccag atttctcctg ctgactgttc 8400

tgatgattca cccccacatc ccagcctttt tacctttact gcagagccgg aaagggtgtg 8460

gggaagagag gagagggagg caggtcttgg gccctggtcc cgccccctgc tcctccccac 8520

ccttctctgg gcctggccac ccagccaaaa ggcaggccaa gagcaggaga gacacagagt 8580

ccggcattgg tcccaggcag cagttagccc gccgcccgcc tgtgtgtccc cagagccatg 8640

gagagagcca gtctgatcca gaaggccaag ctggcagagc aggccgaacg ctatgaggac 8700

atggcagcct tcatgaaagg cgccgtggag aagggcgagg agctctcctg cgaagagcga 8760

aacctgctct cagtagccta taagaacgtg gtgggcggcc agagggctgc ctggagggtg 8820

ctgtccagta ttgagcagaa aagcaacgag gagggctcgg aggagaaggg gcccgaggtg 8880

cgtgagtacc gggagaaggt ggagactgag ctccagggcg tgtgcgacac cgtgctgggc 8940

ctgctggaca gccacctcat caaggaggcc ggggacgccg agagccgggt cttctacctg 9000

aagatgaagg gtgactacta ccgctacctg gccgaggtgg ccaccggtga cgacaagaag 9060

cgcatcattg actcagcccg gtcagcctac caggaggcca tggacatcag caagaaggag 9120

atgccgccca ccaaccccat ccgcctgggc ctggccctga acttttccgt cttccactac 9180

gagatcgcca acagccccga ggaggccatc tctctggcca agaccacttt cgacgaggcc 9240

atggctgatc tgcacaccct cagcgaggac tcctacaaag acagcaccct catcatgcag 9300

ctgctgcgag acaacctgac actgtggacg gccgacaacg ccggggaaga ggggggcgag 9360

gctccccagg agccccagag ctgagtgttg cccgccaccg ccccgccctg ccccctccag 9420

tcccccaccc tgccgagagg actagtatgg ggtgggaggc cccacccttc tcccctaggc 9480

gctgttcttg ctccaaaggg ctccgtggag agggactggc agagctgagg ccacctgggg 9540

ctggggatcc cactcttctt gcagctgttg agcgcaccta accactggtc atgcccccac 9600

ccctgctctc cgcacccgct tcctcccgac cccaggacca ggctacttct cccctcctct 9660

tgcctccctc ctgcccctgc tgcctctgat cgtaggaatt gaggagtgtc ccgccttgtg 9720

gctgagaact ggacagtggc aggggctgga gatgggtgtg tgtgtgtgtg tgtgtgtgtg 9780

tgtgtgcgcg cgcgccagtg caagaccgag actgagggaa agcatgtctg ctgggtgtga 9840

ccatgtttcc tctcaataaa gttcccctgt gacactcctc ctgtctctct tccagttctt 9900

ggcgatgggc tgggagtggg actggaatct gacttagaga ccctgacttt ggacctctga 9960

gttagggccc tgaactccct aggtggctca gtggcccgca cgcaagactt tgagtccagg 10020

tgaggccggg gtcc 10034

103

agcttgcagc cccagcccgg gccagccagg tacaggaggc cggactgcaa ccggttgctt 60

ccctcccgtc gcgcctggcc gtcccacgct gcgccgtcgc tgctgcctcc tggcgcccct 120

gggattttat acgcacctct gaaacacgct ccgctccggc ccccggttct tctccttgcc 180

taggggttgt ttcccaatag atactgactc ctttagaaga tccaaaaacc aaaccaaaac 240

accccctacc cgccccaaac acctgctctg gggcgcgggg gctgccaaac agagactaga 300

cgaagggagt cagatttagc gaantcttcg agctcccaaa gattcgaaca ctaactcgcg 360

cccgtgggcc gatggaggtt ctccctactc cactccttgg tccccttaac tggcttccgc 420

ctcctggtca atcactgagc aaccagaatg gtatcctcga ccagggccac aggcagtgct 480

cggcggagtg gctccaggag ttacccgctc ctgccgggct tcgtatccaa accctcccct 540

tcacccctcc tccccaaact gggcgccagg atgctccggc cggaatatac gcaggctttg 600

ggcgtttgcc caagggtttt cttccctcct aaactagccg ctgttttccc ggcttaaccg 660

tagaagaatt agatattcct cactggaaag ggaaactaag tgctgctgac tccaatttta 720

ggtaggcggc aaccgcttcc gcctggcgca aacctcacca agtaaacaac tactagccga 780

tcgaaatacg cccggcttat aactggtgca actcccggcc acccaactga gggacgttcg 840

ctttcagtcc cgacctctgg aacccacaaa gggccacctc tttccccagt gaccccaaga 900

tcatggccac tcccctaccc gacagttcta gaagcaagag ccagactcaa gggtgcaaag 960

caagggtata cgcttctttg aagcttgact gagttctttc tgcgctttcc tgaagttccc 1020

gccctcttgg agcctacctg cccctccctc caaaccactc ttttagatta acaaccccat 1080

ctctactccc accgcattcg accctgcccg gactcactgc ttacctgaac ggactctcca 1140

gtgagacgag gctcccacac tggcgaaggc caagaagggg aggtgggggg agggttgtgc 1200

cacaccggcc agctgagagc gcgtgttggg ttgaagagga gggtgtctcc gagagggacg 1260

ctccctcgga cccgccctca ccccagctgc gagggcgccc ccaaggagca gcgcgcgctg 1320

cctggccggg cttgggctgc tgagtgaatg gagcggccga gcctcctggc tcctcctctt 1380

ccccgcgccg ccggcccctc ttatttgagc tttgggaagc tgagggcagc caggcagctg 1440

gggtaaggag ttcaaggcag cgcccacacc cgggggctct ccgcaacccg accgcctgtc 1500

cgctccccca cttcccgccc tccctcccac ctactcattc acccacccac ccacccagag 1560

ccgggacggc agcccaggcg cccgggcccc gccgtctcct cgccgcgatc ctggacttcc 1620

tcttgctgca ggacccggct tccacgtgtg tcccggagcc ggcgtctcag cacacgctcc 1680

gctccgggcc tgggtgccta cagcagccag agcagcaggg agtccgggac ccgggcggca 1740

tctgggccaa gttaggcgcc gccgaggcca gcgctgaacg tctccagggc cggaggagcc 1800

gcggggcgtc cgggtctgag cctcagcaaa tgggctccga cgtgcgggac ctgaacgcgc 1860

tgctgcccgc cgtcccctcc ctgggtggcg gcggcggctg tgccctgcct gtgagcggcg 1920

cggcgcagtg ggcgccggtg ctggactttg cgcccccggg cgcttcggct tacgggtcgt 1980

tgggcggccc cgcgccgcca ccggctccgc cgccaccccc gccgccgccg cctcactcct 2040

tcatcaaaca ggagccgagc tggggcggcg cggagccgca cgaggagcag tgcctgagcg 2100

ccttcactgt ccacttttcc ggccagttca ctggcacagc cggagcctgt cgctacgggc 2160

ccttcggtcc tcctccgccc agccaggcgt catccggcca ggccaggatg tttcctaacg 2220

cgccctacct gcccagctgc ctcgagagcc agcccgctat tcgcaatcag ggtaagtagg 2280

ccggggagcg ccccta 2296

104

2495

Homo sapiens

104

actatagggc acgcgtggtc gacggcccgg gctggtattg atagatgcat tttcttcacc 60

ctcacctatc tttttctgcc tgttggctta tggttgaaat tccttcatga cggtttccat 120

ttccagagat atcttgttaa caagtatata ccaccaaatg aagctgattt tttttttttt 180

ttttttttga gacagagtct cgctctgtcg cccaggctgg aatgcagtgg cgcgatcttg 240

gctcactgca acctccgcct cccatgttca agcgattctc ctgcctcagc ctcctgagta 300

gctgggatta ctggcatgtg ccaccgcgtc cagccaattt ttgtattttt agtagagacg 360

aggtttcacc atgttggtca ggctggtctc aaactcctga cctcgtgatc cacctgcctc 420

ggcctcccaa agtgctgaga ttataggtgt gagccaccat gcctggccat gaagctgatt 480

tttttaaacc atcatttaac attttctcca taaggtggca aggaggaaga gcatatgggg 540

actgggtact ttgagagacc ccaggacagg agacagggag gctgagattg gcatgttgtc 600

tgctgcagtt atttgccagc gacacactct ttccgtccaa actaacttct ctgcctcaag 660

gacagggaga ctctgccttt caacctgaga gaaaccagga ctctcagctt taatgaaaat 720

tggacttagg gtggggcagt ggagactttt cacagctatt gtttagctga tgaagcagat 780

gcttctccat ctttggagcc tgtcttcatt acctgtggac ctcatcttta tcaacccaga 840

gcacacttgc gtctctctat tttggctaaa caccaaacag ctgaggctgg tactgtaaaa 900

ctttccctcc aaatgccccc cctcgtcttc ctctattaga gatctggatc acaaccctca 960

aaaaccatgt cccttatgcc acctgagtag atggtttgat gattaattag gcacagatgt 1020

gacactgggg gggtctcaca atggcctgtg ggtcacatgc tactttcctt ttcattttca 1080

tcagcaacag ctgccttaaa gccagttaag actgtggtcc tagtctcgca ccctggggct 1140

cctgctgggg tgggtgaggg gaacacccca ttaagctggg ggaactgggg ctgccaccag 1200

ggggcgcgag gggccttcgc ccgagaagag gggtgggcag gtgcctccag cggagaaggg 1260

cgccgtggcc ggaggcacag gtctccccgg tgccacttca agtgagttcg aggaagtacc 1320

tgggatcttt gatctaacgc gaaaggcctt cccagtgacc tcttgagggc tgagaaccca 1380

ctccctccac ctctagtcca cggctttgcc actccagggc ccgaggttac gtttgctgct 1440

ggggatttga caaacccaaa gcctctctgg tttcaccact ggctccttag aatcagacat 1500

ctgttctgaa tgacacttat gtgagtcagg ggctgaggac gtgatcctcg aagtgtggtc 1560

cccagactgg ctgtatcagt gtcggcatcc cccaggacct ggttggaaat gcatattctc 1620

aggccctact ccagacctct taaatctgag actggggctg cggggagcgc catctgtgcg 1680

ccactatcct tgtgggtgga ccaggagtcg gttcgagggt gctcccactt agaggtcacg 1740

cgcggcgtcg ggcgttcctg agaccgtcgg gctccctggc tcggtcacgt gggctcaggc 1800

actactcccc tctaccctcc tctcggtctt taaaaggaag aaggggctta tcgttaagtc 1860

gcttgtgatc ttttcagttt ctccagctgc tggctttttg gacacccact cccccgccag 1920

gaggcagttg caagcgcgga ggctgcgaga aataactgcc tcttgaaact tgcagggcga 1980

agagcaggcg gcgagcgctg ggccggggag ggaccacccg agctgcgacg ggctctgggg 2040

ctgcggggca gggctggcgc ccggagcctg agctgcagga ggtgcgctcg ctttcctcaa 2100

caggtggcgg cggggcgcgc gccgggagac cccccctaat gcgggaaaag cacgtgtccg 2160

cattttagag aaggcaaggc cggtgtgttt atctgcaagc cattatactt gcccacgaat 2220

ctttgagaac attataatga cctttgtgcc tcttcttgca aggtgttttc tcagctgtta 2280

tctcaagaca tggatataaa aaactcacca tctagcctta attctccttc ctcctacaac 2340

tgcagtcaat ccatcttacc cctggagcac ggctccatat acataccttc ctcctatgta 2400

gacagccacc atgaatatcc agccatgaca ttctatagcc ctgctgtgat gaattacagc 2460

attcccagca atgtcactaa cttggaaggt gggcc 2495

105

1630

Homo sapiens

105

gagctcgagc cacgccatgc ccgctgcacg tgccagcttg cgcagcacat cagggcgctg 60

gtctctcccc ttcctcctgg agtgaaatac accaaagggc gcggtggggg tggggggtga 120

cgggaggaag gaggtgaaga aacgccacca gatcgtatct cctgtaaaga cagccttgac 180

tcaaggatgc gttagagcac gtgtcagggc cgaccgtgct ggcggacttc accgcagtcg 240

gctcccaggg agaaagcctg gcagagtgag gcgcgaaacc ggagggtcgg cgaggatgcg 300

ggcgaaggac cgagcgtgga ggcctcatgc ctccggggaa aggaaggggt ggtggtgttt 360

gcgcaggggg agcgaggggg agccggacct aatccctcac tcgccccctc cccctcccgg 420

gccatttcct agaaagctgc atcggtgtgg ccacgctcag cgcagacacc tcgggcggct 480

tgtcagcaga tgcaggggcg aggaagcggg tttttcctgc gtggccgctg ggcgggggaa 540

ccgctgggag ccctgccccc ggcctgcggc ggccctagac gctgcaccgc gtcgccccac 600

gggccccgaa gagcccccag aaacacgatg gtttctgctc gaggatcaca ttctatccct 660

ccagagaagc accccccttc cttcctaata cccacctctc cctccctctt cttcctctgc 720

acacactctg cagggggggg cagaagggac gttgttctgg tccctttaat cggggctttc 780

gaaacagctt cgaagttatc aggaacacag acttcaggga catgaccttt atctctgggt 840

atgcgaggtt gctattttct aaaatcaccc cctcccttat ttttcactta agggacctat 900

ttctaaattg tctgaggtca ccccatcttc agataatcta ccctacattc ctggatctta 960

aatacaaggg caggaggatt aggatccgtt ttgaagaagc caaagttgga gggtcgtatt 1020

ttggcgtgct acacctacag aatgagtgaa attagagggc agaaatagga gtcggtagtt 1080

ttttgtgggt tgcctgtccg gggcccctgg catgcaggct ggatggaggg agaggggtgg 1140

ggggtggcgg gggaccgcgt ttgaagttgg gtcgggccag ctgctgttct ccttaataac 1200

gagaggggaa aaggagggag ggagggagag attgaaagga ggaggggagg accgggaggg 1260

gaggaaaggg gaggaggaac cagagcgggg aggcgcgggg agagggagga gagctaactg 1320

cccagccagc ttgcgtcacc gcttcagagc ggagaagagc gagcagggga gagcgagacc 1380

agttttaagg ggaggaccgg tgcgagtgag gcagccccga ggctctgctc gcccaccacc 1440

caatcctcgc ctcccttctg ctccaccttc tctctctgcc ctcacctctc ccccgaaaac 1500

cccctattta gccaaaggaa ggaggtcagg ggaacgctct cccctcccct tccaaaaaac 1560

aaaaacagaa aaaccctttt ccaggccggg gaaagcagga gggagagggg ccgccgggct 1620

ggccatggag 1630

106

1800

Homo sapiens

106

cattggactg ggtttccttc caccgaagag tgaacttctg cctctttcga gcaccttccg 60

aggcgtagtc ctttggatgt tggggagcgt cagactgggt cgttgtagag gggaaaggag 120

ggcccagaag ggcgagagag caggccggga cgcaaatcct cagcccccgc ggcgcgccac 180

gtcttcagaa acgccaggac ctccgggctg ggccgccgcg gtttggcctt tggaactcaa 240

gggttcgtct acctgaccat tgggtggctc cgcggttgac acttttcttg gcatgccccc 300

ccaccccgcg ccacaccacc cccccagccc cagcaatcca aatcggcccc acggacctag 360

agggctcttg ggcgagatga gacatcaccc actgtgtaga agctgttgcc attgctgctg 420

tcacagccac tccggatggg gctgccaccg tggccaggac agtctcctcc gaccgcttcc 480

tgggctgcgc tagggttcgg gggcgctgcc cgcacgctcc ggcggggaag gaaatcgccc 540

cgcgcccgcc ggaggaaggc gacggggagg gaagggggag ggcggctagg aggcgggtgg 600

aggggccggc cgcccgggcc aggtcgtttt tgaatggttt gggaggacga attgttagac 660

cccgaggaag ggaggtggga cgggggaggg ggactggaaa gcggaaactt tcctataaaa 720

cttcgaaaag tccctcctcc tcacgtcagg ccaatgacac tgctgccccc aaactttccg 780

cctgcacgga ggtataagag cctccaagtc tgcagctctc gcccaactcc cagacacctc 840

gcgggctctg cagcaccggc accgtttcca ggaggcctgg cggggtgtgc gtccagccgt 900

tgggcgcttt ctttttggga cctcggggcc atccacaccg tcccctcccc ctcccgcctc 960

cctccccgcc tcccccgcgc gccctccccg cggaggtccc tcccgtccgt cctcctgctc 1020

tctcctccgc gggccgcatc gcccgggccg gcgccgcgcc ggggggaagc tggcgggctg 1080

aggcgccccg ctcttctcct ctgccccggg cccgcgaggc cacgcgtcgc cgctcgagag 1140

atgatgcagg acgtgtccag ctcgccagtc tcgccggccg acgacagcct gagcaacagc 1200

gaggaagagc cagaccggca gcagccgccg agcggcaagc gcgggggacg caagcggcgc 1260

acgagcaggc gcacggcggg cggcggcgcg gggcccggcg gagcgggtgg gggcgtcgga 1320

ggcggcgacg agccgggcag cccggcccag ggcaagcgcg gcaagaagtc tgcgggctgt 1380

ggcggcggcg gcggcgcggg cggcggcggc ggcagcagca gcggcggcgg gagtccgcag 1440

tcttacgagg agctgcagac gcagcgggtc atggccaacg tgcgggagcg ccagcgcacc 1500

cagtcgctga acgaggcgtt cgccgcgctg cggaagatca tccccacgct gccctcggac 1560

aagctgagca agattcagac cctcaagctg gcggccaggt acatcgactt cctctaccag 1620

gtcctccaga gcgacgagct ggactccaag atggcaagct gcagctatgt ggctcacgag 1680

cggctcagct acgccttctc ggtctggagg atggaggggg cctggtccat gtccgcgtcc 1740

cactagcagg cggagccccc caccccctca gcagggccgg agacctaggt aaggaccgcg 1800

107

20

Artificial Sequence

Forward primer

107

tttcggatgg ggttgttatc 20

108

20

Artificial Sequence

Reverse primer

108

cctaacccaa acaaccaacc 20

109

21

Artificial Sequence

Forward primer

109

tttggatggg gttgttattg t 21

110

19

Artificial Sequence

Reverse primer

110

aaacgaccta acccgaacg 19

111

23

Artificial sequence

Forward primer

111

agggaagttt tttttatttg gtt 23

112

26

Artificial sequence

Reverse primer

112

gtggttttgt tttgtatgtt ttggtg 26

113

26

Artificial sequence

Reverse primer

113

caccgaaaca tacaaaacaa aaccac 26

114

20

Artificial sequence

Forward primer

114

gtttgttaag aggaagtttt 20

115

26

Artificial sequence

Reverse primer

115

caccgaaaca tacaaaacaa aaccac 26

116

24

Artificial sequence

Forward primer

116

ggtacgggtt ttttacggtt cgtc 24

117

22

Artificial sequence

Reverse primer

117

aacttcttat acccgatcct cg 22

118

24

Artificial sequence

Forward primer

118

ggtatgggtt ttttatggtt tgtt 24

119

25

Artificial sequence

Reverse primer

119

caaaacttct tatacccaat cctca 25

120

ctttgctctc catctgtcat acttctgcag gtgacagacg agtgaggaca tttagagaaa 60

ctcaggaaca aaccaggccc atccactggc gtcgaggcta gtttcgaaga cagaaaaacg 120

ccccacttct gtttgcactg tggctgcctg tggcccggcc ggggaggcgg ccgggagtga 180

ggcctgatcg tccctggcgc ctccacctcc ccaggcgcag aaggcgccca cgaggacccc 240

cagtgcccga cgttgccacg gtctgggatc agaggcacgg gaccagggag ccaggaactg 300

cgccgccccg ccctgcctgg cgcgaggaag ctccctcacc gggccagcct gcagggggng 360

cttctcttcc cttctttccc ccccctttcc gtcctctctc tcctcctcct ctcctcctct 420

tgtcgtatct ttctctccct gtcgctgtct gttcttcctc tccctgctgc tgccgtcggt 480

tctgtctctt ctctgtcgtt cccttctccc cttcgccctc gtgtcgctgc ttatataagt 540

gagaaaaaaa aaaccaaaaa aacagacatg ttgctaagat agtctttccc attattttct 600

tatttcaaac gtgatctggt cttccacgcc tccgctagca ttaacaaaaa caaaacgtca 660

cagtgctggc cgccgtatct cggatagcca ttttccggcc tccaatccca gtccaatggc 720

ccgtggggac cccattccca agtttccact gtcggaatct ttctatgacc aggtacccag 780

ttcttgccct cccctgccct gtcctgtctt cgagggaagc tcccctcacc cggcccagcc 840

ctgacggggg cgctggggtc agaccgcaaa gcgaaggtgc gggccggggt gggcctcgcg 900

gagacaaagg ccgggcctgc ctctctcaga gggccccagc gcctgccaag aggaagtcct 960

cgaggcccgg gcagggaagg gggcacgggc ttcccacggc ccgccggccg cagcaggaag 1020

ttggccaggg cacggccgtg agcggagcgg gcagggcttt ctcaggagcg cgggcgaggc 1080

cggcgctgga ggggcgagga ccgggtataa gaagcctcgt ggccttgccc gggcagccgc 1140

aggttccccg cgcgccccga gcccccgcgc catgaagctc gccgccctcc tggggctctg 1200

cgtggccctg tcctgcatgc tccggtgagc gccccgggct cctggcgcgc acggtgggcc 1260

tgaggcctcg gcgccccgtg cgcccccgcc gctgcctgcg ccgatcgtgg atcccaggtc 1320

cttccagcct cgcccggcgc ccagagggct ccgccacccc ggggccccgc gcctgcagcc 1380

cgcggcctcc ccctctcagg gctgtctcca gcctcgtgcc gggatggagg ccgcccctgg 1440

cctggggaca cccgtctgcc ccccgtctgg agaccggctc ctcatcctgc aaggcgcagc 1500

gcgagtgtcc cctcccttgg gggcctgtcc cggaccctgc acagagttca ccggtccttc 1560

cgccaccctc aggccactcc ggtgaccctg cagcgtctcc tggcggggcc gcctccccag 1620

acccgcctgt gtcccggggg ctcgcacctg gcaggcctcg gcgcaggagg gaggggcggt 1680

cggggagccg ggcagggcgc gacggttcct gggcgcctcc cgcgggggcg cgtcctggac 1740

ctgccttctg gggaccccgg cgcgcaggcg gtgacccctc cctgttcgct tgca 1794

121

900

Homo sapiens

121

tcagcaaacc ggaccaggag ggccagggcc ggatgtgggg accctcttcc tctagcacag 60

taaagctggc ctccagaaac acgggtatct ccgcgtggtg ctttgcggtc gccgtcgttg 120

tggccgtccg gggtggggtg tgaggagggg acgaaggagg gaaggaaggg caaggcgggg 180

ggggctctgc gagagcgcgc ccagccccgc cttcgggccc cacagtccct gcacccaggt 240

ttccattgcg cggctctcct cagctccttc ccgccgccca gtctggatcc tgggggaggc 300

gctgaagtcg gggcccgccc tgtggccccg cccggcccgc gcttgctagc gcccaaagcc 360

agcgaagcac gggcccaacc gggccatgtc gggggagcct gagctcattg agctgcggga 420

gctggcaccc gctgggcgcg ctgggaaggg ccgcacccgg ctggagcgtg ccaacgcgct 480

gcgcatcgcg cggggcaccg cgtgcaaccc cacacggcag ctggtccctg gccgtggcca 540

ccgcttccag cccgcggggc ccgccacgca cacgtggtgc gacctctgtg gcgacttcat 600

ctggggcgtc gtgcgcaaag gcctgcagtg cgcgcgtgag tagtggcccc gcgcgcctac 660

gagagcggaa ggggcagcca aggggcagcg cagtcgccgc gggtcaagtc gcggcagagg 720

gggtcggcgg ggacagctcc cgaggactag gtccgttact ttcgccccat cgctgaagag 780

tgcgcgaaaa tggtttatcc cttgtcgcac tccactcgta tctgggccac agatgagcag 840

aggtggctgc ttatatgtaa aaatacgctg attttaagtt tcttatcttt aaaatgcctt 900

122

21

Artificial sequence

PCR sense primer

122

gggagtttga gtttattgag t 21

123

19

Artificial sequence

PCR antisense primer

123

accccttaac taccccttc 19

124

19

Artificial sequence

PCR sense primer

124

gttggtattc gttgggcgc 19

125

19

Artificial sequence

PCR sense primer

125

gcaccacgta tacgtaacg 19

126

19

Artificial sequence

PCR antisense primer

126

gcaccacgta tacgtaacg 19

127

20

Artificial sequence

PCR sense primer

127

ggttgtattt ggttggagtg 20

128

22

Artificial sequence

PCR antisense primer

128

ctacaaacct ttacacacaa ca 22

129

25

Artificial sequence

PCR sense primer

129

tattttttgt aaagatagtt ttgat 25

130

22

Artificial sequence

PCR antisense primer

130

tacaactttc taaaaataac cc 22

131

23

Artificial sequence

PCR sense primer

131

gagatgagat attatttatt gtg 23

132

23

Artificial sequence

PCR antisense primer

132

aacaacaata tcattaacct aac 23

133

22

Artificial sequence

PCR sense primer

133

gtaggagggt ttattttttg tt 22

134

23

Artificial sequence

PCR antisense primer

134

aattacattt tccaaactta ctc 23

135

19

Artificial sequence

Forward primer

135

gaacgcgagc gattcgagt 19

136

19

Artificial sequence

Reverse primer

136

gaccaatcca accgaaacg 19