DNA METHYLATION DIAGNOSTIC TEST FOR BREAST CANCER

BACKGROUND OF THE INVENTION

Invasive breast cancer represents a quarter of all cancers diagnosed worldwide and its rate increases by 0.3% every year. Ductal carcinoma in situ (DCIS) is considered to be a pre-invasive form of breast cancer. Almost a quarter of all new breast cancers diagnosed in the United States are DCIS. However, patients with DCIS are treated with surgery and radiation, identically to patients presenting with invasive disease. Not all women with untreated DCIS will develop invasive ductal cancer (IDC). At present, there is no tool to predict concurrent or subsequent invasion in DCIS. This results in overtreatment of a significant number of patients who carry the stigma of cancer.

BRIEF SUMMARY OF THE INVENTION

The present subject matter provides, inter alia, methods, compounds, compositions, kits, and systems for diagnosing IDC or detecting IDC risk in a subject who has DCIS, treating breast cancer in a subject detected to have IDS or determined to be at risk of IDC, and detecting invasive competent DCIS and invasive incompetent DCIS in a subject.

In an aspect, included herein is method of detecting methylation or unmethylation of a DCIS cell DNA molecule of a subject. In embodiments, the method comprises (i) contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and (ii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject.

In embodiments, provided herein is a method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. In embodiments, the method comprises (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule; (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and (iii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject.

Also provided herein is a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject. In embodiments, the method comprises (i) contacting a plurality of isolated DCIS cancer cell proliferation DNA molecules from the subject with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; and (ii) detecting the level of uracil in the plurality of reacted DCIS cancer cell proliferation DNA molecules at a plurality of methylation sites set forth in Table 1, thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

Embodiments also provide a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject. In embodiments, the method comprises (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules; (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; and detecting the level of uracil in the plurality of reacted DCIS cancer cell proliferation DNA molecules at a plurality of methylation sites set forth in Table 1, thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, further provided is a method of detecting a risk of developing IDC in a subject who has DCIS. In embodiments, the method comprises (i) contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and (ii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting the risk for developing IDC in the subject. In embodiments, the method comprises (i) contacting a plurality of isolated DCIS cancer cell proliferation DNA molecules from the subject with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; and (ii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a plurality of methylation sites set forth in Table 1, thereby detecting the risk for IDC in the subject.

Also provided is a method of detecting IDC or a risk of developing IDC in a subject. In embodiments, the method includes (i) isolating a DCIS cancer cell proliferation DNA molecule from DCIS tissue of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule; (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and (iii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting the risk for developing IDC in the subject. In embodiments, the method comprises (i) isolating a plurality of DCIS cancer cell proliferation deoxyribonucleic acid (DNA) molecules from DCIS tissue of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules; (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; and (iii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a plurality of methylation sites set forth in Table 1, thereby detecting the risk for IDC in the subject.

In embodiments, the detection of the presence, absence, or level of uracil in a reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1 comprises amplifying the reacted DCIS cancer cell proliferation DNA molecule [e.g., by a polymerase chain reaction (PCR)] to produce an amplicon, and determining whether a thymidine is present at the nucleotide position of the amplicon that corresponds to the nucleotide position of the uracil in the reacted DCIS cancer cell proliferation DNA molecule.

Aspects also include a method of treating or preventing IDC a subject detected to have a risk of IDC or diagnosed as having IDC. In embodiments, the method includes administering to the subject a treatment to treat or prevent IDC or directing the subject to obtain treatment to treat or prevent IDC.

Also included herein is a DNA molecule at least 5 to 100 nucleotides in length comprising a uracil-containing sequence that is identical to a sequence of at least 5 contiguous nucleotides within a sequence chosen from SEQ ID NO:1 to SEQ ID NO:242.

In embodiments, provided herein is an oligonucleotide comprising a uracil-containing sequence that is identical to a sequence of at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 contiguous nucleotides within the sequence chosen from SEQ ID NO:1 to SEQ ID NO:242, or an oligonucleotide that is an amplicon or is identical to an amplicon of a methylation site-containing sequence of at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 contiguous nucleotides within the sequence chosen from SEQ ID NO:1 to SEQ ID NO:242.

Aspects of the present subject matter also include a deoxyribonucleic acid chosen from SEQ ID NO:243 to SEQ ID NO: 356, wherein the nucleic acid is hybridized to a complementary DNA sequence comprising uridine or cytosine.

Also provided is a kit comprising a plurality of nucleic acids each independently comprising SEQ ID NO: 242 to SEQ ID NO: 356, wherein each nucleic acid of the plurality is unique.

In embodiments, included herein is a system for detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. In embodiments, the system provides at least one processor; and at least one memory including program code which when executed by the at least one processor provides operations comprising: contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfate salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject; generating a diagnosis for the subject based at least in part on the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at the methylation site set forth in Table 1; and providing, via a user interface, the diagnosis or prognosis for the subject. In embodiments, the system provides at least one processor; and at least one memory including program code which when executed by the at least one processor provides operations comprising: contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1 thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject; generating a diagnosis for the subject based at least in part on the level of methylation or unmethylation at the plurality of methylation sites set forth in Table 1; and providing, via a user interface, the diagnosis or prognosis for the subject.

Aspects also include a system for detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. In embodiments, the system comprises at least one processor and at least one memory including program code which when executed by the at least one processor provides operations. In embodiments, the operations comprise (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule; (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; (iii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject; (iv) generating a diagnosis for the subject based at least in part on the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at the methylation site set forth in Table 1; and (v) providing, via a user interface, the diagnosis or prognosis for the subject.

Also provided herein is a system for detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject. In embodiments, the system comprises at least one processor and at least one memory including program code which when executed by the at least one processor provides operations. In embodiments, the operations comprise (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules; (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; (iii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1 thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject; (iv) generating a diagnosis for the subject based at least in part on the level of methylation or unmethylation at the plurality of methylation sites set forth in Table 1; and (v) providing, via a user interface, the diagnosis or prognosis for the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a set of images, FIG. 1C is a table, and FIGS. 1B and 1D are heatmap images relating to the epigenetic analysis of DCIS. FIG. 1A: Extraction of cancer cells from DCIS containing ducts. DCIS slide before and after a laser cut. FIG. 1B: DNA methylation patterns at promoters in DCIS. Heatmap was done for promoter regions with available DNA methylation information (20,910 regions). Green represents low level of DNA methylation, red indicates a high level of DNA methylation. FIG. 1C: Pathology data for the samples included in the study. FIG. 1D: DNA methylation status of 140 cytosines separates “invasion incompetent” and “invasion competent” DCIS. Green represents low level of DNA methylation, red indicates a high level of DNA methylation. “ER” means “estrogen receptor” and “PR” means “progesterone receptor.” (+) means positive for estrogen and progesterone receptor staining. (−) means negative for estrogen and progesterone receptor staining. DCIS_IDC_11, DCIS_IDC_13(+), and DCIS_IDC_14(+) are samples from patients with DCIS with adjacent IDC. DCIS_3(−) and DCIS_6(−), INV, are samples from patients without IDC with DCIS negative for estrogen and progesterone receptor staining. DCIS_4, DCIS_5(+), DCIS_7(+), DCIS_8(+), and DCIS_1(+) are samples from patients without IDC with DCIS positive for estrogen and progesterone receptor staining (with the exception that status of estrogen receptor and progesterone receptor staining is not known for the DCIS_4).

FIG. 2 depicts a block diagram illustrating an exemplary breast cancer diagnostics system.

FIG. 3 depicts a flowchart illustrating an exemplary process for diagnosing breast cancer.

FIG. 4 depicts a flowchart illustrating an exemplary process for diagnosing breast cancer.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are, inter alia, compositions, methods, and kits for detecting methylated and/or unmethylated DNA. In some aspects, the present disclosure includes compositions, methods, and kits for detecting methylated and/or unmethylated DNA from ductal carcinoma in situ.

The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in oncology, cell culture, molecular genetics, epigenetics, and biochemistry).

As used herein, the term “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” discloses 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg. Additionally, where two values for a parameter are disclosed, then a range of all values between and including those two values is also disclosed. For example, “1, 2, and 3” discloses, e.g., 1-2, 1-3, and 2-3.

A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.

The term “ductal carcinoma in situ cancer cell proliferation” (also recited as “DCIS cancer cell proliferation” herein) refers to a biological sample comprising one or more cells obtained or provided from DCIS tissue of a subject. In embodiments, the DCIS cancer cell proliferation comprises a biopsy taken, e.g., from an abnormality [such as mammographic or magnetic resonance imaging (MRI) abnormality] in the breast of a subject. Non-limiting examples of mammographic abnormalities may include or be referred to as suspicious calcifications, microcalcifications, clustered microcalcifications, pleomorphic calcifications, pleomorphic branching calcifications, or a cluster of heterogeneous calcifications. In embodiments, a mammographic abnormality is observed as a mass. In embodiments, the mammographic abnormality is other than a mass (e.g., is a proliferation of cells within or along a duct in a string, sheet, plurality, or population of cells). Non-limiting examples of breast MRI abnormalities may include or be referred to as an abnormal enhancement, a mass-like enhancement, a non-mass enhancement, a linear non-mass enhancement, an enhancing intraductal mass, or a suspicious enhancing lesion. In embodiments, an MRI abnormality or enhancement is observed as a mass. In embodiments, during a breast MRI, mammogram, ultrasound, or physical exam, a DCIS cancer cell proliferation presents as a string, sheet, plurality, or population of cells, a mass, an irregular mass, or a mass lesion, or by ultrasound as a hypoechoic mass. In embodiments, the DCIS cancer cell proliferation is substantially free of normal cells. For example, at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more of the cells in the DCIS cancer cell proliferation are dysplastic cells such as DCIS cells. In embodiments, at least 75% of the cells in the DCIS cancer cell proliferation are DCIS cancer cells.

The term “disease” refers to any deviation from the normal health of a mammal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested. According to the present disclosure, the methods disclosed herein are suitable for use in a patient that is a member of the Vertebrate class, Mammalia, including, without limitation, primates, livestock and domestic pets (e.g., a companion animal). Typically, a patient will be a human patient.

The terms “subject,” “patient,” “individual,” and the like as used herein are not intended to be limiting and can be generally interchanged. That is, an individual described as a “patient” does not necessarily have a given disease, but may be merely seeking medical advice.

The term “subject” as used herein includes any mammal. In embodiments, the subject is a primate such as a human. In embodiments, the subject is a female. In embodiments, the subject is a male. Non-limiting examples of mammals include rodents (e.g., mice and rats), elephants, sloths, armadillos, primates (such as lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., pets or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), donkeys, zebras, tapirs, rhinoceroses, cats (e.g., domesticated cats and large cats such as lions, cheetahs, tigers, and leopards), whales, dolphins, porpoises, pigs, cattle, and deer.

It must be noted that as used herein and in the appended embodiments, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease,” “a disease state”, “a nucleic acid” or “a CpG site” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a patient suspected or at risk of having breast cancer and compared to samples from a known breast cancer patient, or a known normal (non-disease) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., breast cancer patients or healthy individuals with a similar medical background, same age, weight, etc. A control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to disease, or prior to treatment. One of skill will recognize that controls can be designed for assessment of any number of parameters.

One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

As used herein, the term “diagnosing” and the like includes detecting a disease, disorder, and/or a symptom or feature (e.g., invasiveness) thereof. Diagnosing also includes determining the stage or degree of a disease or disorder. The term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop a disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, invasiveness, etc.). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.

“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof. With regard to cancer, “treating” or “treatment” may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, invasion, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, invasion, or metastasis, or some combination thereof. With regard to a tumor, “treating” or “treatment” includes eradicating all or part of a tumor, inhibiting or slowing tumor growth, invasion, and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

As used herein, a “symptom” associated with a disorder includes any clinical or laboratory manifestation associated with the disorder, and is not limited to what the subject can feel or observe.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids, including ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

The term “bp” and the like refer, in the usual and customary sense, to the indicated number of base pairs.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids (e.g., genomic sequences or subsequences or coding sequences) or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length.

An example of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. As will be appreciated by one of skill in the art, the software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information.

A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

The term “associated” or “associated with” in the context of a substance (e.g., level of uracil or methylation level in a breast lump or DCIS cancer cell proliferation) does not necessarily mean that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function (i.e., level of uracil in the regions of chromosomes assayed).

The term “unmethylated DNA” or “demethylated DNA” means DNA that lacks a methyl group conjugated to cytosine in a segment of the DNA. DNA methylation typically occurs in a CpG dinucleotide context. In the context of the present disclosure, the DNA can be equivalent to a short (2-50, 5-50, 2-300, 2-350 nucleotides, e.g. 5-350 nucleotides) double stranded or single stranded nucleic acid, a nucleic acid fragment cloned in a plasmid DNA, a nucleic acid fragment amplified from a sample of a subject, and/or synthetically prepared a nucleic acid fragment. DNA methylation at the 5′ position of cytosine may have the specific effect on gene expression in vivo. DNA methylation may also form the basis of epigenetic structure, which typically enables a single cell to grow into multiple organs or perform multiple functions.

The CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” is shorthand for “—C-phosphate-G-”, that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA. The “CpG” notation is used to distinguish this linear sequence from the CG base-pairing of cytosine and guanine. The CpG notation can also be interpreted as the cytosine being 5′ prime to the guanine base.

In embodiments, methylation is detected based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG. However, methylated cytosine is not converted in this process, the methods of the present disclosure allow determination of methylation status as methylated or unmethylated.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide [ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)] is free of the genes or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

Exemplary Epigenetic Signatures

DCIS is the most common type of non-invasive breast cancer. Ductal means that the cancer starts inside the milk ducts (which are the “pipes” that carry milk from the milk-producing lobules to the nipple), carcinoma refers to any cancer arising in the epithelial tissue of the skin or of the lining of the internal organs (including breast tissue), and in situ means “in its original place.” DCIS is called “non-invasive” because it hasn't spread beyond the milk duct into any normal surrounding breast tissue. However, as disclosed herein, DCIS may be predisposed or prone to becoming invasive. DCIS is frequently found to be associated with invasive cancer where the DCIS remains localized in the ducts while the adjacent invasive cancer invades out of the ducts. IDC, which stands for infiltrating ductal carcinoma or invasive ductal carcinoma, is the most common type of invasive breast cancer. Invasive means that the cancer “invades” or spreads to surrounding tissues (e.g., surrounding breast tissues). All together, “invasive ductal carcinoma” refers to cancer that has broken through the wall of the milk duct and invaded the tissues of the breast. Over time, invasive ductal carcinoma can spread to the lymph nodes and possibly to other areas of the body.

Provided herein are methods, compounds, compositions, kits, and systems for detecting invasive competent DCIS and invasive incompetent DCIS in a subject who has DCIS, treating breast cancer in a subject detected to have invasive competent DCIS or invasive incompetent DCIS, monitoring a subject who has DCIS for invasive competent DCIS, and distinguishing invasive competent DCIS from invasive incompetent DCIS in a subject.

In an aspect, included herein are molecular epigenetic diagnostic methods that reduce breast cancer overdiagnosis and overtreatment. In embodiments, the treatment of a subject differs based on whether the subject is determined to have IDC or a risk for IDC. For example, a subject determined to have IDC or a risk of IDC may receive surgery such as a mastectomy, whereas a subject determined to not have IDC or no risk of IDC may receive less invasive surgery, radiation therapy, hormonal therapy, or be monitored periodically (e.g., retested for IDC, or IDC risk according to a method disclosed herein at a future time point, such as 0.5-5 years from the initial test) without receiving surgery, chemotherapy, radiation therapy, or hormonal therapy.

In embodiments, DCIS can be classified into two groups. One group comprises DCIS from patients with potential or concomitant invasion and characterized by cancer specific DNA methylation changes (“invasion competent DCIS” or “IC-DCIS”), the second group with DCIS is without any invasive disease (“invasion incompetent DCIS” or “II-DCIS”) and associated with DNA methylation pattern more similar to normal cells. IC-DCIS has a risk of becoming invasive in the future. In embodiments, a subject with IC-DCIS may have a substantial risk (e.g., at least a 10%, 20%, 30%, 40%, or 50% chance) of having IDC (e.g., within about 1, 2, 3, 4, 5, or 10 years).

Not to be bound by theory, it is hypothesized that there are two different epigenetic programs driving DCIS progression, and patients with the “invasion incompetent” signature are not at risk of developing invasive ductal cancer over time while patients with DCIS from the second epigenetic group having “invasion competent” signature will have a very high chance to develop invasive ductal cancer.

In embodiments, provided herein are epigenetic biomarkers based on DNA methylation that detect DCIS invasive state or potential and/or predict DCIS progression to an invasive state. In embodiments, the DNA methylation of cells in a sample obtained or provided from a subject is detected. In embodiments, the sample comprises DCIS-containing cancer cells isolated by a laser capture procedure from a biopsy. In embodiments, the sample comprises a DCIS cancer cell proliferation that is substantially free of normal cells. For example, at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more of the cells in the DCIS cancer cell proliferation are dysplastic cells (e.g., breast cancer cells such as DCIS cells). In embodiments, the DCIS cancer cell proliferation comprises cancer cells isolated by a laser capture procedure from a biopsy. In embodiments, the DCIS cancer cell proliferation is isolated by surgical excision followed by cancer cell purification by using a laser capture procedure. In embodiments, the DCIS cancer cell proliferation is isolated directly from a subject or from a sample that was previously obtained from the subject. In embodiments, the DCIS cancer cell proliferation is obtained or provided from a section of tissue that has been obtained or provided from the subject. In embodiments, the tissue comprises tissue from a mammographic abnormality or lump in the breast of a subject that comprises or is suspected of comprising breast cancer cells such as DCIS cells. In embodiments, the DCIS cancer cell proliferation is obtained or provided from a tissue section, e.g. a slice or section or a sample that has been stained for DNA and protein detection such as a slice or section that has been mounted on a slide. In embodiments, the DCIS cancer cell proliferation is isolated or captured from a paraffin-embedded sample slide using, e.g., a laser. In embodiments, DNA methylation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, 100, 125, or 140, or 1-50, 1-100, 1-140, 50-100, or 50-140 (inclusive) of the sites in Table 1 identifies the DCIS of a subject as IC-DCIS or II-DCIS.

Method of Detection Methylation Status of a Ductal Carcinoma DNA

In an aspect, provided herein is a method for detecting the presence, absence, and/or level of methylation at one or more sites set forth in Table 1. In embodiments, the level of methylation at a site set forth in Table 1 is detected. In embodiments, determining the level of methylation comprises bisulfite salt treatment, methyl-sensitive cut counting, and/or any method for a single base DNA methylation detection.

In embodiments, the present disclosure provides a method of detecting methylation or unmethylation of a breast lump DNA molecule of a subject, the method including: (i) isolating DNA from cancer cells obtained from a mammographic or Mill ductal abnormality, a breast nodule, a tumor, or a lump of said subject (e.g., a DCIS cancer cell proliferation or tissue) thereby forming an isolated DNA molecule, (ii) contacting said isolated DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DNA molecule, (iii) detecting the presence or absence of uracil in said reacted DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of said DNA molecule of said subject.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA of a subject. The method includes: (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule, (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule, (iii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated single stranded DNA.

In embodiments, the method of detecting methylation or unmethylation of a DCIS DNA molecule of a subject, includes determining alteration (e.g., compared to normal or II-DCIS cancer cell proliferation DNA) in methylation at a plurality of methylation sites set forth in Table 1.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with the bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a plurality of methylation sites set forth in Table 1, thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with the bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a plurality of methylation sites set forth in Table 1, thereby detecting methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. The method includes: (i) contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule, (ii) amplifying the reacted DCIS cancer cell proliferation DNA molecule thereby forming a reacted DCIS cancer cell proliferation DNA amplicon molecule, (iii) detecting the presence or absence of thymidine in a reacted DCIS cancer cell proliferation DNA amplicon molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated single stranded DNA.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. The method includes: (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule, (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule, (iii) amplifying the reacted DCIS cancer cell proliferation DNA molecule thereby forming a reacted DCIS cancer cell proliferation DNA amplicon molecule, (iv) detecting the presence or absence of thymidine in a reacted DCIS cancer cell proliferation DNA amplicon molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated single stranded DNA.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject comprising (i) contacting a plurality of isolated cancer DCIS cancer cell proliferation DNA molecules from a subject with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (ii) amplifying the plurality of reacted DCIS cancer cell proliferation DNA molecules thereby forming a plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules, (iii) detecting one or more DCIS cancer cell proliferation DNA amplicon molecules within the plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules having a thymidine at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of detecting methylation or unmethylation of a plurality of DCIS cancer cell proliferation DNA molecules of a subject comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfate salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) amplifying the plurality of reacted DCIS cancer cell proliferation DNA molecules thereby forming a plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules, (iv) detecting one or more DCIS cancer cell proliferation DNA amplicon molecules within the plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules having a thymidine at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In embodiments, detecting one or more DCIS cancer cell proliferation DNA amplicon molecules comprises detecting the level of one or more one or more DCIS cancer cell proliferation DNA amplicon molecules. In embodiments, detecting one or more DCIS cancer cell proliferation DNA amplicon molecules comprises detecting the level of reacted DCIS cancer cell proliferation DNA amplicon molecules in the plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules having a thymidine at a methylation site set forth in Table 1, thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In embodiments, detecting a level includes determining the number (e.g. quantitating) or molecules having, e.g., a thymidine or a uracil. In embodiments, detecting a level includes detecting the portion or proportion of a population or plurality of molecules having, e.g., a thymidine or a uracil.

In embodiments, the isolated DCIS cancer cell proliferation DNA sample is treated with a bisulfite reagent, e.g., a bisulfite salt (i.e., a process called DNA bisulfite conversion). Non-limiting examples of bisulfite salts include sodium bisulfite, potassium bisulfite, ammonium bisulfite, magnesium bisulfite, sodium metabisulfite, potassium metabisulfite, ammonium metabisulfite and magnesium metabisulfite. Bisulfite salts such as sodium bisulfite or ammonium bisulfite can convert cytosine to uracil and leave 5-methylcytosine (5-mC) the same. Thus after bisulfite treatment methylated cytosine in the DNA remains the same and unmodified cytosines will be changed to uracil. The bisulfate treatment can be performed by using the methods disclosed herein or in the art, and/or with commercial kits such as the BisulFlash DNA Modification Kit (EpiGentek) and Imprint DNA Modification Kit (Sigma). For achieving the optimal bisulfite conversion, the bisulfite reaction should be carried out in an appropriate concentration of bisulfite reagents, appropriate temperature and appropriate reaction time period. A reagent such as potassium chloride that reduces thermophilic DNA degradation could also be used in bisulfite treatment so that the DNA bisulfite process can be much shorter without interrupting a completed conversion of unmethylated cytosine to uracil and without a significant thermodegradation of DNA resulted from depurination. In embodiments, a commercially available bisulfite treatment kit is used. A non-limiting example of such a kit is EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, Calif., USA).

In embodiments, once DNA bisulfite conversion is complete, DNA is captured, desulphonated and washed. In embodiments, the bisulfite-treated DNA can be captured by, e.g., a solid matrix selected from silica salt, silica dioxide, silica polymers, magnetic beads, glass fiber, celite diatoms and nitrocellulose in the presence of high concentrations of chaotropic or non-chaotropic salts. In embodiments, the bisulfite-treated DNA is further desulphonated with an alkalized solution, preferably sodium hydroxide at concentrations from 10 mM to 300 mM. In embodiments, the DNA is then eluted and collected into a capped microcentrifuge tube. Non-limiting examples of elution solutions include DEPC-treated water and TE buffer (10 mM Tris-HCL, pH 8.0, and 1 mM EDTA).

In embodiments, the reacted DCIS cancer cell proliferation DNA resulting from bisulfite treatment is amplified. In embodiments, detecting the presence or absence of uracil in reacted DCIS cancer cell proliferation DNA molecule at a methylation site comprises amplifying the reacted DCIS cancer cell proliferation DNA molecule thereby forming a reacted DCIS cancer cell proliferation DNA amplicon molecule, and detecting the presence or absence of thymidine in a reacted DCIS cancer cell proliferation DNA amplicon molecule at the methylation site. In embodiments, a polymerase chain reaction (PCR) method is used for amplifying the reacted DCIS cancer cell proliferation DNA. PCR methods are known to those of ordinary skill in the art. In general, the PCR reactions can be set up by adding sample, dNTPs, and appropriate polymerase such as Taq polymerase, primers, and a buffer.

In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA of a subject, includes detecting methylation or unmethylation at a plurality of methylation sites set forth in Table 1. In embodiments, the plurality of methylation sites comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites comprises less than about 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. In embodiments, the plurality of methylation sites is about 2, 3, 4, 5, 10, 25, 50, 75, 80, 85, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites includes one, two, or more methylation sites set forth in Table 1 and no other methylation sites.

In embodiments, the method includes detecting methylation or unmethylation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 1 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 5 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 10 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 50 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 100 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of at least 120 of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

In embodiments, the method includes detecting methylation or unmethylation of each of the following sites: Chromosome 1 (Chr1) position 4714314, Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chromosome 3 (Chr3) position 14852857, Chr3 position 121903470, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chromosome 4 (Chr4) position 44449864, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chromosome 7 (Chr7) position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477232, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chromosome 12 (Chr12) position, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 95941925, Chr12 position 128750309, Chr12 position 129338355, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chromosome 19 (Chr19) position 30016170, Chr19 position 30017283, Chr19 position 30717013, Chr19 position 30719659, Chromosome 20 (Chr20) position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 23016002, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, and Chromosome 22 position 43807517.

Aspects provide a method for monitoring whether the risk of IDC in a subject who has DCIS. In embodiments, a method provided herein is practiced for a subject more than once over time to determine whether the DCIS is progressing from II-DCIS to IC-DCIS or from IC-DCIS to IDC. In embodiments, the methylation of one or more methylation sites in a subject becomes less indicative of II-DCIS over time. In embodiments, the methylation of one or more methylation sites in a subject becomes more indicative of IC-DCIS over time. In embodiments, methylation or unmethylation of DCIS cancer cell proliferation DNA from a subject is assessed using a method provided herein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In embodiments, the method is repeated at least once every 4, 6, 8, 12 or 18 months, or at least once every 2, 3, 4, or 5 more years.

In embodiments, the method includes: (i) isolating DNA from multiple cells of a DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) detecting the proportion of DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of said DCIS cancer cell proliferation DNA of the subject.

The methylation of a CpG site of interest may vary between individual cells (and even between chromosome pairs of individual cells) in a biological sample. When DNA is obtained from a biological sample and treated with a bisulfite salt to convert unmethylated cytosines to uracils, the bisulfite-treated DNA will typically contain (i) a proportion of DNA molecules with a cytosine at the site of interest (indicating that the site was methylated); and (ii) a proportion of DNA molecules with a uracil at the site of interest (indicating that the site was unmethylated). Since a uracil at a site of interest in bisulfite-treated DNA indicates that the site was unmethylated in the untreated DNA, a thymidine at the corresponding site in an amplicon of the bisulfite-treated DNA (e.g., an amplicon obtained by PCR) also indicates that the site was unmethylated in the untreated DNA.

In embodiments, the level of methylation at a site of interest is the proportion of bisulfite-treated DNA molecules having a cytosine rather than a uracil at that site of interest. In embodiments, the level of methylation at a site of interest is the proportion of amplicons of bisulfite-treated DNA molecules having a cytosine rather than a thymidine at that site of interest.

In embodiments, the level of unmethylation at a site of interest is the proportion of bisulfite-treated DNA molecules having a uracil rather than a cytosine at that site of interest. In embodiments, the level of unmethylation at a site of interest is the proportion of amplicons of bisulfite-treated DNA molecules having a thymidine rather than a cytosine at that site of interest. In Table 1, an indicated level of uracil is the proportion of bisulfite-treated DNA molecules having a uracil rather than a cytosine at the specified methylation site. The same levels listed in Table 1 also apply to the thymidine levels at a site of interest in an amplicon, i.e., the proportion of amplicons (derived from the PCR amplification of bisulfite-treated DNA molecules) having a thymidine rather than a cytosine at the specified methylation site.

The level of DNA methylation at a site of interest (e.g., a methylation site listed in Table 1) may be determined using sequencing technology. Sequencing technology can reveal nucleotide sequence variations in a plurality of DNA molecules at a single nucleotide base resolution. For example, the proportions of corresponding DNA molecules having a uracil, a thymidine, and/or a cytosine at a site may be determined. A non-limiting example of a sequencing-based method for determining the methylation level at a site of interest is described in Masser et al. (2015) Targeted DNA Methylation Analysis by Next-generation Sequencing, J Vis Exp. (96): 52488, the entire content of which is incorporated herein by reference.

The chromosomal positions listed in Tables 1-3 relate to the human genome that is publically accessible in the University of California Santa Cruz (UCSC) genome browser database under accession number HG19, the entire content of which is incorporated herein by reference in its entirety. Non-limiting information regarding the UCSC Genome Browser is provided in Kent W J, Sugnet C W, Furey T S, Roskin K M, Pringle T H, Zahler A M, Haussler D. The human genome browser at UCSC. Genome Res. 2002 June; 12(6):996-1006, the entire content of which is incorporated herein by reference. Each methylation site of interest listed in Table 1 may be located in other human genomes (e.g., within the genome of a specific subject or group of subjects) by replacing every U and R in the corresponding sequence with a C and then searching for the location of the X within a reference genome by aligning the sequence against the reference genome. For example, the methylation site of interest “X” in SEQ ID NO:1 may be located within a genome by replacing each U and R in SEQ ID NO: 1 with a C (to obtain the pre-bisulfite-modified sequence having an X at the site of interest) and then aligning the sequence against the genome using a BLAST algorithm. Also expressly provided, disclosed, and incorporated herein is the non-bisulfite-modified sequence corresponding to each of SEQ ID NOS: 1-242. The non-bisulfite-modified sequence corresponding to each of SEQ ID NOS:1-242 is each respective sequence in which each U and R is replaced with a C, where X is the methylation site of interest. For example, the non-bisulfite-modified sequence corresponding to SEQ ID NO:1 provided herein is a modified version of SEQ ID NO:1 in which each U and R in SEQ ID NO:1 is replaced with a C, where X is the methylation site of interest; the non-bisulfite-modified sequence corresponding to SEQ ID NO:2 provided herein is a modified version of SEQ ID NO:2 in which each U and R in SEQ ID NO:2 is replaced with a C, where X is the methylation site of interest; the non-bisulfite-modified sequence corresponding to SEQ ID NO:3 provided herein is a modified version of SEQ ID NO:3 in which each U and R in SEQ ID NO:3 is replaced with a C, where X is the methylation site of interest, and so on.

The chromosome positions listed herein correspond to the cytosine (of the CpG methylation site) that is methylated on the forward strand. However, the cytosine of the reverse complement of the forward strand's CpG methylation site may also or alternatively be methylated. At each chromosomal position provided, methylation may be determined for the forward strand, the reverse strand, or both the forward strand and the reverse strand. In embodiments, each chromosomal position listed herein may refer to (i) the cytosine of the methylation site on the forward strand, (ii) the corresponding cytosine on the reverse strand of the methylation site, or (iii) both the cytosine of the methylation site on the forward strand and the corresponding cytosine on the reverse strand of the methylation site.

TABLE 1

Uracil level
Uracil level

in invasive-
in invasive-
SEQ
SEQ

competent DCIS
competent DCIS
ID NO:
ID NO:

Chromosomal
is about above
is about below
Forward
Reverse

Site
Chromosome
position
indicated level*
indicated level*
Strand
Strand

1
chr1
4714314
N/A
74.32
1
2

2
chr1
11413742
76.09
N/A
3
4

3
chr1
39957798
N/A
63.81
5
6

4
chr1
46951513
N/A
85.11
7
8

5
chr1
47904912
N/A
73.08
9
10

6
chr1
62660691
N/A
71.20
11
12

7
chr1
63785800
N/A
46.94
13
14

8
chr1
67600465
N/A
42.86
15
16

9
chr1
91183172
N/A
48.28
17
18

10
chr1
166853786
N/A
64.29
19
20

11
chr1
179545096
N/A
88.89
21
22

12
chr1
207669851
N/A
89.29
23
24

13
chr1
237205704
N/A
82.61
25
26

14
chr1
237205705
N/A
78.57
25
26

15
chr1
240161215
N/A
60.00
27
28

16
chr1
240934954
78.57
N/A
29
30

17
chr10
8076277
N/A
75.00
31
32

18
chr10
50818610
N/A
76.30
33
34

19
chr10
77157527
N/A
50.43
35
36

20
chr10
123778639
74.49
N/A
37
38

21
chr10
123778640
79.31
N/A
37
38

22
chr10
124902829
N/A
67.35
39
40

23
chr10
124909545
N/A
80.00
41
42

24
chr10
130085373
N/A
76.74
43
44

25
chr10
134598235
N/A
34.43
45
46

26
chr10
134599080
N/A
79.31
47
48

27
chr11
1215978
74.47
N/A
49
50

28
chr11
9025912
N/A
65.85
51
52

29
chr11
15963013
N/A
51.72
53
54

30
chr11
66187593
N/A
80.95
55
56

31
chr11
71318977
N/A
51.52
57
58

32
chr11
101453451
N/A
57.89
59
60

33
chr12
49726711
N/A
60.00
61
62

34
chr12
50297756
N/A
69.81
63
64

35
chr12
50297763
N/A
82.98
63
64

36
chr12
50297768
N/A
81.13
63
64

37
chr12
50297774
N/A
75.47
63
64

38
chr12
50297776
N/A
77.36
63
64

39
chr12
50444766
N/A
54.17
65
66

40
chr12
75601447
N/A
52.00
67
68

41
chr12
95941925
N/A
36.84
69
70

42
chr12
128750309
83.33
N/A
71
72

43
chr12
129338355
N/A
57.14
73
74

44
chr12
129338471
N/A
60.71
75
76

45
chr13
28502190
N/A
65.85
77
78

46
chr13
79181509
N/A
79.84
79
80

47
chr13
92051154
N/A
70.70
81
82

48
chr13
95363553
N/A
73.91
83
84

49
chr13
95363592
N/A
78.95
83
84

50
chr14
29236052
N/A
72.36
85
86

51
chr14
29236065
N/A
62.60
85
86

52
chr14
101543886
N/A
92.45
87
88

53
chr15
29407958
N/A
69.84
89
90

54
chr15
45403826
N/A
73.68
91
92

55
chr15
76630094
N/A
62.30
93
94

56
chr15
89951787
N/A
39.68
95
96

57
chr16
1255253
84.62
N/A
97
98

58
chr17
3211643
82.35
N/A
99
100

59
chr17
30244229
70.45
N/A
101
102

60
chr17
35294171
N/A
71.43
103
104

61
chr17
64831307
N/A
56.52
105
106

62
chr17
74136562
N/A
43.48
107
108

63
chr17
74865566
N/A
62.79
109
110

64
chr18
19745047
N/A
67.01
111
112

65
chr18
19745054
N/A
64.10
111
112

66
chr18
19747206
N/A
80.00
113
114

67
chr18
44774403
N/A
68.29
115
116

68
chr18
55103840
N/A
66.20
117
118

69
chr18
55106910
N/A
50.00
119
120

70
chr18
70534832
N/A
66.67
121
122

71
chr18
72880039
56.52
N/A
123
124

72
chr18
77547934
N/A
33.33
125
126

73
chr19
30016170
N/A
38.89
127
128

74
chr19
30017283
N/A
60.00
129
130

75
chr19
30717013
N/A
54.24
131
132

76
chr19
30719659
N/A
66.67
133
134

77
chr2
20870821
N/A
12.20
135
136

78
chr2
45156764
N/A
34.25
137
138

79
chr2
74743346
N/A
34.69
139
140

80
chr2
80549703
N/A
28.21
141
142

81
chr2
95989474
84.21
N/A
143
144

82
chr2
105471544
N/A
63.64
145
146

83
chr2
115919663
N/A
64.44
147
148

84
chr2
115920004
N/A
79.31
149
150

85
chr2
118982006
N/A
47.06
151
152

86
chr2
177001540
N/A
63.16
153
154

87
chr20
1294019
N/A
55.43
155
156

88
chr20
3073503
N/A
62.96
157
158

89
chr20
10198305
N/A
62.44
159
160

90
chr20
23015989
N/A
80.70
161
162

91
chr20
23016002
N/A
65.59
161
162

92
chr20
26189258
N/A
55.56
163
164

93
chr20
48626669
N/A
36.00
165
166

94
chr20
53092916
N/A
72.13
167
168

95
chr20
59827619
N/A
58.82
169
170

96
chr20
59828325
N/A
76.47
171
172

97
chr21
9825842
N/A
37.32
173
174

98
chr21
9826150
N/A
50.00
175
176

99
chr21
9826934
N/A
53.36
177
178

100
chr22
43807517
N/A
40.68
179
180

101
chr3
14852857
N/A
57.54
181
182

102
chr3
121903470
N/A
71.96
183
184

103
chr3
170303393
N/A
68.63
185
186

104
chr3
170303422
N/A
67.65
185
186

105
chr3
170303423
N/A
81.82
185
186

106
chr3
170303424
N/A
56.52
185
186

107
chr3
170303425
N/A
69.77
185
186

108
chr4
44449864
N/A
78.38
187
188

109
chr4
54976099
N/A
72.00
189
190

110
chr4
56023880
N/A
55.10
191
192

111
chr5
71014951
N/A
65.00
193
194

112
chr5
72677229
N/A
80.82
195
196

113
chr5
87981177
N/A
55.26
197
198

114
chr5
140743998
N/A
71.83
199
200

115
chr5
178421786
N/A
56.25
201
202

116
chr6
41337153
N/A
57.25
203
204

117
chr6
85484102
N/A
68.18
205
206

118
chr6
157557787
N/A
55.56
207
208

119
chr6
160769248
N/A
75.51
209
210

120
chr7
1282082
N/A
70.45
211
212

121
chr7
32467637
N/A
31.03
213
214

122
chr7
71801896
N/A
63.89
215
216

123
chr7
71801905
N/A
63.89
215
216

124
chr7
100946148
N/A
55.17
217
218

125
chr7
100946151
N/A
43.66
217
218

126
chr7
121957003
N/A
75.00
219
220

127
chr7
150038502
N/A
84.83
221
222

128
chr7
157477232
N/A
26.45
223
224

129
chr7
157477399
N/A
71.43
225
226

130
chr7
157477401
N/A
92.59
225
226

131
chr8
9764011
N/A
70.91
227
228

132
chr8
11566080
N/A
85.51
229
230

133
chr8
11566102
N/A
65.22
229
230

134
chr8
11566125
N/A
65.22
229
230

135
chr8
56015232
N/A
53.85
231
232

136
chr8
65281933
N/A
95.00
233
234

137
chr8
145105472
N/A
44.93
235
236

138
chr9
126780185
N/A
85.19
237
238

139
chr9
127239956
N/A
66.96
239
240

140
chr9
140772369
N/A
84.62
241
242

*Level values provided are the proportion (percentage) of reacted DCIS cancer cell proliferation DNA molecules having a uracil at the methylation site of interest. When amplicons generated from reacted DCIS cancer cell proliferation DNA molecules (e.g., by PCR) are used to assess the level of methylation, the values provided correspond to the proportion of amplicons having a thymidine at the nucleotide position that corresponds to the methylation site of interest.

In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA of a subject detects an alteration in methylation including increase or loss of uracil level at one methylation site or a plurality of methylation sites. In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA of a subject detects an alteration in methylation including increase or loss of thymidine level at plurality of methylation sites. In embodiments, the indicated levels in Tables 1, 2, and 3 are approximate indicated levels, and include values that are within about 15%, about 10%, or about 5% above and below the indicated levels.

In embodiments, the uracil level is above a threshold as set forth in Table 2 in subjects with invasive competent DCIS cancer cell proliferations. In embodiments, the thymidine level is above a threshold as set forth in Table 2 in subjects with invasive competent DCIS cancer cell proliferations. In embodiments, the uracil level is below a threshold as set forth in Table 2 in subjects with invasive incompetent DCIS cancer cell proliferations. In embodiments, the thymidine level is below a threshold as set forth in Table 2 in subjects with invasive incompetent DCIS cancer cell proliferations. In embodiments, subject with invasive competent DCIS are identified as at risk of IDC. In embodiments, subject with invasive incompetent DCIS are identified as not at risk of IDC.

TABLE 2

Methylation threshold for invasive competent DCIS

Uracil level in invasive

Chromosomal
competent DCIS is about

Chromosome
position
above indicated level*

chr1
11413742
76.09

chr1
240934954
78.57

chr10
123778639
74.49

chr10
123778640
79.31

chr11
1215978
74.47

chr12
128750309
83.33

*Level values provided are the proportion (percentage) of reacted DCIS cancer cell proliferation DNA molecules having a uracil at the methylation site of interest. When amplicons generated from reacted DCIS cancer cell proliferation DNA molecules (e.g., by PCR) are used to assess the level of methylation, the values provided correspond to the proportion of amplicons having a thymidine at the nucleotide position that corresponds to the methylation site of interest.

In embodiments, the uracil level is above a threshold as set forth in Table 3 in subjects with invasive incompetent DCIS cancer cell proliferations. In embodiments, the thymidine level is above a threshold as set forth in Table 3 in subjects with invasive incompetent DCIS cancer cell proliferations. In embodiments, the uracil level is below a threshold as set forth in Table 3 in subjects with invasive competent DCIS cancer cell proliferations. In embodiments, the thymidine level is below a threshold as set forth in Table 3 in subjects with invasive competent DCIS cancer cell proliferations.

TABLE 3

Methylation threshold for invasive-incompetent DCIS

Uracil level in invasive-

Chromosomal
competent DCIS is about

Chromosome
position
below indicated level*

chr1
4714314
74.32

chr1
39957798
63.81

chr1
46951513
85.11

chr1
47904912
73.08

chr1
62660691
71.20

chr1
63785800
46.94

chr1
67600465
42.86

chr1
91183172
48.28

chr1
166853786
64.29

chr1
179545096
88.89

chr1
207669851
89.29

chr1
237205704
82.61

chr1
237205705
78.57

chr1
240161215
60.00

chr10
8076277
75.00

chr10
50818610
76.30

chr10
77157527
50.43

chr10
124902829
67.35

chr10
124909545
80.00

chr10
130085373
76.74

chr10
134598235
34.43

chr10
134599080
79.31

chr11
9025912
65.85

chr11
15963013
51.72

chr11
66187593
80.95

chr11
71318977
51.52

chr11
101453451
57.89

chr12
49726711
60.00

chr12
50297756
69.81

chr12
50297763
82.98

chr12
50297768
81.13

chr12
50297774
75.47

chr12
50297776
77.36

chr12
50444766
54.17

chr12
75601447
52.00

chr12
95941925
36.84

chr12
129338355
57.14

chr12
129338471
60.71

chr13
28502190
65.85

chr13
79181509
79.84

chr13
92051154
70.70

chr13
95363553
73.91

chr13
95363592
78.95

chr14
29236052
72.36

chr14
29236065
62.60

chr14
101543886
92.45

chr15
29407958
69.84

chr15
45403826
73.68

chr15
76630094
62.30

chr15
89951787
39.68

chr17
35294171
71.43

chr17
64831307
56.52

chr17
74136562
43.48

chr17
74865566
62.79

chr18
19745047
67.01

chr18
19745054
64.10

chr18
19747206
80.00

chr18
44774403
68.29

chr18
55103840
66.20

chr18
55106910
50.00

chr18
70534832
66.67

chr18
77547934
33.33

chr19
30016170
38.89

chr19
30017283
60.00

chr19
30717013
54.24

chr19
30719659
66.67

chr2
20870821
12.20

chr2
45156764
34.25

chr2
74743346
34.69

chr2
80549703
28.21

chr2
105471544
63.64

chr2
115919663
64.44

chr2
115920004
79.31

chr2
118982006
47.06

chr2
177001540
63.16

chr20
1294019
55.43

chr20
3073503
62.96

chr20
10198305
62.44

chr20
23015989
80.70

chr20
23016002
65.59

chr20
26189258
55.56

chr20
48626669
36.00

chr20
53092916
72.13

chr20
59827619
58.82

chr20
59828325
76.47

chr21
9825842
37.32

chr21
9826150
50.00

chr21
9826934
53.36

chr22
43807517
40.68

chr3
14852857
57.54

chr3
121903470
71.96

chr3
170303393
68.63

chr3
170303422
67.65

chr3
170303423
81.82

chr3
170303424
56.52

chr3
170303425
69.77

chr4
44449864
78.38

chr4
54976099
72.00

chr4
56023880
55.10

chr5
71014951
65.00

chr5
72677229
80.82

chr5
87981177
55.26

chr5
140743998
71.83

chr5
178421786
56.25

chr6
41337153
57.25

chr6
85484102
68.18

chr6
157557787
55.56

chr6
160769248
75.51

chr7
1282082
70.45

chr7
32467637
31.03

chr7
71801896
63.89

chr7
71801905
63.89

chr7
100946148
55.17

chr7
100946151
43.66

chr7
121957003
75.00

chr7
150038502
84.83

chr7
157477232
26.45

chr7
157477399
71.43

chr7
157477401
92.59

chr8
9764011
70.91

chr8
11566080
85.51

chr8
11566102
65.22

chr8
11566125
65.22

chr8
56015232
53.85

chr8
65281933
95.00

chr8
145105472
44.93

chr9
126780185
85.19

chr9
127239956
66.96

chr9
140772369
84.62

*Level values provided are the proportion (percentage) of reacted DCIS cancer cell proliferation DNA molecules having a uracil at the methylation site of interest. When amplicons generated from reacted DCIS cancer cell proliferation DNA molecules (e.g., by PCR) are used to assess the level of methylation, the values provided correspond to the proportion of amplicons having a thymidine at the nucleotide position that corresponds to the methylation site of interest.

In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA is of a candidate breast cancer patient. In embodiments, the subject is suspected of having IDC, invasive competent DCIS, invasive incompetent DCIS, or DCIS. In embodiments, the subject has IDC, invasive competent DCIS, invasive incompetent DCIS, or DCIS.

In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA is based on the level of uracil as set forth Table 2, in which the uracil level above the threshold identifies the DCIS cancer cell proliferation as invasive competent. In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA is based on a level of thymidine indicated in Table 2, in which the thymidine level above the threshold identifies the DCIS cancer cell proliferation as invasive competent. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method. Non-limiting examples of quantitation methods include sequencing and microarray methods.

In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA is based on the level of uracil as set forth Table 3, in which the uracil level above the threshold identifies the DCIS cancer cell proliferation as invasive incompetent. In embodiments, the method of detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA is based on a level of thymidine indicated in Table 3, in which the thymidine level above the threshold identifies the DCIS cancer cell proliferation as invasive incompetent. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the DCIS cancer cell proliferation is a specimen obtained by laser capture procedure from a biopsy or from surgical resection of a subject.

In embodiments, the subject has undergone lumpectomy or mastectomy, radiation therapy, chemotherapy, and administration of an active agent before the subject undergoes the method of detecting methylation or unmethylation of a breast cancer DNA of the present disclosure.

In embodiments, the method of the present disclosure includes a determination of prognosis for invasive ductal carcinoma.

In embodiments, the method of detecting DNA methylation level in DNA of breast lump may lead to changes in therapeutic regimen for treating the subject. In embodiments, a subject identified as having invasive competent DCIS with a method of the present disclosure may be treated with lumpectomy, mastectomy, radiation therapy, chemotherapy, hormonal therapy (such as but not limited to tamoxifen), or targeted therapy (such as but not limited to trastuzumab and everolimus), or a combination thereof. In embodiments, a subject identified as having invasive incompetent DCIS with a method of the present disclosure is not treated with lumpectomy, mastectomy, radiation therapy, chemotherapy, hormonal therapy, targeted therapy, or a combination thereof.

In embodiments, the active agent administered to a subject before or after detecting the level of methylation or unmethylation is: trastuzumab (e.g., Herceptin®), trastuzumab emtansine (e.g., Kadcyla®), lapatinib (e.g., Tykerb®), pertuzumab (e.g., Perjeta®), bevacizumab (e.g., Avastin®), tamoxifen (e.g., Nolvadex®), exemestane Aromasin®), anastrozole Arimidex), letrozole (e.g., Femara®), doxorubicin (e.g., Adriamycin®), epirubicin (e.g., Ellence®), cyclophosphamide (e.g., Cytoxan®), docetaxel (e.g., Taxotere®), paclitaxel (e.g., Taxol®), nab paclitaxel (e.g., Abraxane®), eribulin (e.g., Halaven®), everolimus (e.g., Afinitor®), palbociclib (e.g., Ibrance®), capecitabine (e.g., Xeloda®), ixabepilone (e.g., Ixempra®), methotrexate (e.g., Trexall®), or fluorouracil (also called 5-fluorouracil or 5-FU; e.g., Adrucil®).

Method of Determining Breast Cancer or Risk of Developing Invasive Ductal Carcinoma In Situ

Aspects further provide a method of detecting a risk of developing IDC in a subject who has DCIS. In embodiments, the method comprises

- (i) contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and
- (ii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting the risk for developing IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject. The method involves:

- (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule;
- (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and
- (iii) detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1; thereby diagnosing IDC or detecting risk of IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof, comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with the bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1; thereby diagnosing IDC or detecting risk of IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof, comprising (i) contacting a plurality of isolated DCIS cancer cell proliferation DNA molecules from the subject with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; and (ii) detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a plurality of methylation sites set forth in Table 1, thereby detecting the risk for IDC in the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof, comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with the bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) detecting the presence or absence of uracil in a reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the plurality DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof. The method includes: (i) contacting an isolated DCIS cancer cell proliferation DNA molecule from said subject with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule, (ii) amplifying the reacted DCIS cancer cell proliferation DNA molecule thereby forming a reacted DCIS cancer cell proliferation DNA amplicon molecule, (iii) detecting the presence or absence of thymidine in a reacted DCIS cancer cell proliferation DNA amplicon molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated single stranded DNA.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof. The method includes: (i) isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule, (ii) contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule, (iii) amplifying the reacted DCIS cancer cell proliferation DNA molecule thereby forming a reacted DCIS cancer cell proliferation DNA amplicon molecule, (iv) detecting the presence or absence of thymidine in a reacted DCIS cancer cell proliferation DNA amplicon molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated single stranded DNA.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof, comprising (i) contacting a plurality of isolated DCIS cancer cell proliferation DNA molecules from said subject with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) amplifying the plurality of reacted DCIS cancer cell proliferation DNA molecules thereby forming a plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules, (iv) detecting one or more DCIS cancer cell proliferation DNA amplicon molecules within the plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules having a thymidine at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In an aspect, provided herein is a method of diagnosing IDC or detecting risk of IDC in a subject in need thereof, comprising (i) isolating a plurality of DCIS cancer cell proliferation DNA molecules from the DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) amplifying the plurality of reacted DCIS cancer cell proliferation DNA molecules thereby forming a plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules, (iv) detecting one or more DCIS cancer cell proliferation DNA amplicon molecules within the plurality of reacted DCIS cancer cell proliferation DNA amplicon molecules having a thymidine at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the plurality of DCIS cancer cell proliferation DNA molecules of the subject.

In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated DCIS cancer cell proliferation DNA.

In embodiments, the method of determining diagnosing IDC or detecting risk of IDC in a subject includes selecting a subject that has or is at risk for having or developing diagnosing IDC or detecting risk of IDC. In embodiments, the subject (a) is a woman; (b) is about 30 to about 75 years old; (c) has at least one mutant breast cancer 1 (BRCA1), breast cancer 2 (BRCA2), Partner and localizer of BRCA2 (PALB2), phosphatase and tensin homolog (PTEN), or p53 allele; (d) has a parent, sibling, or child who has been diagnosed with breast cancer; (e) has had the non-cancerous breast diseases atypical ductal hyperplasia or lobular carcinoma in situ; (f) has had previous radiation treatment to the chest or a breast before the age of 30; (g) has received a combination hormone therapy with estrogen and progestin for at least five years; and/or (h) has or has had breast cancer.

In embodiments, the method includes detecting methylation or unmethylation at a plurality of methylation sites set forth in Table 1. In embodiments, the plurality of methylation sites comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites comprises less than about 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. In embodiments, the plurality of methylation sites is about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites includes one, two, or more methylation sites set forth in Table 1 and no other methylation sites.

In embodiments, a method provided herein is practiced for a subject more than once over time. In embodiments, methylation or unmethylation of DCIS cancer cell proliferation DNA from a subject is assessed using a method provided herein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In embodiments, the method is repeated at least once every 4, 6, 8, 12 or 18 months, or at least once every 2, 3, 4, or 5 more years.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining alteration in methylation at a plurality of methylation sites set forth in Table 1. In embodiments, the method comprises: (i) isolating DNA from multiple cells of a DCIS cancer cell proliferation of the subject thereby forming a plurality of isolated DCIS cancer cell proliferation DNA molecules, (ii) contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfate salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules, (iii) detecting the proportion of DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes alteration, i.e., increase or loss of uracil level at plurality of methylation sites. In embodiments, the method of determining a diagnosing IDC or detecting risk of IDC in a subject in need thereof includes alteration, i.e., increase or loss of thymidine level at plurality of methylation sites.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a uracil level which is above a threshold as set forth in Table 2. In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a thymidine level which is above a threshold indicated in Table 2. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a uracil level which is below a threshold as set forth in Table 3. In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a thymidine level which is below a threshold indicated in Table 3. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC involves a candidate IDC patient. In embodiments, the subject is suspected of having IDC or IC-DCIS. In embodiments, the subject has IC-DCIS.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a uracil level in which a threshold above the threshold set forth in Table 2 identifies the DCIS cancer cell proliferation as IC-DCIS or IDC. In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a thymidine level in which a threshold above the threshold indicated in Table 2 identifies the DCIS cancer cell proliferation as a IC-DCIS or IDC. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a uracil level in which a threshold above the threshold set forth in Table 3 identifies the DCIS cancer cell proliferation as II-DCIS. In embodiments, the method of determining a diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a thymidine level in which a threshold above the threshold indicated in Table 3 identifies the DCIS cancer cell proliferation as II-DCIS. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a uracil level in DNA of a DCIS cancer cell proliferation specimen obtained by biopsy or by surgical resection of a subject. In embodiments, the method of diagnosing IDC or detecting risk of IDC in a subject in need thereof includes determining a thymidine level in DNA of a DCIS cancer cell proliferation specimen obtained by biopsy or by surgical resection of a subject.

Aspects provide methods of determining a prognosis or selecting a treatment for a subject comprising detecting IDC or a risk of IDC in the subject. In embodiments, the subject has previously undergone treatment for breast cancer. In embodiments, the subject was in remission after the previous treatment for breast cancer. In embodiments, breast cancer tissue has been resected from the subject. In embodiments, breast cancer tissue has been resected from the subject, but the subject is not in remission for breast cancer.

In embodiments, the method of diagnosing IDC or detecting risk of IDC may lead to a prognostic assessment of the subject. In embodiments, the method of diagnosing IDC or detecting risk of IDC may lead to a change or a particular choice or set of choices in the therapeutic regimen for treating the subject. For example, a diagnosis of IDC of risk of IDC may lead to a different treatment regimen (such as surgery, hormone therapy, radiation therapy, chemotherapy, targeted therapy, or a combination thereof) compared to a subject who is not diagnosed with IDC or a risk of IDC (such as monitoring or a treatment other than surgery, hormone therapy, radiation therapy, chemotherapy, or targeted therapy). In embodiments, a method of diagnosing IDC or detecting risk of IDC may lead to a determination of susceptibility (i.e., likelihood to respond) to treatment such as surgery, hormone therapy, radiation therapy, chemotherapy, targeted therapy, or a combination thereof. In embodiments a subject identified as having IDC or IDC risk may be treated with surgery, hormone therapy, radiation therapy, chemotherapy, targeted therapy, or any combination thereof. In embodiments, a subject identified as having IDC or being at risk of developing IDC according to a method disclosed herein is advised and/or directed to receive additional screening and/or treatment for breast cancer.

In embodiments, a subject having IDC or at risk of developing IDS is administered an active agent such as trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.

In embodiments, the method of determining an invasive ductal carcinoma may lead to changes in therapeutic regimen (e.g., treatment and/or dose) for treating the subject. In embodiments a subject identified as having IDC with a method disclosed herein may be treated with trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.

In embodiments, the active agent administered to a subject after determining IDC is: trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.

Method of Treating Breast Cancer

The present disclosure provides a method of treating breast cancer in a subject by administering to the subject an active agent for treating breast cancer (such as IDC or IC-DCIS), in which the subject is identified for treatment by a method including contacting an isolated breast cellular proliferation DNA with sodium bisulfite thereby forming a reacted breast cellular proliferation DNA; and detecting the presence or absence of uracil in the reacted breast cellular proliferation DNA at a methylation site set forth in Table 1; thereby determining ductal carcinoma invasion in the subject. In embodiments, the DCIS is not present as a mass or a lump. In embodiments, the DCIS is found as a mammographic abnormality, such as a microcalcification in a ductal pattern.

In an aspect, provided herein is a method of treating breast cancer in a subject by administering to the subject an active agent for treating breast cancer (such as IDC or IC-DCIS), in which the subject is identified for treatment by a method including isolating DNA from a breast cellular proliferation of the subject thereby forming isolated breast cellular proliferation DNA; contacting the isolated breast cellular proliferation DNA with sodium bisulfite thereby forming a reacted breast cellular proliferation DNA; and detecting the presence or absence of uracil in the reacted breast cellular proliferation DNA at a methylation site set forth in Table 1; thereby determining ductal carcinoma invasion in the subject.

In embodiments, the method of treating a ductal carcinoma in a subject in need thereof includes determining alteration in methylation at a plurality of methylation sites set forth in Table 1.

In embodiments, the method of treating a ductal carcinoma in a subject in need thereof includes alteration which includes increase or loss of uracil level at plurality of methylation sites.

In an aspect, included herein is a method of treating breast cancer in a subject by administering to the subject an active agent for treating breast cancer, in which the subject is identified for treatment by a method including contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1; thereby detecting the breast cancer in the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated DCIS cancer cell proliferation DNA.

Also provided herein is a method of treating breast cancer in a subject by administering to the subject an active agent for treating breast cancer, in which the subject is identified for treatment by a method including isolating a DCIS cancer cell proliferation DNA molecule from a DCIS cancer cell proliferation of the subject thereby forming an isolated DCIS cancer cell proliferation DNA molecule; contacting the isolated DCIS cancer cell proliferation DNA molecule with a bisulfite salt (such as sodium bisulfite) thereby forming a reacted DCIS cancer cell proliferation DNA molecule; and detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1; thereby detecting the breast cancer in the subject. In embodiments, contacting the isolated DCIS cancer cell proliferation DNA with a bisulfite salt comprises adding a solution comprising the bisulfite salt to a solution comprising the isolated DCIS cancer cell proliferation DNA.

In embodiments, the method includes detecting methylation or unmethylation at a plurality of methylation sites set forth in Table 1. In embodiments, the plurality of methylation sites comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites comprises less than about 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 methylation sites. In embodiments, the plurality of methylation sites is about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites includes two or more methylation sites set forth in Table 1 and no other methylation sites.

In embodiments, the method of treating breast cancer in a subject in need thereof includes determining alteration in methylation at a plurality of methylation sites set forth in Table 1.

In embodiments, the method of treating breast cancer in a subject in need thereof includes alteration which includes increase or loss of uracil level at plurality of methylation sites.

In embodiments, the method of treating breast cancer in a subject in need thereof includes determining a uracil level which is above a threshold as set forth in Table 2. In embodiments, the method of treating breast cancer in a subject in need thereof includes determining a thymidine level which is above a threshold indicated in Table 2. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of treating breast cancer in a subject in need thereof includes determining a uracil level which is below a threshold as set forth in Table 3. In embodiments, the method of treating breast cancer in a subject in need thereof includes determining a thymidine level which is below a threshold indicated in Table 3. In embodiments, the level is the proportion of molecules (e.g., in a plurality of reacted DCIS cancer cell proliferation DNA molecules or a plurality of reacted DCIS cancer cell proliferation DNA amplicons) having a uracil or thymidine as determined by a quantitation method.

In embodiments, the method of treating breast cancer includes administering surgery, radiation therapy, chemotherapy, targeted therapy, or hormone therapy, before the detection of IDC.

In embodiments, the method of treating breast cancer includes administering an active agent such as trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil, before the detection of IDC.

Target Sites for Methylation Level of Breast Lump

In embodiments, the present disclosure includes a deoxyribonucleic acid 5 to 100 nucleotides in length including a uracil-containing sequence identical to at least a 5 contiguous nucleotide sequence within a sequence including SEQ ID NO:1 to SEQ ID NO: 242.

Included herein are about 300 bp length sequences which are surrounding the target sites (e.g., 149 or 150 bp from each site). The sequences are after bisulfite conversion. Therefore “C” in the non-CpG context becomes “U”, and C in the CpG context is designated as R (either “U” either “C”). The DNA strands (sense and antisense) are no longer complementary after bisulfite conversion. Therefore, each DNA strand are identified here with their unique sequence, and designated as “forward” and “reverse”, respectively.

In embodiments, the present disclosure includes a DNA molecule which includes a methylation site set forth in Table 1.

Also provided herein is a DNA molecule comprising a nucleotide sequence that is identical to 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 of contiguous nucleotide sequence of the sequence including a sequence of SEQ ID NO:1 to SEQ ID NO: 242. Also included is a plurality of such DNA molecules.

In embodiments, included herein is a plurality of DNA molecules comprising methylation sites set forth in Table 1 are methylated or unmethylated. A plurality of bisulfite-converted DNA molecules comprising methylation sites set forth in Table 1 are also included. In embodiments, the plurality of methylation sites comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 80, 90, 100, 110, 120, 130, or 140 methylation sites. In embodiments, the plurality of methylation sites comprises between 1-50, 50-100, 100-250, 100-300, 100-400, 100-500, 100-550, 250-550, or 350-500 methylation sites (e.g., methylation sites included herein and others). In embodiments, the plurality of methylation sites does not comprise a methylation site other than the sites listed in Table 1.

SEQ ID NO:1 to SEQ ID NO:242 are sequences that include the target sites (i.e., methylation sites of interest). The sequences provided are as modified after bisulfite conversion. Therefore “C” in the non-CpG context becomes “U”, and C in the CpG context is designated as R or X (either “U” either “C”), where X is the target site. The DNA strands (sense and antisense) are no longer complementary after bisulfite conversion. Therefore, each DNA strand is identified with its unique sequence, and is designated as “forward” and “reverse” respectively, in Table 1.

The sequences listed in Table 1 are provided below with their respective sequence identification number.

TABLE 4

Sequences Listed in Table 1

SEQ ID

NO:
Sequence

1
GGUURGRGTGGUTUAGUTURGUUUUAGGAAAUTTTTRGRGUAGUUUURGUTUUATGRG

UUUUUAUUUAGTUUTTUTTRGGGGURGUUUUUTUUUUAAAGTAGUTUTURGGGTUUAU

TGGGRGUUUURGTAAURGGGTRGGAAUUTRGAAXGGUTTRGRGTGUUATURGGTTAUU

UTGGUAAUAUUATURGGRGGRGUURGGRGGTUUAATUUTAGUUTGRGGUUTUTUTRGA

GUUTTTRGUAAGGTGGGGAURGGGARGRGAURGGGGATGGGGAAGGGGGUTGUAGGAG

GUUURGUUTG

2
UAGGRGGGGUUTUUTGUAGUUUUUTTUUUUATUUURGGTRGRGTUURGGTUUUUAUUT

TGRGAAAGGUTRGAGAGAGGURGUAGGUTAGGATTGGAURGURGGGRGURGURGGATG

GTGTTGUUAGGGTAAURGGATGGUARGRGAAGUXGTTRGAGGTTURGAUURGGTTARG

GGGGRGUUUAGTGGAUURGGAGAGUTAUTTTGGGGAGGGGGRGGUUURGAAGAAGGA

UTGGGTGGGGGRGUATGGAGRGGGGGUTGRGRGAAAAGTTTUUTGGGGRGGAGUTGAG

UUARGRGGGUU

3
TTTTUTTTTTUTTTTUTTTTTTUUTTAAGGUAGAGTUTGGTTUTGTRGUUUAGAGAGRGGT

AAUARGATUTUAGUTUAUTGUAAUUTUTGUUTUURGGGTTUAAGUAATTTTUATGUUTU

AGGUTUUTGAGTAGUTGGAGTTAUAGGUAXGTGUTAUUATGUURGGUTAATGTTTTGTA

TTTTTAGTAGAGAUAGGGTTTUAUUATGTTGGUTGGGUTGGTUTTGAAUTUUUTAUUTU

AAGRGATUTGUTUAUUTUAGAUTUUUAAAATGUTGGGATTAUAGGTGTGAGUUAUUAU

AUTG

4
UAGTGTGGTGGUTUAUAUUTGTAATUUUAGUATTTTGGGAGTUTGAGGTGAGUAGATRG

UTTGAGGTAGGGAGTTUAAGAUUAGUUUAGUUAAUATGGTGAAAUUUTGTUTUTAUTA

AAAATAUAAAAUATTAGURGGGUATGGTAGUAXGTGUUTGTAAUTUUAGUTAUTUAGG

AGUUTGAGGUATGAAAATTGUTTGAAUURGGGAGGUAGAGGTTGUAGTGAGUTGAGAT

RGTGTTAURGUTUTUTGGGRGAUAGAAUUAGAUTUTGUUTTAAGGAAAAAAGAAAAGA

AAAAGAAAA

5
RGUUUURGGUURGUUATGGURGRGRGUUURGGAURGUTUTGGUTTUTGGGUUTGARGT

TGTGRGRGUTGGGRGGGGGRGGUUURGGUUTGRGAUUUURGUURGGUTGTUUUUAGRG

ARGTUTGGGRGRGRGRGAGRGURGGGARGTGUAGXGRGAGATUUTGGRGGTGUTRGGG

UTAUURGGGRGGUUURGGUUURGRGRGUUAUURGURGUUTUURGGUTGUURGRGTURG

RGURGUTUTTUATGUTGGAUUTGTAUUARGUUATGGUTGGRGARGARGARGAGGARGG

RGRGUURGRG

6
RGRGGGRGRGURGTUUTRGTRGTRGTRGUUAGUUATGGRGTGGTAUAGGTUUAGUATGA

AGAGRGGRGRGGARGRGGGUAGURGGGAGGRGGRGGGTGGRGRGRGGGGURGGGGUR

GUURGGGTAGUURGAGUAURGUUAGGATUTRGXGUTGUARGTUURGGRGUTRGRGRGR

GUUUAGARGTRGUTGGGGAUAGURGGGRGGGGGTRGUAGGURGGGGURGUUUURGUU

UAGRGRGUAUAARGTUAGGUUUAGAAGUUAGAGRGGTURGGGGRGRGRGGUUATGGR

GGGURGGGGGRG

7
GTUAGRGRGRGUUATGGATUAAGATGATGAATRGUTGRGGARGGRGUAGATGRGGGRG

GUURGRGGURGGGUUUURGGGTAGGGGTGGGAGGTGGAGGGGGURGRGGGGGGUURG

GURGURGUUATTAAUTURGGAATTAGGTUTAAGUXGUUUAUUAUUUAGUUAUTGUUUR

GGGGAGRGUUAGURGTTGGGGRGGGAGRGGGUUUAGGATGGGGAUTGAGAUURGRGT

UUUUUAUURGAAUUTGGAUUTAGUUTUUTUTGAARGUAGAGGGUAGTGGGURGUURG

GAAGGGGRGGGGA

8
TUUURGUUUUTTURGGGRGGUUUAUTGUUUTUTGRGTTUAGAGGAGGUTAGGTUUAGG

TTRGGGTGGGGGARGRGGGTUTUAGTUUUUATUUTGGGUURGUTUURGUUUUAARGGU

TGGRGUTUUURGGGGUAGTGGUTGGGTGGTGGGXGGUTTAGAUUTAATTURGGAGTTA

ATGGRGGRGGURGGGUUUUURGRGGUUUUUTUUAUUTUUUAUUUUTAUURGGGGGUU

RGGURGRGGGURGUURGUATUTGRGURGTURGUAGRGATTUATUATUTTGATUUATGGR

GRGRGUTGAU

9
UAUURGGAUUTURGARGGUUTRGGTGTTRGUAGGRGRGGGATRGGUUUUAGUTUUTGR

GUUTGUUTUAGGUTRGGGUURGGGUURGGGUUURGUAGGUUTGUURGUUTTUUTGGGR

GRGGAGUTGGGUTGRGUUAAAGUUTTUTARGRGGXGTUUUTGAGTUUTUURGUAGURG

GUAURGRGGRGGGTUTGUUUAURGUAUTTUTGRGUUAGGGUUTUAAGARGGARGRGGG

RGGTGGTGUAGGRGGRGGGGGRGURGGGGUAGGGUAGAGGUUTTUUTTUTUTATAGAU

UAUATUATGG

10
UUATGATGTGGTUTATAGAGAAGGAAGGUUTUTGUUUTGUUURGGRGUUUURGURGUU

TGUAUUAURGUURGRGTURGTUTTGAGGUUUTGGRGUAGAAGTGRGGTGGGUAGAUUR

GURGRGGTGURGGUTGRGGGAGGAUTUAGGGAXGURGRGTAGAAGGUTTTGGRGUAGU

UUAGUTURGRGUUUAGGAAGGRGGGUAGGUUTGRGGGGUURGGGUURGGGUURGAGU

UTGAGGUAGGRGUAGGAGUTGGGGURGATUURGRGUUTGRGAAUAURGAGGURGTRGG

AGGTURGGGTG

11
ATUUTUAUTTTGTTRGUTUUTUAGTRGTUUAGGRGGATTUUTTTTTRGUUAGGTAAGGUT

GGUURGGGTGUATGGGGUURGGRGTGUUUTGGGTAAGGUTGGUUUAGGRGRGTGGGGT

URGGGGRGRGURGGGTAAGGUTGGGUUAGGRGXGTGGGGTURGGGGTGUUURGGGTAA

GGUTGGTUUAGGGGRGTGGGGTURGGGGTGUUURGGGTAAGGUTGGTUTAGGRGRGTG

GGGTURGUAGRGUUUUAAGTAAGGUTGGTUUAGGAGRGTGAGGTUUAGGGTGUUURGG

ATAAGGUT

12
AGUUTTATURGGGGUAUUUTGGAUUTUARGUTUUTGGAUUAGUUTTAUTTGGGGRGUT

GRGGAUUUUARGRGUUTAGAUUAGUUTTAUURGGGGUAUUURGGAUUUUARGUUUUT

GGAUUAGUUTTAUURGGGGUAUUURGGAUUUUARGXGUUTGGUUUAGUUTTAUURGG

RGRGUUURGGAUUUUARGRGUUTGGGUUAGUUTTAUUUAGGGUARGURGGGUUUUAT

GUAUURGGGUUAGUUTTAUUTGGRGAAAAAGGAATURGUUTGGARGAUTGAGGAGRGA

AUAAAGTGAGGAT

13
UAAGAGATTAGUAUAATAGATUTUTAAURGAGGGGAAGRGTTGUTTTTUARGUTARGRG

URGTAATTAATGGTATGAATUAATTAATTTGAUTTTTATTGTGTRGAAGGAAAAAAGRGU

AAUAAATGGAAURGGUAGUTGGGAGTTGTTXGTUUTUUAUUUUUTTUUUUAGGGAGGT

TUUAAGGAGAUAURGGGGAATGGARGGATUAGGUTGGGURGTGGUAGAGGGAGGGTA

GGAGGUAGRGAUUAGUAGRGTGGAGGGAGTUUAGAGAGUTAGUUTUTGRGGARGGRG

GAATRGAAA

14
TTTRGATTURGURGTURGUAGAGGUTAGUTUTUTGGAUTUUUTUUARGUTGUTGGTRGU

TGUUTUUTAUUUTUUUTUTGUUARGGUUUAGUUTGATURGTUUATTUUURGGTGTUTUU

TTGGAAUUTUUUTGGGGAAGGGGGTGGAGGAXGAAUAAUTUUUAGUTGURGGTTUUAT

TTGTTGRGUTTTTTTUUTTRGAUAUAATAAAAGTUAAATTAATTGATTUATAUUATTAAT

TARGGRGRGTAGRGTGAAAAGUAARGUTTUUUUTRGGTTAGAGATUTATTGTGUTAATU

TUTTG

15
UATGGGAAAGUAAAATTAAGGGUAAUATTGAGGAAGUTUATTAATATTTAGTTTAGAAG

ATGUAAAGGGTTUTTURGRGAAUUTGGAAGAGUUUUURGGGUTRGUUTUTRGURGRGG

TUUUAUAUTTGUUTGAUUTUAAUUAUAUTUTUUXGGTUUAGGUUURGUTGGAGAARGT

UUUAURGAUTURGGGGAUAGAAAGGURGTTTATGTAAAARGARGTTTTTUUTATTTRGU

UTUUUAUUUTUATGUAAATTTTGATTTTUAAUTUTTUUAAUUUTTURGUAUUUTGAAAU

AAAUUTU

16
GAGGTTTGTTTUAGGGTGRGGAAGGGTTGGAAGAGTTGAAAATUAAAATTTGUATGAGG

GTGGGAGGRGAAATAGGAAAAARGTRGTTTTAUATAAARGGUUTTTUTGTUUURGGAGT

RGGTGGGARGTTUTUUAGRGGGGUUTGGAUXGGGAGAGTGTGGTTGAGGTUAGGUAAG

TGTGGGAURGRGGRGAGAGGRGAGUURGGGGGGUTUTTUUAGGTTRGRGGAAGAAUUU

TTTGUATUTTUTAAAUTAAATATTAATGAGUTTUUTUAATGTTGUUUTTAATTTTGUTTT

UUUATG

17
GAAUTTGRGUTUUAGGAARGAUTGRGUARGTGGRGRGGRGGTGGRGGRGRGGAGGAUU

UAGGRGAAGGRGAAGGRGAAGGRGAAGGRGUAGGRGAAGGRGAAGGRGUAGGRGGRG

GGAAGUTARGUUAAAGURGURGURGURGURGUTGUXGGGGTUTGUUUAUAGUUTGGU

AURGGGRGGUAGRGGTGGRGGRGGRGGARGRGGUAGGTGUAGRGAURGRGAAGUURG

GGRGGURGRGRGUUUTRGGAAUTUUUURGRGGUTUURGAGGTGGUAGURGRGRGUUAU

TRGGUAGUUURGT

18
ARGGGGUTGURGAGTGGRGRGRGGUTGUUAUUTRGGGAGURGRGGGGGAGTTURGAGG

GRGRGRGGURGUURGGGUTTRGRGGTRGUTGUAUUTGURGRGTURGURGURGUUAURG

UTGURGUURGGTGUUAGGUTGTGGGUAGAUUUXGGUAGRGGRGGRGGRGGRGGUTTTG

GRGTAGUTTUURGURGUUTGRGUUTTRGUUTTRGUUTGRGUUTTRGUUTTRGUUTTRGU

UTTRGUUTGGGTUUTURGRGURGUUAURGURGRGUUARGTGRGUAGTRGTTUUTGGAGR

GUAAGTTU

19
GUAURGUTTGUUTTRGUTAGAGGAAGAAGAGARGGUUUTGAGRGUAGUATUAUUTRGG

URGGTTGGAGTUTGUURGGUTGURGUUAGGGGGTGATGRGUUUTUAUUTTTUUTUUAG

GATGUURGGGATUUUAAGGAGGGTRGTGGGTUTUXGRGTUUUAAUUTGGAGUUTTGGU

URGGGATGATAUUUTTGGUUAURGGUAGUAGGTUTUTGGTGGUUAUUTGARGUTTGAG

UATTTUTTUTGRGGAUUUTGGGAAGTUUTAUUUUTTGGAUTUATAAAATATUTTGGTTG

TUUAAAGGT

20
AUUTTTGGAUAAUUAAGATATTTTATGAGTUUAAGGGGTAGGAUTTUUUAGGGTURGUA

GAAGAAATGUTUAAGRGTUAGGTGGUUAUUAGAGAUUTGUTGURGGTGGUUAAGGGTA

TUATUURGGGUUAAGGUTUUAGGTTGGGARGXGGAGAUUUARGAUUUTUUTTGGGATU

URGGGUATUUTGGAGGAAAGGTGAGGGRGUATUAUUUUUTGGRGGUAGURGGGUAGA

UTUUAAURGGURGAGGTGATGUTGRGUTUAGGGURGTUTUTTUTTUUTUTAGRGAAGGU

AAGRGGTGU

21
UTGURGUUTRGUURGRGGGAUTUUUTGGAGGAGUTURGRGUUUTUUTUTUUATUUTUA

GAGUTGURGGGRGGUTGGAGUAGUAGRGRGGGAGRGUTAGGGGUARGGGAGRGUAGT

UUUTGTGGAGTRGUTGRGGGTURGRGTGGRGTGUUXGGGGGAUUUTAAAGAURGTRGG

GTGGGGGUTGAGGGRGAGGGGRGGGAUAURGGGGURGRGGGRGGGGRGUAUURGGAA

UUURGAUAGUTGTGTUTTGGTGGAGUTGTGGAUTGRGUURGURGAUTUUUARGGURGG

GGRGGRGUTGAA

22
TTUAGRGURGUUURGGURGTGGGAGTRGGRGGGRGUAGTUUAUAGUTUUAUUAAGAUA

UAGUTGTRGGGGTTURGGGTGRGUUURGUURGRGGUUURGGTGTUURGUUUUTRGUUU

TUAGUUUUUAUURGARGGTUTTTAGGGTUUUUXGGGUARGUUARGRGGAUURGUAGRG

AUTUUAUAGGGAUTGRGUTUURGTGUUUUTAGRGUTUURGRGUTGUTGUTUUAGURGU

URGGUAGUTUTGAGGATGGAGAGGAGGGRGRGGAGUTUUTUUAGGGAGTUURGRGGGR

GAGGRGGUAG

23
TGTGGTGUTGUTTGRGUTGURGGTGGUUTGGGGTGAGAGGRGGGRGGGRGTGGGGAGG

RGUURGGGRGGARGAGGAAUURGGGGUUURGUAGAGAAUTRGRGTGUAGRGUTGAGUT

GRGUTGUTUTGRGRGUURGGGTURGAAGGUAGRGXGATGGGTGGGUTGAGRGRGRGAU

URGGUAGGGRGGRGGGTGTAGGATUUTTUTGRGUAUTGGAGAUUUTRGUTGUTTUTGG

GTAAGRGTGGAGTTUUUAGGTGUAGGGGUTTAAGTRGTGARGAGRGUAGTGGAAGGRG

UAGATGUTGA

24
TUAGUATUTGRGUUTTUUAUTGRGUTRGTUARGAUTTAAGUUUUTGUAUUTGGGAAUTU

UARGUTTAUUUAGAAGUAGRGAGGGTUTUUAGTGRGUAGAAGGATUUTAUAUURGURG

UUUTGURGGGTRGRGRGUTUAGUUUAUUUATXGRGUTGUUTTRGGAUURGGGRGRGUA

GAGUAGRGUAGUTUAGRGUTGUARGRGAGTTUTUTGRGGGGUUURGGGTTUUTRGTUR

GUURGGGRGUUTUUUUARGUURGUURGUUTUTUAUUUUAGGUUAURGGUAGRGUAAG

UAGUAUUAUA

25
GUTTUUURGRGTUUTURGGGUURGGGURGUUUTUUTUURGUAUAGTGRGGAGUAGGGA

GGUUURGRGUUTRGAUUAUURGRGUURGAGRGTURGRGUUTUUTUUTURGUTUTGUAG

GRGGGGAURGUURGGRGUTRGGUAUURGGUAGXGRGGUUUUUTUUAGUUUURGGUTU

URGGUAGUAGAAGUAGAAGGUAGRGUUAGGGGURGURGURGURGURGAGUTURGRGG

GGUTRGGGAGURGGUUURGGRGAGGAGGRGRGGAAUUATGGURGATGGGGGRGAGGG

RGAAGARGAGATU

26
GATUTRGTUTTRGUUUTRGUUUUUATRGGUUATGGTTURGRGUUTUUTRGURGGGGURG

GUTUURGAGUUURGRGGAGUTRGGRGGRGGRGGRGGUUUUTGGRGUTGUUTTUTGUTT

UTGUTGURGGGAGURGGGGGUTGGAGGGGGURGXGUTGURGGGTGURGAGRGURGGGR

GGTUUURGUUTGUAGAGRGGAGGAGGAGGRGRGGARGUTRGGGRGRGGGTGGTRGAGG

RGRGGGGUUTUUUTGUTURGUAUTGTGRGGGAGGAGGGRGGUURGGGUURGGAGGARG

RGGGGAAGU

27
GUUUUUUAGGAUTGGRGGUUURGGGUUURGUTUUUAUUUAUUUAUUTARGUAGGGGG

RGTUUTGUTUAGGUAATTUUTUURGRGRGUUURGTRGGGGAURGGGRGGGGARGGGAG

AAGGAAAAGGGUUUUTGGUTURGGGAUUAGGGUTUXGGAGGGTGURGGGRGGGGAGR

GGAAUAGGGAARGGGUTGGTGGRGGUUUUAAGRGGGAGGGARGGAURGAUARGRGGU

UUUUTGGRGGUUTTGRGAUTRGURGAUUTGRGGAAUUTRGTRGURGUUUTUAUAGUUU

RGRGGUUAURGUU

28
GGRGGTGGURGRGGGGUTGTGAGGGRGGRGARGAGGTTURGUAGGTRGGRGAGTRGUA

AGGURGUUAGGGGGURGRGTGTRGGTURGTUUUTUURGUTTGGGGURGUUAUUAGUUR

GTTUUUTGTTURGUTUUURGUURGGUAUUUTUXGGAGUUUTGGTUURGGAGUUAGGGG

UUUTTTTUUTTUTUURGTUUURGUURGGTUUURGARGGGGRGRGRGGGAGGAATTGUUT

GAGUAGGARGUUUUUTGRGTAGGTGGGTGGGTGGGAGRGGGGUURGGGGURGUUAGTU

UTGGGGGGU

29
AAAATGTTTGGUAUUTUATTGATUATGTTTAGUTGATUATUUUTGGGGUTGGGGAGGGU

AGUUAGGUUAGUUAGRGRGUAGGGUAGGGGRGGGRGGAGGAGAAGUUTGGAGGAGGU

TGARGGGAAGUUTRGAGGAGGGGUAUUURGGTUXGAGGUTGGGUTGGGAARGUTGUTU

UAUAGRGUTGTGAGTGGURGGGAGGAUTTAURGUUAUUAURGUTGUUTUTGGGGUATU

TUUUAUURGUURGGGATGUTGURGUUTUUTTTUUAUAATGUUTGTTGATUAATTTTTTA

ATUAAGAAG

30
UTTUTTGATTAAAAAATTGATUAAUAGGUATTGTGGAAAGGAGGRGGUAGUATUURGG

GRGGGTGGGAGATGUUUUAGAGGUAGRGGTGGTGGRGGTAAGTUUTUURGGUUAUTUA

UAGRGUTGTGGAGUAGRGTTUUUAGUUUAGUUTXGGAURGGGGTGUUUUTUUTRGAGG

UTTUURGTUAGUUTUUTUUAGGUTTUTUUTURGUURGUUUUTGUUUTGRGRGUTGGUTG

GUUTGGUTGUUUTUUUUAGUUUUAGGGATGATUAGUTAAAUATGATUAATGAGGTGUU

AAAUATTTT

31
GAGUUUAGTTRGUATUTUTAUAGRGUUAUAUATUTAGGUUAGUUUAGUTURGGTUUUA

GTRGGTUTUTGRGGAGUUUUAGUUAGUTURGRGTUTTGGTUUTGGUTTGRGGGTGGUAU

AUAGAUAAGRGAURGGGGTGGUURGGAGAGGTXGTGUUUUTGGUTGRGAGARGARGRG

AUAGUAGGGRGUTURGGGGGURGUAGRGGRGTRGAAGGRGGTUUUTGGGGGRGGGGUT

UTGGUARGAGGGGGAUUUTUURGGGTUAAGTTUAUAGAGGRGGAGGGGUUTGGGUTGR

GUUUUAGAG

32
UTUTGGGGRGUAGUUUAGGUUUUTURGUUTUTGTGAAUTTGAUURGGGAGGGTUUUUU

TRGTGUUAGAGUUURGUUUUUAGGGAURGUUTTRGARGURGUTGRGGUUUURGGAGRG

UUUTGUTGTRGRGTRGTUTRGUAGUUAGGGGUAXGAUUTUTURGGGUUAUUURGGTRG

UTTGTUTGTGTGUUAUURGUAAGUUAGGAUUAAGARGRGGAGUTGGUTGGGGUTURGU

AGAGAURGAUTGGGAURGGAGUTGGGUTGGUUTAGATGTGTGGRGUTGTAGAGATGRG

AAUTGGGUTU

33
GRGGRGGGGGUTGUTUTGGGRGRGUUURGGGRGAAGTGRGUUUAGTUTURGGUUURGG

UUUUTRGGRGRGUURGAUTTUURGGURGUUUUTGAGUUUAGUAGURGRGGGTUURGGG

ATRGGUTAAGAGTAGUTGUAARGUUTRGURGGAXGGAGTUUTTTUUTTTUURGGGARGU

TGGGUUATGAGUTURGRGGUUAUUTGAGGUAUAGGGGAGTUTGUTRGGUUAGGAUAGU

UTUUURGAAGTUURGTGUUUTRGUUTUTGUAUTGRGGGARGUUAGRGUTRGGUUUTGG

RGGAGGRGT

34
ARGUUTURGUUAGGGURGAGRGUTGGRGTUURGUAGTGUAGAGGRGAGGGUARGGGAU

TTRGGGGAGGUTGTUUTGGURGAGUAGAUTUUUUTGTGUUTUAGGTGGURGRGGAGUT

UATGGUUUAGRGTUURGGGAAAGGAAAGGAUTUXGTURGGRGAGGRGTTGUAGUTAUT

UTTAGURGATUURGGGAUURGRGGUTGUTGGGUTUAGGGGRGGURGGGAAGTRGGGRG

RGURGAGGGGURGGGGURGGAGAUTGGGRGUAUTTRGUURGGGGRGRGUUUAGAGUA

GUUUURGURGU

35
AAATGGTGAAATATUUTUTAAAAATATGTTUUUUAAGGUUAAUTTRGRGGUTGGTAGUU

UUTTURGARGUUTTTGUUTUUUAGAAAATUAUAAUAAAGRGATRGGAAATTUAGUUAR

GGTUURGGGAAGAAGGAGTAGUAGTGAGGUUUXGGAAUUUAUTGRGGURGAAAUTGU

UATGUTUTUTTTAAUUAAAATAAAAAAGATAAGAAGAAGAAGTAAAAUUUTTTAATAU

ATUAAATATARGGAATTTTAATUTTTAAAGRGATAUATTGTUTATTATTTTAGTAUATGA

RGTAAAUU

36
GGTTTARGTUATGTAUTAAAATAATAGAUAATGTATRGUTTTAAAGATTAAAATTURGTA

TATTTGATGTATTAAAGGGTTTTAUTTUTTUTTUTTATUTTTTTTATTTTGGTTAAAGAGA

GUATGGUAGTTTRGGURGUAGTGGGTTUXGGGGUUTUAUTGUTAUTUUTTUTTUURGGG

AURGTGGUTGAATTTURGATRGUTTTGTTGTGATTTTUTGGGAGGUAAAGGRGTRGGAA

GGGGUTAUUAGURGRGAAGTTGGUUTTGGGGAAUATATTTTTAGAGGATATTTUAUUAT

TT

37
GTGGUAGGGGTAAGGRGUTTUUATTTGUUTGAAAGGUTUTGUUUTAUAUAAAAATAGA

AAUTGGTTGUATGUATGUAAAGGATGGUTGAGTGGTAATAAUTGGAGGAAUTGRGGAU

AGURGGAGGUTGRGGGGURGGAGGGTUTTTGGGTXGGTAUTUTGUUUUAURGGAGGTU

UAUAGGGUUTUUTUUATUUAGGGTGGGURGGUAAGTUTGUUTATUAGRGATUTGGGUA

AGUUTGUTAUTTTTTATGAUUTUAUTTGURGAAAUTUAAGUUARGUUTGGGTAAGTAUA

TTUTGUAUA

38
TGTGUAGAATGTAUTTAUUUAGGRGTGGUTTGAGTTTRGGUAAGTGAGGTUATAAAAAG

TAGUAGGUTTGUUUAGATRGUTGATAGGUAGAUTTGURGGUUUAUUUTGGATGGAGGA

GGUUUTGTGGAUUTURGGTGGGGUAGAGTAUXGAUUUAAAGAUUUTURGGUUURGUA

GUUTURGGUTGTURGUAGTTUUTUUAGTTATTAUUAUTUAGUUATUUTTTGUATGUATG

UAAUUAGTTTUTATTTTTGTGTAGGGUAGAGUUTTTUAGGUAAATGGAAGRGUUTTAUU

UUTGUUAU

39
AUTUTGGAAGTGTGUARGGURGTTGTGUAGGGTGGATGGTGTTGAUUTTTTGAUUTGAA

AAUAGTTGGGGGUTGGGGAGRGGAGGAAGGATGGRGGAAGAGAGGAAAGAGUUARGA

GAAUAAUTAGGRGGGATGTAUTTTTGAGUUUTGUXGGGTGTUTURGATRGGAGTUTGGG

GTTGAGATTTGGGUTGUAUTTGTUUURGGTGTGTUTUTURGGRGGAGTAUUUTGAAGGT

GUARGAGGTGGGGAGUATAGGUTGAGGTGGGTAATRGGGTUUTGGATAGAAAUAUAAU

UUTRGTUU

40
GGARGAGGGTTGTGTTTUTATUUAGGAUURGATTAUUUAUUTUAGUUTATGUTUUUUAU

UTRGTGUAUUTTUAGGGTAUTURGURGGAGAGAUAUAURGGGGAUAAGTGUAGUUUAA

ATUTUAAUUUUAGAUTURGATRGGAGAUAUUXGGUAGGGUTUAAAAGTAUATUURGUU

TAGTTGTTUTRGTGGUTUTTTUUTUTUTTURGUUATUUTTUUTURGUTUUUUAGUUUUUA

AUTGTTTTUAGGTUAAAAGGTUAAUAUUATUUAUUUTGUAUAARGGURGTGUAUAUTT

UUAGAGT

41
AAAGAUTTGGTTUUAGAAURGURGUAAUAAGTGGAAGRGGUAGUTUTRGGUTGAGUTG

GAGGRGGUUAAUATGGRGUARGRGTRGGRGUAGAUTUTGGTGAGUATGURGUTGGTGT

TURGGGAUAGTTRGUTGUTGRGRGTGURGGTGUXGRGUTRGUTRGUUTTTUURGRGURG

UTUTAUTAUURGGGAAGUAAUUTUTRGGUUTTAUUTUTUTAUAAUUTATAUAAUAAGU

TRGAUTAUTGAURGGUURGURGUUURGRGURGUUUUUAGUTGUURGUAGAGURGGGRG

RGTAUTGTA

42
TAUAGTARGRGUURGGUTUTGRGGGUAGUTGGGGGRGGRGRGGGGRGGRGGGURGGTU

AGTAGTRGAGUTTGTTGTATAGGTTGTAGAGAGGTAAGGURGAGAGGTTGUTTUURGGG

TAGTAGAGRGGRGRGGGAAAGGRGAGRGAGRGXGGUAURGGUARGRGUAGUAGRGAA

UTGTUURGGAAUAUUAGRGGUATGUTUAUUAGAGTUTGRGURGARGRGTGRGUUATGT

TGGURGUUTUUAGUTUAGURGAGAGUTGURGUTTUUAUTTGTTGRGGRGGTTUTGGAAU

UAAGTUTTT

43
UUAGRGUAUAGUURGGUUAGGGGRGUUUTUUUTGURGURGURGGUUUTTTGATRGUUR

GRGGUURGRGGUURGUAGAUUAUAGUUAGRGUTGTGUTGGGUAGGTGGRGGUAGRGG

GGRGRGGRGRGUTUAGGRGUARGGGTUUURGGGUXGRGGRGRGUTUUURGUUARGURG

UAUATUAAGGUURGGURGGURGGRGGGRGUUTTUATTAGUAGUUTGAAATTATAATAT

TATGTTAAAGAAUAAAGUTGUTUUURGGAAAAATATGTGUTGUATATUTGAUAAAGAT

AAATTGGATTA

44
TAATUUAATTTATUTTTGTUAGATATGUAGUAUATATTTTTURGGGGAGUAGUTTTGTTU

TTTAAUATAATATTATAATTTUAGGUTGUTAATGAAGGRGUURGURGGURGGURGGGUU

TTGATGTGRGGRGTGGRGGGGAGRGRGURGXGGUURGGGGAUURGTGRGUUTGAGRGR

GURGRGUUURGUTGURGUUAUUTGUUUAGUAUAGRGUTGGUTGTGGTUTGRGGGURGR

GGGURGRGGGRGATUAAAGGGURGGRGGRGGUAGGGAGGGRGUUUUTGGURGGGUTG

TGRGUTGG

45
URGTUURGGGUTUUTGGRGGUTGTRGUTGRGGTTUUTTUURGRGGGURGGGUUUUTTUU

UTGRGUUTTRGURGUUTUUTRGRGUUTGUURGGGGUURGUAGUUTURGUAURGGGAAU

URGGAGGAUURGAGGRGGGRGUAGGGGRGAAGUXGGGGURGGGGAGGGGURGUUTRG

UTURGGGTTRGAGARGGAAGAAAUARGRGGRGUAGGUTURGGAGRGARGGUTURGARG

GGGAUURGTTAAATAATTTATTGATGATAUAAAGRGAUTRGRGUUUAUURGGGGURGU

UUURGGATTU

46
GAATURGGGGGRGGUUURGGGTGGGRGRGAGTRGUTTTGTATUATUAATAAATTATTTA

ARGGGTUUURGTRGGAGURGTRGUTURGGAGUUTGRGURGRGTGTTTUTTURGTUTRGA

AUURGGAGRGAGGRGGUUUUTUUURGGUUUXGGUTTRGUUUUTGRGUURGUUTRGGGT

UUTURGGGTTUURGGTGRGGAGGUTGRGGGUUURGGGUAGGRGRGAGGAGGRGGRGAA

GGRGUAGGGAAGGGGUURGGUURGRGGGAAGGAAURGUAGRGAUAGURGUUAGGAGU

URGGGARGG

47
UTTGTUUAGGARGURGURGGURGGGGUTGUAAGGGAGGGGAAGGGAGGGAGGTUAGR

GGURGGRGGGGTUUUUUTURGRGUUUAUURGUUURGUAUUUUURGRGRGGGUUAUTU

AUURGGGUUAGUUAGARGRGGGTUUUTUUAGGGRGXGUUUTGUAUUARGURGGGUUA

GAAGATGGGRGGGRGUUURGGUAGUTRGGUUAGGGGUTTGGGGTAGURGRGRGUUAUA

GRGGURGRGGGUURGAAGTAAARGURGGRGGARGARGRGAGUURGTTGAGURGGGGUA

GUUUUUUUAGGAG

48
UTUUTGGGGGGGUTGUUURGGUTUAARGGGUTRGRGTRGTURGURGGRGTTTAUTTRGG

GUURGRGGURGUTGTGGRGRGRGGUTAUUUUAAGUUUUTGGURGAGUTGURGGGGRGU

URGUUUATUTTUTGGUURGGRGTGGTGUAGGGXGRGUUUTGGAGGGAUURGRGTUTGG

UTGGUURGGGTGAGTGGUURGRGRGGGGGGTGRGGGGRGGGTGGGRGRGGAGGGGGAU

UURGURGGURGUTGAUUTUUUTUUUTTUUUUTUUUTTGUAGUUURGGURGGRGGRGTU

UTGGAUAAG

49
UTURGGUUTUATUTTUTURGTGGAGGTGUUUTTUAGUAAGTTTGUUAAUAAUAURGAGG

GUUAGTGRGGTGAGGUUAUAGGGUTUURGGGUATRGTUTGGUATTRGRGGGGGRGGGG

GTGURGGGUAGGGGRGAGGUUAUUARGTGURGXGTGTGURGGTGTUTUTGUTTTUTGG

UTGUTUTGUTGAGTGUAGGUUAUAGGUATGAGGUTTARGUUTGURGGTAUUTGUAGUT

UUUUAGTAUUATAGGGAUTGTUUUAGGGTTGTTUTRGGGGGAUAGTGAGGUAUAGGUA

GGGUURGUT

50
AGRGGGUUUTGUUTGTGUUTUAUTGTUUUURGAGAAUAAUUUTGGGAUAGTUUUTATG

GTAUTGGGGAGUTGUAGGTAURGGUAGGRGTAAGUUTUATGUUTGTGGUUTGUAUTUA

GUAGAGUAGUUAGAAAGUAGAGAUAURGGUAUAXGRGGUARGTGGTGGUUTRGUUUU

TGUURGGUAUUUURGUUUURGRGAATGUUAGARGATGUURGGGAGUUUTGTGGUUTUA

URGUAUTGGUUUTRGGTGTTGTTGGUAAAUTTGUTGAAGGGUAUUTUUARGGAGAAGA

TGAGGURGGAG

51
UUAURGRGGUUTTTTUAUUUAAURGUUUUUTUUTGRGTGGGGGUUURGUATUUUUTGG

AUTGGRGTGGGUTUTGGGGUURGATGUUUTGGUAGUUURGGUUATUTUTGURGUTGGU

RGRGGGTAUUTGUTUAUUTGTUUARGURGTGGUXGAUURGGGGATUAURGAUAGURGG

GUUATGUUTRGAGTUURGURGUURGRGURGUTGUTRGGUTTUTUAGAAARGUAUUTUU

ARGURGUUAGURGGGGUTUUAGGGTUUAURGUURGGGUURGGAGGAUTGGRGAUTUU

UTURGAGGTTG

52
UAAUUTRGGAGGGAGTRGUUAGTUUTURGGGUURGGGRGGTGGAUUUTGGAGUUURGG

UTGGRGGRGTGGAGGTGRGTTTUTGAGAAGURGAGUAGRGGRGRGGGRGGRGGGAUTR

GAGGUATGGUURGGUTGTRGGTGATUUURGGGTXGGUUARGGRGTGGAUAGGTGAGUA

GGTAUURGRGGUUAGRGGUAGAGATGGURGGGGUTGUUAGGGUATRGGGUUUUAGAG

UUUARGUUAGTUUAGGGGATGRGGGGUUUUUARGUAGGAGGGGGRGGTTGGGTGAAA

AGGURGRGGTGG

53
ATGUTAGTGUAGTTUUUTGRGUAGURGUUAGGTGGRGTUAAGGURGURGUUTGGGGGG

URGGGUAGGURGAGGUUUUTGUURGTRGUAGTUUUAGUUTRGUTUAUUAUTTGGUURG

GGAGTTGGUTGRGGGUAGRGGUTTGGUAGUUTGXGGURGGGGGRGGARGGTGGGGRGG

TGTGGGTTTUAGUUTUUURGGAGGGUUUTUARGGUTGAGUAAARGTTRGGGUTGATGTR

GGUAAUATGRGGAATUAATTTTRGGGGAAUTUAGUAGUUAAAUUATUUAUUTTTGGGR

GGGAAGUAG

54
UTGUTTUURGUUUAAAGGTGGATGGTTTGGUTGUTGAGTTUUURGAAAATTGATTURGU

ATGTTGURGAUATUAGUURGAARGTTTGUTUAGURGTGAGGGUUUTURGGGGAGGUTG

AAAUUUAUAURGUUUUAURGTURGUUUURGGUXGUAGGUTGUUAAGURGUTGUURGU

AGUUAAUTUURGGGUUAAGTGGTGAGRGAGGUTGGGAUTGRGARGGGUAGGGGUUTRG

GUUTGUURGGUUUUUUAGGRGGRGGUUTTGARGUUAUUTGGRGGUTGRGUAGGGAAUT

GUAUTAGUAT

55
GGAGRGGTGRGGAGGUAGUUAGGUUURGUURGURGUAGURGRGGUAGURGURGGAGG

ATTUUTGTUUTAATATGGAGUTGGGATTUUUURGGUUURGUUUURGUUUURGGUURGR

GGGGAGAUAGAGGUTGGUAGUAGGGRGGGGGGAAGXGUTRGUTTGGGGGURGGUAAR

GGGGGGAAGGGATGUUTAAGTGUAGAUUUAGGTUUTRGURGTGUUUUUARGTUUUTGU

UTUAGTTTUUUUTTUAGTAAGGTTAATTAGUTGAGAGGGAAAUUARGAATUAUTGUAG

AUTAUAGRGUTG

56
UAGRGUTGTAGTUTGUAGTGATTRGTGGTTTUUUTUTUAGUTAATTAAUUTTAUTGAAG

GGGAAAUTGAGGUAGGGARGTGGGGGUARGGRGAGGAUUTGGGTUTGUAUTTAGGUAT

UUUTTUUUUURGTTGURGGUUUUUAAGRGAGXGUTTUUUUURGUUUTGUTGUUAGUUT

UTGTUTUUURGRGGGURGGGGGRGGGGGRGGGGURGGGGGAATUUUAGUTUUATATTA

GGAUAGGAATUUTURGGRGGUTGURGRGGUTGRGGRGGGRGGGGUUTGGUTGUUTURG

UAURGUTUU

57
RGGAGTGGAGUTTTGGGAAUUUUTRGGUUAAGUAUAGRGGTTRGAAAATAUAGUTGAA

AUUUAGRGGGUUUTTAGUARGRGUUUUAGRGURGGAGUAGGGTUAGGGTUTTUTTGRG

AUURGGUTURGUTUUAGATUUUUUUAGUTUTRGGXGGRGGAUURGGGURGRGTGTGAG

RGRGUTTTGUAUUTUUTATUUUUAGGGTURGURGAGAGUUARGATTTTTTAUAGAAAAT

GAGUAATAAAGAGATTTTGTAUTGTUUTGAUTGGGGAGTUUUAGGURGRGGGGGARGG

AGRGUUUUT

58
AGGGGRGUTURGTUUUURGRGGUUTGGGAUTUUUUAGTUAGGAUAGTAUAAAATUTUT

TTATTGUTUATTTTUTGTAAAAAATRGTGGUTUTRGGRGGAUUUTGGGGATAGGAGGTG

UAAAGRGRGUTUAUARGRGGUURGGGTURGUXGURGAGAGUTGGGGGGATUTGGAGRG

GAGURGGGTRGUAAGAAGAUUUTGAUUUTGUTURGGRGUTGGGGRGRGTGUTAAGGGU

URGUTGGGTTTUAGUTGTATTTTRGAAURGUTGTGUTTGGURGAGGGGTTUUUAAAGUT

UUAUTURG

59
AAAAATAUUUTAGATTUATAAAUAAAAGUTTGUAGTTAUUUAUAGUUUAATAUAAARG

AUURGARGUUUTUAUTTUTATAGUURGTGAGTUTUUUAGUUUUTAAUAGGUTRGTUTU

UUUAGARGUUURGGGTGAAAAGGTTRGRGURGUXGGTGGAGAGTUUTATTGGTTUTATT

TUTGTUTTUAUTUUAAGGUUUUAGGAURGGGGAGAGGTUUTGGTUTAAUTTTGGUTGTU

UUARGGTUUAAGGAUUTUAGRGUTUAGUURGAAARGUTAAGUUAAGRGTGGAGAURG

URGGGRGUA

60
TGRGUURGGRGGTUTUUARGUTTGGUTTAGRGTTTRGGGUTGAGRGUTGAGGTUUTTGG

AURGTGGGAUAGUUAAAGTTAGAUUAGGAUUTUTUUURGGTUUTGGGGUUTTGGAGTG

AAGAUAGAAATAGAAUUAATAGGAUTUTUUAUXGGRGGRGRGAAUUTTTTUAUURGGG

GRGTUTGGGGAGARGAGUUTGTTAGGGGUTGGGAGAUTUARGGGUTATAGAAGTGAGG

GRGTRGGGTRGTTTGTATTGGGUTGTGGGTAAUTGUAAGUTTTTGTTTATGAATUTAGGG

TATTTTT

61
AGUUARGAUUTUTGUUUUTATGGGGTUUAGRGRGUAUTTGGGTGRGGGTGATUUUTUR

GGAAGTRGUTUTGUTUUTUTGGURGGGTUTUUTUUTUTTUURGGUUAUTTATAUUUTTG

AGUAUUAAGAGTTRGUUUATTTAGGURGUUTUXGGGAARGGAAGGGTUUAUUUUAURG

UUAGGUTUTUAAAGGGTGGGGTGGRGUUTRGGGTGGGGRGGGUAGGAGGTGGGTGAGG

ARGGGAGAGAAGGGGRGAGRGGGGGUARGGGGRGGGGURGGUTUAGTRGGGGTAGAT

GATGAAGURG

62
RGGUTTUATUATUTAUUURGAUTGAGURGGUUURGUUURGTGUUUURGUTRGUUUUTT

UTUTUURGTUUTUAUUUAUUTUUTGUURGUUUUAUURGAGGRGUUAUUUUAUUUTTTG

AGAGUUTGGRGGTGGGGTGGAUUUTTURGTTUUXGGAGGRGGUUTAAATGGGRGAAUT

UTTGGTGUTUAAGGGTATAAGTGGURGGGAAGAGGAGGAGAUURGGUUAGAGGAGUA

GAGRGAUTTURGGAGGGATUAUURGUAUUUAAGTGRGRGUTGGAUUUUATAGGGGUAG

AGGTRGTGGUT

63
URGUTTGGGTAAURGUAGUUTGUUTGRGTUTUTTUUTTUUTURGRGTGGGTTUTAGUAA

UATUUAUTGUAGURGGGUUAGGRGAGURGGRGRGTAUUATRGGRGRGGGGGGAGGAG

AGGGURGGGUUTGGGAAGATGUTGXGGAGGAXGUTGXGGATTXGXGAGUURGGGGTA

AGGRGGRGGRGUAURGUUUUUTUURGURGUTTUUUUUUUAUUURGUUUUUUAURGUR

GUUUTTAGUUUTUUUURGGGATGAGAGAGAGTRGRGUTGRGGAGUAAUUUUAGTGGAT

GGGTURGRGGGG

64
UUURGRGGAUUUATUUAUTGGGGTTGUTURGUAGRGRGAUTUTUTUTUATUURGGGGG

AGGGUTAAGGGRGGRGGTGGGGGGRGGGGTGGGGGGGAAGRGGRGGGAGGGGGRGGT

GRGURGURGUUTTAUUURGGGUTXGXGAATUXGUAGXGTUUTUXGUAGUATUTTUUUA

GGUURGGUUUTUTUUTUUUUURGRGURGATGGTARGRGURGGUTRGUUTGGUURGGUT

GUAGTGGATGTTGUTAGAAUUUARGRGGAGGAAGGAAGAGARGUAGGUAGGUTGRGGT

TAUUUAAGRGG

65
ATUTUUUUTTUTGUUTGTGATATGGRGGGUTGUATTAUAAGUTGGGRGTRGAGATAAAG

RGGGGAGURGRGGGARGGRGGUUUAGRGURGGGGUUURGGGTGGGGURGGUUAGGGA

GATAAGGGUUTUUUAGUUUUATGAATTATTUAUXGGUAUTGUUTUUTUTGGUTGGGRG

GURGUUUTGGGRGUTGAGATTGRGTUTTUUAGAGGGGUUTGGGTAGGGTGGGAGGGGG

GTURGTGGGGAAGGGUAGUAGGUTGTAGAGGGGTUTTUUTATGGGGGATGGGGAGAGG

GGAGGAUTGT

66
AUAGTUUTUUUUTUTUUUUATUUUUUATAGGAAGAUUUUTUTAUAGUUTGUTGUUUTT

UUUUARGGAUUUUUUTUUUAUUUTAUUUAGGUUUUTUTGGAAGARGUAATUTUAGRG

UUUAGGGRGGURGUUUAGUUAGAGGAGGUAGTGUXGGTGAATAATTUATGGGGUTGG

GAGGUUUTTATUTUUUTGGURGGUUUUAUURGGGGUUURGGRGUTGGGURGURGTUUR

GRGGUTUUURGUTTTATUTRGARGUUUAGUTTGTAATGUAGUURGUUATATUAUAGGU

AGAAGGGGAGAT

67
AGGTUATUUAGUAGUAGGGUTUUARGTRGGTUTRGTRGATGUUUUAGAAGGUUAGUTU

UTUUTRGAAGAGRGGUURGUAUARGTUTGRGGGGUAGTGUAGUTTGURGGTGRGGTAG

TAATTGAGUAUATAGGRGAAGARGUURGGGTGUXGGTRGAAGAAGAAUTRGRGGURGU

UAURGGGATGGTRGUTGGUUUTGURGURGRGGGAAUTGUAGTTGUURGRGURGUUUTR

GAAGUAGURGUUTGGUURGGGGGAUAGRGGGGGRGUTUTRGGRGGRGGRGAUAGTGG

AGGRGGRGARG

68
RGTRGURGUUTUUAUTGTRGURGURGURGAGAGRGUUUURGUTGTUUUURGGGUUAGG

RGGUTGUTTRGAGGGRGGRGRGGGUAAUTGUAGTTUURGRGGRGGUAGGGUUAGRGAU

UATUURGGTGGRGGURGRGAGTTUTTUTTRGAUXGGUAUURGGGRGTUTTRGUUTATGT

GUTUAATTAUTAURGUAURGGUAAGUTGUAUTGUUURGUAGARGTGTGRGGGURGUTU

TTRGAGGAGGAGUTGGUUTTUTGGGGUATRGARGAGAURGARGTGGAGUUUTGUTGUT

GGATGAUUT

69
RGTATTAAUAGGTTUUUUTURGRGUAUAUTGAUATATTTUTTATUUUUUATAATGAATT

UAGUUATATGGUATTUTTTUUUATRGAAGGUUATRGGGAATGGUTTTAGGAAGUTGATT

TTUAAGUTTTAAGRGGUAGUAGGTGURGGUAGXGRGGGGAURGATRGATGGAGAGAAG

GRGGGUAAGARGURGGGAAGRGUATTUUTUUTUAAURGAGTGUUAUAAURGUUUTUUR

GAAGTGUUURGGGGUTTRGAGUATUAUUTRGRGGTAATURGGGAGGGTGGAGGGATGR

GGUTGGAU

70
GTUUAGURGUATUUUTUUAUUUTUURGGATTAURGRGAGGTGATGUTRGAAGUUURGG

GGUAUTTRGGGAGGGRGGTTGTGGUAUTRGGTTGAGGAGGAATGRGUTTUURGGRGTUT

TGUURGUUTTUTUTUUATRGATRGGTUUURGXGUTGURGGUAUUTGUTGURGUTTAAAG

UTTGAAAATUAGUTTUUTAAAGUUATTUURGATGGUUTTRGATGGGAAAGAATGUUATA

TGGUTGAATTUATTATGGGGGATAAGAAATATGTUAGTGTGRGRGGAGGGGAAUUTGTT

AATARG

71
GTUTUTGUTATUUAGGUUTATGAGAATUAAGUTTTTAURGTTUUUTTTGGGGUUTGGAT

AGTTTTUAAGUTGUUTTUTUUTUAUATUATGTGAGTAGAGURGGUUAAGAAAAUUAGG

GAGAUATUTUUUUUUAGUUTGGUUUAGAUTTUXGGGUUUUAUUUUAGGAGAGAGUUT

UUUAUTUTGGUUAGTGUUUTGUAAAGUUTTRGUUUUAUUURGUUUUAATTTUTUUTTA

TGUUTGGUTUATTUUTTAGGGGUTGAGTTGGGAGATAAGAUTTGGGGTTGUAGTTGUTG

GGGATUATT

72
AATGATUUUUAGUAAUTGUAAUUUUAAGTUTTATUTUUUAAUTUAGUUUUTAAGGAAT

GAGUUAGGUATAAGGAGAAATTGGGGRGGGGTGGGGRGAAGGUTTTGUAGGGUAUTGG

UUAGAGTGGGAGGUTUTUTUUTGGGGTGGGGUUXGGAAGTUTGGGUUAGGUTGGGGGG

AGATGTUTUUUTGGTTTTUTTGGURGGUTUTAUTUAUATGATGTGAGGAGAAGGUAGUT

TGAAAAUTATUUAGGUUUUAAAGGGAARGGTAAAAGUTTGATTUTUATAGGUUTGGAT

AGUAGAGAU

73
GRGURGGGGUUTARGAAGUUTGGGURGGGGGURGGGGGGRGGGGARGRGGGAUURGG

GGARGRGGGGRGUTUAGUUAGGUUUUUTUUAGURGRGURGGGGURGTUURGAGURGR

GRGUAUAAARGGATGGGURGGTGURGUUTGURGGGXGRGRGGGGGTRGGTGUUTTUTG

RGTGGRGRGRGTGTUUURGGGTUTURGTGRGGURGGRGUATTGGUUTRGRGUTUTURGG

AGGGGAUTGAGUAGGTGAAUAGGUURGGAGUUTGTRGTGGAGGGGUURGGGAAGGUU

TRGUTGUAGAAG

74
UTTUTGUAGRGAGGUUTTUURGGGUUUUTUUARGAUAGGUTURGGGUUTGTTUAUUTG

UTUAGTUUUUTURGGAGAGRGRGAGGUUAATGRGURGGURGUARGGAGAUURGGGGA

UARGRGRGUUARGUAGAAGGUAURGAUUUURGRGXGUURGGUAGGRGGUAURGGUUU

ATURGTTTGTGRGRGRGGUTRGGGARGGUUURGGRGRGGUTGGAGGGGGUUTGGUTGA

GRGUUURGRGTUUURGGGTUURGRGTUUURGUUUUURGGUUUURGGUUUAGGUTTRGT

AGGUUURGGRGU

75
GUAUAAARGGATGGGURGGTGURGUUTGURGGGRGRGRGGGGGTRGGTGUUTTUTGRG

TGGRGRGRGTGTUUURGGGTUTURGTGRGGURGGRGUATTGGUUTRGRGUTUTURGGAG

GGGAUTGAGUAGGTGAAUAGGUURGGAGUUTGTXGTGGAGGGGUURGGGAAGGUUTR

GUTGUAGAAGUAGGATGGGAGUAGGATURGUAGGGATGRGUAGRGGGGTURGRGGAG

UTUURGGRGGGGGRGRGTTTUUAGGURGGGGAURGRGGTGUUAGUUUTGUUUTRGRGG

ATRGGGTUTRG

76
RGAGAUURGATURGRGAGGGUAGGGUTGGUAURGRGGTUUURGGUUTGGAAARGRGUU

UURGURGGGAGUTURGRGGAUUURGUTGRGUATUUUTGRGGATUUTGUTUUUATUUTG

UTTUTGUAGRGAGGUUTTUURGGGUUUUTUUAXGAUAGGUTURGGGUUTGTTUAUUTG

UTUAGTUUUUTURGGAGAGRGRGAGGUUAATGRGURGGURGUARGGAGAUURGGGGA

UARGRGRGUUARGUAGAAGGUAURGAUUUURGRGRGUURGGUAGGRGGUAURGGUUU

ATURGTTTGTGU

77
UTTRGGGUAGURGAGGGRGRGGGATGAUTRGGGUAGGRGTGTGGUUTURGAUTGAUUA

GGGTTGGGUURGGGATUAGAGUTTTGUTTGGAAGTTUTUTGGGTGGUAGAGAUTRGGGU

AAURGGAGUUTGAGUUTUURGRGGGGAAGAGUTXGGAUTURGGAGGTRGRGURGUUTT

GGGUTTGAATTUAGTUUTTUUTTAUTAUTRGTGGUTTTGUTTAUATUATTTAAUTUTUUA

TTTUUTTGTUTATAUAGTGGGUTUATAAGGAGGRGGTAAGGAGATGATGATGTUTTTGA

AGAGTT

78
AAUTUTTUAAAGAUATUATUATUTUUTTAURGUUTUUTTATGAGUUUAUTGTATAGAUA

AGGAAATGGAGAGTTAAATGATGTAAGUAAAGUUARGAGTAGTAAGGAAGGAUTGAAT

TUAAGUUUAAGGRGGRGRGAUUTURGGAGTUXGAGUTUTTUUURGRGGGAGGUTUAGG

UTURGGTTGUURGAGTUTUTGUUAUUUAGAGAAUTTUUAAGUAAAGUTUTGATUURGG

GUUUAAUUUTGGTUAGTRGGAGGUUAUARGUUTGUURGAGTUATUURGRGUUUTRGGU

TGUURGAAG

79
URGUAGUUAUTAUTRGUUUUAUUTTUTUAUUTURGAGGGGTTGAAUURGAGTTGTUTGT

TGGGGTTTAGGGAUTUAGGATRGGGTUTGAGGGAUTTRGAUATAAGTGRGTGGRGGTAG

TGGGTATAUAGGGGAGGGGGGURGGGUUUUTXGUTUUTTUTGGAAAURGGGUUUUAUT

TGUAGGUURGGUUAUUTTGGGTTUTGGTGGURGAAGURGGAGUTGTGTTTUTRGUAGAU

TRGGGGAGUTAUATTGTGRGTAGGUAATTGTTTAGTTTGAAAGGAGGUAUATTTUAUUA

RGUAGU

80
GUTGRGTGGTGAAATGTGUUTUUTTTUAAAUTAAAUAATTGUUTARGUAUAATGTAGUT

UUURGAGTUTGRGAGAAAUAUAGUTURGGUTTRGGUUAUUAGAAUUUAAGGTGGURGG

GUUTGUAAGTGGGGUURGGTTTUUAGAAGGAGXGAGGGGUURGGUUUUUUTUUUUTGT

ATAUUUAUTAURGUUARGUAUTTATGTRGAAGTUUUTUAGAUURGATUUTGAGTUUUT

AAAUUUUAAUAGAUAAUTRGGGTTUAAUUUUTRGGAGGTGAGAAGGTGGGGRGAGTAG

TGGUTGRGG

81
UTUUUTGRGGUTUUAUTAGTTTTUTTRGUUURGUUUAGURGUUUAUTUTTUTRGGUTAG

GGAAGAAGAUUAGAGGGTGUTUAGUTGGAAAAUTUTGGTGTUTUAGUTTAGGGUUTUU

TURGGGAAGAGUTAAUTGUTUUUAGGTGAAGUXGGTGUURGRGGGRGGTURGTAUAUU

URGUAGURGGUTRGUAURGUTRGAGAGUUTRGGURGUTGTGTUTTUUARGTUTGUAGUT

UAGUUAGGGRGRGUAGGGRGAGTGGGGTUUAUTGGRGGGTAAAGGGGAUUAGGARGG

RGAGGATGG

82
UUATUUTRGURGTUUTGGTUUUUTTTAUURGUUAGTGGAUUUUAUTRGUUUTGRGRGU

UUTGGUTGAGUTGUAGARGTGGAAGAUAUAGRGGURGAGGUTUTRGAGRGGTGRGAGU

RGGUTGRGGGGTGTARGGAURGUURGRGGGUAUXGGUTTUAUUTGGGAGUAGTTAGUT

UTTUURGGAGGAGGUUUTAAGUTGAGAUAUUAGAGTTTTUUAGUTGAGUAUUUTUTGG

TUTTUTTUUUTAGURGAGAAGAGTGGGRGGUTGGGRGGGGRGAAGAAAAUTAGTGGAG

URGUAGGGAG

83
TAUATATGTAUARGTGTGUAUAURGGTUUTRGRGAGGRGGUUURGRGGGGTUATAGRGR

GGUAGRGTAGGURGRGGGGTAGGGGTUUAGUTGGGGUTTGUUUATGUURGGUAGUAGG

ATGTAGGRGAGRGGRGGUTGUAGUUXGGGGUTGGGUUAXGRGUTGUAGTTGUARGGGA

TUATGTAGUURGGGTTGUURGGGUTGGGGTGRGAGTGRGTGTGUUUUURGGRGGRGGR

GGURGURGUTGUAGURGURGURGURGRGURGTGGAAGGRGUURGRGUURGRGGTRGGG

TAGUUUAGU

84
GUTGGGUTAUURGAURGRGGGRGRGGGRGUUTTUUARGGRGRGGRGGRGGRGGUTGUA

GRGGRGGURGURGURGURGGGGGGUAUARGUAUTRGUAUUUUAGUURGGGUAAUURG

GGUTAUATGATUURGTGUAAUTGUAGRGXGTGGUUUAGUUUXGGGUTGUAGURGURGU

TRGUUTAUATUUTGUTGURGGGUATGGGUAAGUUUUAGUTGGAUUUUTAUUURGRGGU

UTARGUTGURGRGUTATGAUUURGRGGGGURGUUTRGRGAGGAURGGTGTGUAUARGT

GTAUATATGTA

85
ARGURGUUAGUTUTGATTGGUUUAGRGGTAGGAAAGGTTAAAUUAAAAATTTTTTTAUA

GUUUTAGTGTGRGUUTGTAGUTRGGAAAATTAATTGTGGUTATAGURGUUTRGATRGUT

GTUTUUUUAGUUTRGURGRGGURGUTURGGGAXGRGUURGUURGURGUURGGUTUTUU

UUUUUTTTGGGUTGUTGUTGUTGUTGUTGTGAUTGUTGUTGRGAGAGGAGGAGGAGGA

GGAGGAAGUAGRGGGGGGGGGAGRGGGGGGTGGGGGGGGAGAUUAAGAAGTAUAGTT

GGGAGRGAG

86
UTRGUTUUUAAUTGTAUTTUTTGGTUTUUUUUUUUAUUUUURGUTUUUUUUUURGUTG

UTTUUTUUTUUTUUTUUTUUTUTRGUAGUAGUAGTUAUAGUAGUAGUAGUAGUAGUUU

AAAGGGGGGGAGAGURGGGRGGRGGGRGGGRGXGTUURGGAGRGGURGRGGRGAGGU

TGGGGAGAUAGRGATRGAGGRGGUTATAGUUAUAATTAATTTTURGAGUTAUAGGRGU

AUAUTAGGGUTGTAAAAAAATTTTTGGTTTAAUUTTTUUTAURGUTGGGUUAATUAGAG

UTGGRGGRGT

87
AGGUAGAATAAAAUUUUUTUUUUTAGAGURGGGGTGGUTUAGRGGAATUATRGAGAAT

GAGAURGUTGGTTGUTAATGGGUTTGGGGAAAATGGGATGUAATTTUUURGGTGTTTTT

UAGGUUUAGAGUTATTGAATAAATGAAGTGRGXGURGGRGGAGTUAGTAAUTUAUTGR

GRGGUTUURGGUAGGRGGGGGRGGAGTGGGGGURGUAGAAURGGARGTGUUTGGRGA

GGTTUAGAGGRGUTAGRGGTGTGGTGGGTGGGUAGRGURGGGATGGUTGGAGGGAGGG

GUTARGGGGG

88
UUUURGTAGUUUUTUUUTUUAGUUATUURGGRGUTGUUUAUUUAUUAUAURGUTAGRG

UUTUTGAAUUTRGUUAGGUARGTURGGTTUTGRGGUUUUUAUTURGUUUURGUUTGUR

GGGAGURGRGUAGTGAGTTAUTGAUTURGURGGXGRGUAUTTUATTTATTUAATAGUTU

TGGGUUTGAAAAAUAURGGGGAAATTGUATUUUATTTTUUUUAAGUUUATTAGUAAUU

AGRGGTUTUATTUTRGATGATTURGUTGAGUUAUUURGGUTUTAGGGGAGGGGGTTTTA

TTUTGUUT

89
UUUTGURGAGGTTATGGTGUUTGTTTUUAGUAGUTUTGAGUUUARGRGGAAUATGAGTR

GUTGATAAAUAAGGUTTGUAGTTGRGAGGGGUUTRGGGGTGGGTTTUTGGTTGAGGAAR

GURGGGGAATGGUTGATGGAGGAAGUUUUTGUXGGGAGUAGGAGUAGRGGAAAUUTA

UATRGGUUAUAUAGATUURGGRGGGGGAGGGAGGGUAARGUTUAGAAGUAGAGUTGR

GRGATGTGUUTTTUTGGGUTGUTUAGUUUAGGGUURGAGAUUTGUTTGUUTGAGGAGG

GGUAGATGUA

90
TGUATUTGUUUUTUUTUAGGUAAGUAGGTUTRGGGUUUTGGGUTGAGUAGUUUAGAAA

GGUAUATRGRGUAGUTUTGUTTUTGAGRGTTGUUUTUUUTUUUURGURGGGATUTGTGT

GGURGATGTAGGTTTURGUTGUTUUTGUTUUXGGUAGGGGUTTUUTUUATUAGUUATTU

UURGGRGTTUUTUAAUUAGAAAUUUAUUURGAGGUUUUTRGUAAUTGUAAGUUTTGTT

TATUAGRGAUTUATGTTURGRGTGGGUTUAGAGUTGUTGGAAAUAGGUAUUATAAUUT

RGGUAGGG

91
GGUUURGARGUUAGUTGTUUUUURGAGAAGUTURGUAGRGRGTRGUTUUAGGAGTGRG

AGGAGUUATAGATGGRGUTGURGTUUAGUUAGUURGTUAUUTGGTTGGUUTGRGGGGU

ARGRGGRGGGTGAGUURGGGTRGAGAGGRGGRGGXGGGUUAGGGAAGGURGGGGUUR

GGRGGGTGTURGRGGUTGGGGAGTGGGRGUUTUTUUUUTUUAGGUUUTGUUAGGUURG

AAGUUUAGGUUUUAUUTGGUTGGGGTGRGGTUUUTTUURGURGUUTTUUURGUUTUAU

UAGGTUURGGG

92
UURGGGAUUTGGTGAGGRGGGGAAGGRGGRGGGAAGGGAURGUAUUUUAGUUAGGTG

GGGUUTGGGUTTRGGGUUTGGUAGGGUUTGGAGGGGAGAGGRGUUUAUTUUUUAGUR

GRGGAUAUURGURGGGUUURGGUUTTUUUTGGUUXGURGURGUUTUTRGAUURGGGUT

UAUURGURGRGTGUUURGUAGGUUAAUUAGGTGARGGGUTGGUTGGARGGUAGRGUU

ATUTATGGUTUUTRGUAUTUUTGGAGRGARGRGUTGRGGAGUTTUTRGGGGGGAUAGUT

GGRGTRGGGGUU

93
GTTTTTRGTTTUUAATGUUUTGGAUUTTTAGAGAGUTUATUATUTTTUUTTATGGTRGUA

AGTUTTGAAAGAAAGAGUAAGAGAGRGAGRGGGTTGGGTTGGGGUTGGAGTAGURGAG

GURGGUUTGGGTURGGGUAGTUAGGUUTGAXGRGGUUURGRGUUUTTUUURGGUAGAG

AAGUURGGGARGGUUATGTGRGTGGGUTGRGGGAGTUAGATUUARGAUUAGTTTATUU

TGRGGGTGTRGUURGAUUTRGAGTGGUARGRGGUUTGUUTUAAGTGTGURGAGTGUAG

UUAGTAUU

94
GGTAUTGGUTGUAUTRGGUAUAUTTGAGGUAGGURGRGTGUUAUTRGAGGTRGGGRGA

UAUURGUAGGATAAAUTGGTRGTGGATUTGAUTUURGUAGUUUARGUAUATGGURGTU

URGGGUTTUTUTGURGGGGAAGGGRGRGGGGURGXGTUAGGUUTGAUTGUURGGAUUU

AGGURGGUUTRGGUTAUTUUAGUUUUAAUUUAAUURGUTRGUTUTUTTGUTUTTTUTTT

UAAGAUTTGRGAUUATAAGGAAAGATGATGAGUTUTUTAAAGGTUUAGGGUATTGGAA

ARGAAAAAU

95
TGTAUUUTGGGTTUUUUTAUTTTUUUUUAGTTGGGUUUUAUATUTGGGTUTGGUTUTTA

AGTUTTGGGUATATGGGATAGAGAATTTAAGGATUUAGATAUTTTGUAATUUURGGGUT

AGTAGGAGGAAAAGTGGUTUUTUTGGGAAUUXGGATGGGURGAAUAGUAGUTTURGGU

AGAGGUUUTUAGGTTTUAGAGTTUTUUAAGTGGGGAGAAGGGRGGUUTGUTUAGUURG

RGUAUURGTGGUAGUAUUAUAGAGGGAATAAAAGUAGGGTAAAUAATTTGAAGATUU

UUARGAAGG

96
UUTTRGTGGGGATUTTUAAATTGTTTAUUUTGUTTTTATTUUUTUTGTGGTGUTGUUARG

GGTGRGRGGGUTGAGUAGGURGUUUTTUTUUUUAUTTGGAGAAUTUTGAAAUUTGAGG

GUUTUTGURGGAAGUTGUTGTTRGGUUUATUXGGGTTUUUAGAGGAGUUAUTTTTUUTU

UTAUTAGUURGGGGATTGUAAAGTATUTGGATUUTTAAATTUTUTATUUUATATGUUUA

AGAUTTAAGAGUUAGAUUUAGATGTGGGGUUUAAUTGGGGGAAAGTAGGGGAAUUUA

GGGTAUA

97
UTGUUTGUAGUURGAGGAGUTGAUTAATGUTUTGGAGATUAGUAAUATRGTGTTUAUU

AGUATGTTTGUUUTGGAGATGUTGUTGAAGUTGUTGGUUTGRGGUUUTUTGGGUTAUAT

URGGAAUURGTAUAAUATUTTRGARGGUATUATXGTGGTUATUAGGTGGGTUUUUAUU

UTUTUUUUAGGAAGAGGGGUURGGGAAGUTUUAUTUTUTGGUAGAAATUUUAUUTGUA

GAGUAAAAUUUAGAGUAUAGGAGGAAGTARGARGATAGUTUTTTATGAUAGGURGTGG

GAAGUAGGT

98
AUUTGUTTUUUARGGUUTGTUATAAAGAGUTATRGTRGTAUTTUUTUUTGTGUTUTGGG

TTTTGUTUTGUAGGTGGGATTTUTGUUAGAGAGTGGAGUTTUURGGGUUUUTUTTUUTG

GGGAGAGGGTGGGGAUUUAUUTGATGAUUAXGATGATGURGTRGAAGATGTTGTARGG

GTTURGGATGTAGUUUAGAGGGURGUAGGUUAGUAGUTTUAGUAGUATUTUUAGGGUA

AAUATGUTGGTGAAUARGATGTTGUTGATUTUUAGAGUATTAGTUAGUTUUTRGGGUTG

UAGGUAG

99
UUUUUTGGAAGGTGUTAATTTUTGAGGUUTUUAGGGGAAUAATGAGGAGGAGAAAAAT

UUUAGTGTGAAAATGATGTUAGRGGAGTTGUTTGTUUUTTTGTUTGTUTGTUUTTTATAA

GUUATUUUUTUTGUAGAGGUTGUTGGGURGGXGGGAUTGTUUUTUUTUUTUTTTGUUU

ATUTUTTTGGURGGGTGAGTUTUATUTUAAGAUTUUTUAUTGTAUTUTUAUAGUTTGGT

UTUUATAUAAATGAAGTTUAUUUTUTGGGUTUTGAAAUAGUTTUTUAUTUUAUUUAAA

AAGATUU

100
GGATUTTTTTGGGTGGAGTGAGAAGUTGTTTUAGAGUUUAGAGGGTGAAUTTUATTTGT

ATGGAGAUUAAGUTGTGAGAGTAUAGTGAGGAGTUTTGAGATGAGAUTUAUURGGUUA

AAGAGATGGGUAAAGAGGAGGAGGGAUAGTUUXGURGGUUUAGUAGUUTUTGUAGAG

GGGATGGUTTATAAAGGAUAGAUAGAUAAAGGGAUAAGUAAUTURGUTGAUATUATTT

TUAUAUTGGGATTTTTUTUUTUUTUATTGTTUUUUTGGAGGUUTUAGAAATTAGUAUUT

TUUAGGGGG

101
RGTRGTAGTTGGGGURGAGGTURGTGTGGTAGGGGAUTGGGAUAGGAGTRGGAGTRGU

AGGGRGAGGGUUUAGATRGGGGUTGGAAGUUAUAGGUUTUTUAUUTRGUTTGGURGAG

RGGURGGGAUTURGGGUURGUAGRGGATGGAAGXGGGRGUTGUUUTGUURGGRGGRGG

URGGGGUUTTUTGGGGTTTTUURGRGGGRGGUTRGGUAGGGRGGUAGUAGUTGURGRG

GAGAGGARGAURGUUAURGGTGUAAUUAGGTUUURGRGUUUUTGUURGGUUTUTGGG

GRGUUUAAGRG

102
RGUTTGGGRGUUUUAGAGGURGGGUAGGGGRGRGGGGAUUTGGTTGUAURGGTGGRGG

TRGTUUTUTURGRGGUAGUTGUTGURGUUUTGURGAGURGUURGRGGGAAAAUUUUAG

AAGGUUURGGURGURGURGGGUAGGGUAGRGUUXGUTTUUATURGUTGRGGGUURGG

AGTUURGGURGUTRGGUUAAGRGAGGTGAGAGGUUTGTGGUTTUUAGUUURGATUTGG

GUUUTRGUUUTGRGAUTURGAUTUUTGTUUUAGTUUUUTAUUAUARGGAUUTRGGUUU

UAAUTARGARG

103
RGUAGGURGUAGTGARGTRGGRGGGRGUTGUATTAAGGRGAGGGGGUTTUUAGAGUTU

AGUUAAUAGUUAGGAGUAGTGAUUAAGURGURGGAGUTGGGGAGAGARGUAURGGGG

RGGRGAUTGGGUUAGGAGAUUAGGGAUTGAGGGAXGRGUURGGGGAGGGUUAGUAAU

AAGURGRGGUURGGGRGRGAUAGRGGRGGTURGRGURGAGURGTTUUAGURGURGGUU

TAUTGTAGURGUTGRGUUAGGAUATTTTTTTTTAAAGUTUTUUAAGUTGUUUUUUTUUT

UUURGAUTUUT

104
AGGAGTRGGGGAGGAGGGGGGUAGUTTGGAGAGUTTTAAAAAAAAATGTUUTGGRGUA

GRGGUTAUAGTAGGURGGRGGUTGGAARGGUTRGGRGRGGAURGURGUTGTRGRGUUR

GGGURGRGGUTTGTTGUTGGUUUTUUURGGGRGXGTUUUTUAGTUUUTGGTUTUUTGGU

UUAGTRGURGUUURGGTGRGTUTUTUUUUAGUTURGGRGGUTTGGTUAUTGUTUUTGGU

TGTTGGUTGAGUTUTGGAAGUUUUUTRGUUTTAATGUAGRGUURGURGARGTUAUTGRG

GUUTGRG

105
RGUUUTRGGUUURGUUUUUTGUURGUUUUTUUUTUTRGGGGTURGGGGRGRGAGUTGR

GGRGGRGGRGGUUARGGTUATTGGRGURGAGRGGTTURGGUTGAUTGGARGGGGRGGG

RGTUURGGGUAGUUTAGRGRGGTAUUTUURGUUUXGRGRGUUUAGURGGRGAGGGAUA

TTGGAUUAGGGTRGGGGGTRGRGGURGUTUUAGRGAGGTAAGAGURGGGAAGAURGGG

AGAGAUUAUUTUTTUUATUUTGGGGGGGTUUUTGGGGGARGGTUTUUUAURGGTGUTG

GGURGRGGRG

106
RGURGRGGUUUAGUAURGGTGGGAGAURGTUUUUUAGGGAUUUUUUUAGGATGGAAG

AGGTGGTUTUTUURGGTUTTUURGGUTUTTAUUTRGUTGGAGRGGURGRGAUUUURGAU

UUTGGTUUAATGTUUUTRGURGGUTGGGRGRGXGGGGRGGGAGGTAURGRGUTAGGUT

GUURGGGARGUURGUUURGTUUAGTUAGURGGAAURGUTRGGRGUUAATGAURGTGGU

RGURGURGURGUAGUTRGRGUUURGGAUUURGAGAGGGAGGGGRGGGUAGGGGGRGG

GGURGAGGGRG

107
RGUUUTURGGUURGGUUTUUTURGURGGUURGGUTUURGAGAGGRGUAGUUAGUTUTU

RGUUATGTUTGRGGGGAUTUTURGAGGGGGRGGTUAGUATUUARGGGUTGAGURGGGG

GTGGUURGURGRGUTUTUTGGUURGUTGAGTUUXGUAGUTUURGTTAUARGGUUTUUR

GGARGRGRGUTTUUATUTRGRGAUUURGGGGGRGUUTUUTURGAATAAGTATGTGGTGU

UTGRGAGGAUUARGGTGGGAGUTGAGUAUTAUUUUAUUUURGAGGGGAUAGTGTGTGT

ARGGGGARG

108
RGTUUURGTAUAUAUAUTGTUUUUTRGGGGGTGGGGTAGTGUTUAGUTUUUAURGTGG

TUUTRGUAGGUAUUAUATAUTTATTRGGAGGAGGRGUUUURGGGGTRGRGAGATGGAA

GRGRGRGTURGGGAGGURGTGTAARGGGAGUTGXGGGAUTUAGRGGGUUAGAGAGRGR

GGRGGGUUAUUUURGGUTUAGUURGTGGATGUTGAURGUUUUUTRGGAGAGTUUURGU

AGAUATGGRGGAGAGUTGGUTGRGUUTUTRGGGAGURGGGURGGRGGAGGAGGURGGG

URGGAGGGRG

109
RGGGGTGUAGAGGTAAGAATGGGGGUAGAAAAUUUUUAGGTRGTGGTUUTGGGUUUAG

AAAGTTGUUUUAGUUTGRGRGUUUUTTUUUAGUUUUTUAGGGTUUTTUUTUAUUURGR

GGARGGTUUUATURGGGTGGUAAAGTTAGTGTGXGGRGUUTTGGAGUTUUUUUTURGG

TUUUTUURGTUURGUURGTUTGUUUUTAGGTRGGUTAUUUUUUAAUUUUTURGUUTGT

GUUAUUUTUTUUUUAGUUTTTGGTGGUAUTGUTUTUUTUUURGRGGGGUTRGGGUUTG

GUTURGARGA

110
TRGTRGGAGUUAGGUURGAGUUURGRGGGGAGGAGAGUAGTGUUAUUAAAGGUTGGG

GAGAGGGTGGUAUAGGRGGAGGGGTTGGGGGGTAGURGAUUTAGGGGUAGARGGGRG

GGARGGGAGGGAURGGAGGGGGAGUTUUAAGGRGUXGUAUAUTAAUTTTGUUAUURG

GATGGGAURGTURGRGGGGTGAGGAAGGAUUUTGAGGGGUTGGGAAGGGGRGRGUAG

GUTGGGGUAAUTTTUTGGGUUUAGGAUUARGAUUTGGGGGTTTTUTGUUUUUATTUTTA

UUTUTGUAUUURG

111
GGTAATTTRGGUUUUUTGAGUTTGGUTTAGTTTTTURGRGAAGTGUARGGGGGURGTTTT

AGGATAUUUAGUTUUUAUUTGGAGUTUUAGAGUTUURGGGGAUUUUUTTGTURGUURG

TUTUUTAGGGUUTGGUAUTUUUTGGUUUXGUAGUUXGGGGAUUTUUAUUTTUUUUAGG

RGGUAGUUAUAGGTUURGURGGGUURGTURGAGGTUTGRGGURGURGAAGTRGGGGTU

TUAGGGRGTUAGGGAGUAAUUAGGURGRGGGGAGGGAGGURGGRGURGGRGRGGAAT

TTUTTTATU

112
GATAAAGAAATTURGRGURGGRGURGGUUTUUUTUUURGRGGUUTGGTTGUTUUUTGA

RGUUUTGAGAUUURGAUTTRGGRGGURGUAGAUUTRGGARGGGUURGGRGGGAUUTGT

GGUTGURGUUTGGGGAAGGTGGAGGTUUUXGGGUTGXGGGGUUAGGGAGTGUUAGGU

UUTAGGAGARGGGRGGAUAAGGGGGTUUURGGGAGUTUTGGAGUTUUAGGTGGGAGUT

GGGTATUUTAAAARGGUUUURGTGUAUTTRGRGGAAAAAUTAAGUUAAGUTUAGGGGG

URGAAATTAUU

113
AGATTGGGAATUTGGAGGGTAAAATGURGGGTUUUTTUURGAGUUUTTAGAGUURGAA

RGTTGTRGAATRGGTUAATGTUUAUUURGUTGUTUAUUTUTGTUUUAGUAGRGURGGGG

UUAGRGRGUUUTUURGURGRGTUTGRGGAGUTGRGGGAAAAGUAGGTUUURGGGGGGT

ATRGAGTGTUUAGGGATATUUARGUAGAUATUUTTGTAUTTGUAAATAUAAAGAAAUA

UAUAGGAUAGAGATUAGGUTRGRGUUAGGGUTRGATUTUUTRGGGGUTUAGGGTTGGA

GGURGTAUA

114
TGTARGGUUTUUAAUUUTGAGUUURGAGGAGATRGAGUUUTGGRGRGAGUUTGATUTU

TGTUUTGTGTGTTTUTTTGTATTTGUAAGTAUAAGGATGTUTGRGTGGATATUUUTGGAU

AUTRGATAUUUUURGGGGAUUTGUTTTTUURGUAGUTURGUAGARGRGGRGGGAGGGR

GRGUTGGUUURGGRGUTGUTGGGAUAGAGGTGAGUAGRGGGGTGGAUATTGAURGATT

RGAUAARGTTRGGGUTUTAAGGGUTRGGGAAGGGAUURGGUATTTTAUUUTUUAGATT

UUUAATUT

115
AGGGUUTUAGRGGRGGUAUURGUATUUTUTTTUTTATGGUAUAAGUTGAAGGRGGRGG

URGRGGUAGGGAAGGTGTAGGGGTGRGGGAAGAGUURGURGUAGUURGGGAAUTTUTG

UAGGARGUUUURGAARGAGUUUTTGUTGGGUAUXGGGATGAUTGGGTAGUTGRGRGGG

UUTTTGGUUUUTGUUURGGUAUURGUUUURGRGUURGRGUUUARGUUUARGURGGUUA

UAUAGUUAUUUUUAGRGURGUUUUUAUUAGTUUURGRGURGUURGAAGUAGUAGUUA

UAGAGAGGUTG

116
UAGUUTUTUTGTGGUTGUTGUTTRGGGRGGRGRGGGGAUTGGTGGGGGRGGRGUTGGG

GGTGGUTGTGTGGURGGRGTGGGRGTGGGRGRGGGRGRGGGGGRGGGTGURGGGGUAG

GGGUUAAAGGUURGRGUAGUTAUUUAGTUATUUXGGTGUUUAGUAAGGGUTRGTTRGG

GGGRGTUUTGUAGAAGTTUURGGGUTGRGGRGGGUTUTTUURGUAUUUUTAUAUUTTU

UUTGURGRGGURGURGUUTTUAGUTTGTGUUATAAGAAAGAGGATGRGGGTGURGURG

UTGAGGUUUT

117
AUTARGGTURGURGGGUUARGAUAAAATGUTUAGUUUUAAUTTRGARGRGUAUUAUAU

TGUUATGUTGAUURGRGGTGAGUAAUAUUTGTUURGRGGUUTGGGUAUUUUAUUTGRG

GUUATGATGTRGUAUUTGAARGGUUTGUAUUAUUXGGGUUAUAUTUAGTUTUARGGGU

RGGTGUTGGUAUUUAGTRGRGAGRGGUUAUUUTRGTUUTUATRGGGUTRGUAGGTGGU

UARGTRGGGUUAGUTGGAAGAAATUAAUAUUAAAGAGGTGGUUUAGRGUATUAUAGR

GGAGUTGAAGU

118
GUTTUAGUTURGUTGTGATGRGUTGGGUUAUUTUTTTGGTGTTGATTTUTTUUAGUTGGU

URGARGTGGUUAUUTGRGAGUURGATGAGGARGAGGGTGGURGUTRGRGAUTGGGTGU

UAGUAURGGUURGTGAGAUTGAGTGTGGUUXGGGTGGTGUAGGURGTTUAGGTGRGAU

ATUATGGURGUAGGTGGGGTGUUUAGGURGRGGGAUAGGTGTTGUTUAURGRGGGTUA

GUATGGUAGTGTGGTGRGRGTRGAAGTTGGGGUTGAGUATTTTGTRGTGGUURGGRGGA

URGTAGT

119
TGGGGTUAGTAGATUAGTUTUTTUAGAUAUTGATGUAGAAGUTGGGAUTGGTAAGTAG

GTATTATGTGUTRGGAGRGUTAGGGGAUAGGAGUAAATGGAGAAGAAAAGRGGAGGUT

TTUTURGUURGGAGTATRGATRGGAATUUURGUXGGTARGURGUAGAGGGUUUTRGUR

GTTGGGUUURGGGGGTTTAAUAAGUUUAGURGUTURGUAGGRGGUTRGGURGGAUTUT

UAGAURGGTGUUTGGAAGAUAURGTUUUTGUUUUUUTUURGUUAAAUUTGUUTUTTUT

UTTTUTUTUA

120
TGAGAGAAAGAGAAGAGGUAGGTTTGGRGGGAGGGGGGUAGGGARGGTGTUTTUUAGG

UAURGGTUTGAGAGTURGGURGAGURGUUTGRGGAGRGGUTGGGUTTGTTAAAUUUUR

GGGGUUUAARGGRGAGGGUUUTUTGRGGRGTAUXGGRGGGGATTURGATRGATAUTUR

GGGRGGAGAAAGUUTURGUTTTTUTTUTUUATTTGUTUUTGTUUUUTAGRGUTURGAGU

AUATAATAUUTAUTTAUUAGTUUUAGUTTUTGUATUAGTGTUTGAAGAGAUTGATUTAU

TGAUUUUA

121
AGUTURGAGUUUARGUTGUAGUUAGATURGGATGAGTURGTUUTURGUUURGGGRGGG

UTUTRGUTUTRGUTGGUUUTUAGRGURGRGUAGUUAGUAGUATUUUUAURGTGARGUT

RGUATUAUAUURGGGRGURGGURGUUAUUATUXGRGURGURGURGTUAGGAUUUTUUT

UURGGGUATRGTRGURGURGRGGGGTRGGGAGGARGRGGRGRGRGGGAGGRGGRGGTR

GUAGGGRGAGUUURGGGARGUUURGAGURGGGGURGGGGURGGGGAGAGGGRGUAGR

GAGGTGGGGGU

122
GUUUUUAUUTRGUTGRGUUUTUTUUURGGUUURGGUUURGGUTRGGGGRGTUURGGGG

UTRGUUUTGRGAURGURGUUTUURGRGRGURGRGTUUTUURGAUUURGRGGRGGRGAR

GATGUURGGGAGGAGGGTUUTGARGGRGGRGGRGXGGATGGTGGRGGURGGRGUURGG

GTGTGATGRGAGRGTUARGGTGGGGATGUTGUTGGUTGRGRGGRGUTGAGGGUUAGRG

AGAGRGAGAGUURGUURGGGGRGGAGGARGGAUTUATURGGATUTGGUTGUAGRGTGG

GUTRGGAGUT

123
AGUUUUTGGRGTUTTTUTRGGGGUUAGAGUTTTGUUTUTAAATGGTUTTTGTTTGUAGA

RGATUTGUTGTGUAGATUATGAAURGTRGURGUAGTGUUAGAUAGAUTUTTUATURGG

AURGGURGTUUUAUTGGGTUUUUTUAGGAAAGXGUAGAAGURGGGGAUUUUAAAURG

RGTUUAGUTUUTUUUAUURGUUUTTUTRGGAGURGRGUAUTTGGTAAUTTGRGTGTGAA

GAAUTUARGGURGTGTTGUUAGGAAAUTGRGGGGAGUAGUUTTTGTGGTUTUAUATAT

GRGTAAUAT

124
ATGTTARGUATATGTGAGAUUAUAAAGGUTGUTUUURGUAGTTTUUTGGUAAUARGGU

RGTGAGTTUTTUAUARGUAAGTTAUUAAGTGRGRGGUTURGAGAAGGGRGGGTGGGAG

GAGUTGGARGRGGTTTGGGGTUUURGGUTTUTGXGUTTTUUTGAGGGGAUUUAGTGGG

ARGGURGGTURGGATGAAGAGTUTGTUTGGUAUTGRGGRGARGGTTUATGATUTGUAUA

GUAGATRGTUTGUAAAUAAAGAUUATTTAGAGGUAAAGUTUTGGUUURGAGAAAGARG

UUAGGGGUT

125
GUTTGAAUAGTTTTTTUUAATGTTRGATATTTAUATTTTTTGTUAUTTTTATTTTAGTUUT

URGRGAAGGAGUUTUTUAAATATTUURGGAGUAUAGGUTAATTTAGAARGTGTTATUUT

GAUAAGGRGGUUUAGUUAGGGAGGAGARGUXGGGGTURGRGURGRGGGGRGGGRGUT

TUUURGGAUURGGGGGAGGRGGGARGUAGGRGAAGGURGUUURGGGAGAGRGGGGTU

URGGGAGAGRGGGGTUURGGUTGTGGGGGARGRGGGURGAGGUTGTRGRGAAGURGUT

GARGGURG

126
RGGURGTUAGRGGUTTRGRGAUAGUUTRGGUURGRGTUUUUUAUAGURGGGAUUURGU

TUTUURGGGAUUURGUTUTUURGGGGRGGUUTTRGUUTGRGTUURGUUTUUUURGGGT

URGGGGAAGRGUURGUUURGRGGRGRGGAUUUXGGRGTUTUUTUUUTGGUTGGGURGU

UTTGTUAGGATAAUARGTTUTAAATTAGUUTGTGUTURGGGAATATTTGAGAGGUTUUT

TRGRGGAGGAUTAAAATAAAAGTGAUAAAAAATGTAAATATRGAAUATTGGAAAAAAU

TGTTUAAGU

127
UUTTRGGGGAUUAGAURGRGUTUUUTUAGATGGUTGGGGAGGGAGUAGAUTURGGGTG

AAGGUTGGGGGAGGTAURGGGATGURGAGTARGUUAGAGUAGGRGGGGGATGGGTTUR

GGUTTUUTGURGUUTRGGUATUTRGUTTTGUAUXGGGUAAAGAAGGGGUUARGAURGG

RGAAGAGRGRGTGGAGAUAUAGGGAUURGRGATUUAGGGGUAGGAAUUURGUUUUUT

UURGGAAAUUTUURGGGUUUTGAGTRGTGUURGGRGUTUUUUAUUUUAUUUUAUUUU

RGURGUUUUTGU

128
GUAGGGGRGGRGGGGGTGGGGTGGGGTGGGGAGRGURGGGUARGAUTUAGGGUURGG

GAGGTTTURGGGAGGGGGRGGGGTTUUTGUUUUTGGATRGRGGGTUUUTGTGTUTUUAR

GRGUTUTTRGURGGTRGTGGUUUUTTUTTTGUUXGGTGUAAAGRGAGATGURGAGGRGG

UAGGAAGURGGAAUUUATUUUURGUUTGUTUTGGRGTAUTRGGUATUURGGTAUUTUU

UUUAGUUTTUAUURGGAGTUTGUTUUUTUUUUAGUUATUTGAGGGAGRGRGGTUTGGT

UUURGAAGG

129
GGATGGGTUURGGRGRGGUUUAGUUUUTGUURGGUURGURGGGUAGAGAUTGAAURGR

GGATUUUUAURGTUUTGTGGARGAURGGAUAGAGAGAGGUAUTGAURGATRGUUAGUA

GUUTUURGGTGGGAURGRGTUTUUTGUAUAUUUXGRGUAGRGUUUUURGURGGAGURG

UAURGGGUAAGURGGRGAGGGAGRGGGGUTGATTGGRGGURGURGGRGGUUAGGGGA

GGGGGRGURGRGRGGGGUUATGGUAGGUTRGGAGGRGTUUTAGUURGAGURGGAGURG

ATURGAGUUUA

130
TGGGUTRGGATRGGUTURGGUTRGGGUTAGGARGUUTURGAGUUTGUUATGGUUURGR

GRGGRGUUUUUTUUUUTGGURGURGGRGGURGUUAATUAGUUURGUTUUUTRGURGGU

TTGUURGGTGRGGUTURGGRGGGGGGRGUTGRGXGGGGTGTGUAGGAGARGRGGTUUU

AURGGGAGGUTGUTGGRGATRGGTUAGTGUUTUTUTUTGTURGGTRGTUUAUAGGARGG

TGGGGATURGRGGTTUAGTUTUTGUURGGRGGGURGGGUAGGGGUTGGGURGRGURGG

GAUUUATUU

131
AGAAGAGGAAAAURGAGURGGRGGUTGRGGURGGGUATRGTATGGRGUTAGAGAAUTT

RGGRGGRGAGRGGGAUUTGRGUUTGGGURGURGUUTUUURGUURGRGGTUURGGGAUR

GTTAUTTTGAAAAGGAGTUURGAGGUUTGGRGUUXGGRGRGRGATRGGGAUUURGRGT

UUAGUTURGGGGAUURGGURGGRGUUUUUUAUURGRGAGRGGUURGRGAGUUAUTUT

UAGGUUURGGGAAAUTTTUAAGAGGGTUTGGGGGGTGGGGGGAGTAAAAAGGGGAGG

GGGTAGGUTGGG

132
UUUAGUUTAUUUUUTUUUUTTTTTAUTUUUUUUAUUUUUUAGAUUUTUTTGAAAGTTT

UURGGGGUUTGAGAGTGGUTRGRGGGURGUTRGRGGGTGGGGGGRGURGGURGGGTUU

URGGAGUTGGARGRGGGGTUURGATRGRGRGUXGGGRGUUAGGUUTRGGGAUTUUTTT

TUAAAGTAARGGTUURGGGAURGRGGGRGGGGAGGRGGRGGUUUAGGRGUAGGTUURG

UTRGURGURGAAGTTUTUTAGRGUUATARGATGUURGGURGUAGURGURGGUTRGGTTT

TUUTUTTUT

133
TTUTUUUTGAAAUTTGTTTGAAAAUUUAAGTGAAAAURGRGARGATUTGUAUAUTUAAA

AGUAAGTGUUAAGTAAGTGUUUTGRGGTGGUUTRGGRGRGUUURGGGGTGAGRGRGUA

AAGURGGGAGRGAGGTGURGRGAGUTGRGRGUXGUUURGGGURGGUUTRGRGTGTGGG

RGGUTGGGRGGUTGGGRGGRGGGAGGARGGUUTURGRGGGUTURGGGAUTGUURGGUT

GGUTGUTRGUAUAAUTTTTTTTTTTTUUUURGTUTGUTGAUTTTTRGGGUUAGGTGAAGT

GTTTGGG

134
UUUAAAUAUTTUAUUTGGUURGAAAAGTUAGUAGARGGGGGAAAAAAAAAAAGTTGTG

RGAGUAGUUAGURGGGUAGTUURGGAGUURGRGGAGGURGTUUTUURGURGUUUAGU

RGUUUAGURGUUUAUARGRGAGGURGGUURGGGGXGGRGRGUAGUTRGRGGUAUUTR

GUTUURGGUTTTGRGRGUTUAUUURGGGGRGRGURGAGGUUAURGUAGGGUAUTTAUT

TGGUAUTTGUTTTTGAGTGTGUAGATRGTRGRGGTTTTUAUTTGGGTTTTUAAAUAAGTT

TUAGGGAGAA

135
ATTURGGGAGATURGRGUUUAGGUURGRGRGUTRGGGGURGUTUTGGUUTUAGAGURG

UTGUURGAUUUAGGAAURGGUAURGRGTRGUUAAGGGUAGTUATTGGRGGURGUAGAR

GGAGGAGGARGGRGTTGGURGGGARGRGGAUAGXGUAGGGUAGRGGRGGGGGRGRGG

GURGGGGUUARGGGRGUAGGGGURGGAGURGUTGUAGURGUAAGURGTTGUARGTGG

AUTTUAAGGAGUTRGGUTGGGARGAUTGGATUATRGRGURGUTGGAUTARGAGGRGTA

UUAUTGRGAGGG

136
UUUTRGUAGTGGTARGUUTRGTAGTUUAGRGGRGRGATGATUUAGTRGTUUUAGURGA

GUTUUTTGAAGTUUARGTGUAARGGUTTGRGGUTGUAGRGGUTURGGUUUUTGRGUUR

GTGGUUURGGUURGRGUUUURGURGUTGUUUTGXGUTGTURGRGTUURGGUUAARGUR

GTUUTUUTURGTUTGRGGURGUUAATGAUTGUUUTTGGRGARGRGGTGURGGTTUUTGG

GTRGGGUAGRGGUTUTGAGGUUAGAGRGGUUURGAGRGRGRGGGUUTGGGRGRGGATU

TUURGGAAT

137
GGGGURGUAGGGGGUTGGAAGGAAGTGRGUAGTGTGGRGAGAGRGRGAAUAAAGUUU

TUTURGGAGUUURGTUAUUUUTRGGTGAUUUUAGGUURGUUURGUTGAGURGRGGGGU

TUURGGGUUTRGUUTURGAGUAGGURGUAURGUUXGGTGGGRGUARGTUAGGUUTURG

RGGURGUURGGGUUAGTGUTUUUUTRGGTUUURGUAGGURGAGUURGRGGURGGUUUR

GAAGGRGRGAGGGAUAGRGRGGURGGRGGTGGAGUUTUAUTUAGGUARGGAGUUUAU

AGAGRGAGGUTG

138
UAGUUTRGUTUTGTGGGUTURGTGUUTGAGTGAGGUTUUAURGURGGURGRGUTGTUU

UTRGRGUUTTRGGGGURGGURGRGGGUTRGGUUTGRGGGGAURGAGGGGAGUAUTGGU

URGGGRGGURGRGGAGGUUTGARGTGRGUUUAUXGGGRGGTGRGGUUTGUTRGGAGGR

GAGGUURGGGAGUUURGRGGUTUAGRGGGGRGGGUUTGGGGTUAURGAGGGGTGARG

GGGUTURGGAGAGGGUTTTGTTRGRGUTUTRGUUAUAUTGRGUAUTTUUTTUUAGUUUU

UTGRGGUUUU

139
URGUTGUUUURGGAUUUTUTUTGUUTGUAUAAUTRGTRGUTUTTRGRGUTGUAGAAUUT

GUAGUUUTGGGURGAGGAUAAUAAAGTGGUTTUAGTGTURGGGUTRGUUTRGGTGGTG

TGAGRGARGUURGTURGATRGGRGTGGAGRGUXGGGUURGGAGRGGTGGAGRGRGRGG

UTGUUTGRGTUUATGGTUTAGTGGUAGURGGGRGRGTGAGGAGRGGUAGGUUTTGAGG

UTGTRGTRGAGGGUTUUTUUAUUAURGGURGGUTUUUAAGUUAGRGTTGRGUAGATGU

ARGGUUAGU

140
GUTGGURGTGUATUTGRGUAARGUTGGUTTGGGAGURGGURGGTGGTGGAGGAGUUUT

RGARGAUAGUUTUAAGGUUTGURGUTUUTUARGRGUURGGUTGUUAUTAGAUUATGGA

RGUAGGUAGURGRGRGUTUUAURGUTURGGGUUXGGRGUTUUARGURGATRGGARGGG

RGTRGUTUAUAUUAURGAGGRGAGUURGGAUAUTGAAGUUAUTTTGTTGTUUTRGGUU

UAGGGUTGUAGGTTUTGUAGRGRGAAGAGRGARGAGTTGTGUAGGUAGAGAGGGTURG

GGGGUAGRGG

141
AUUATTGTUATAGTAAUAUAUAATTRGGGUUUARGTAGAUTTAATUURGAGAGGUAATT

GTTUUUTTGUTTGGGRGGUTARGUTUUURGRGGGGUTGUUTGUAGUUUURGGGUUUTT

GUAGUTURGGGARGGRGRGRGGRGGRGGAGGUXGRGGGGUUUUAAGRGUUAGUUTGG

UUURGGRGUAGATGRGUTGUUURGGURGUAGUUUAGURGURGRGTGTGTTGTUAGGAR

GATRGGAAARGRGTGTGTGGGGAGATGGGTGUUAGTGTRGUUTTGTUUATTATUUAAAA

UUUAGTRGU

142
GRGAUTGGGTTTTGGATAATGGAUAAGGRGAUAUTGGUAUUUATUTUUUUAUAUARGR

GTTTURGATRGTUUTGAUAAUAUARGRGGRGGUTGGGUTGRGGURGGGGUAGRGUATU

TGRGURGGGGUUAGGUTGGRGUTTGGGGUUURGXGGUUTURGURGURGRGRGURGTUU

RGGAGUTGUAAGGGUURGGGGGUTGUAGGUAGUUURGRGGGGAGRGTAGURGUUUAA

GUAAGGGAAUAATTGUUTUTRGGGATTAAGTUTARGTGGGUURGAATTGTGTGTTAUTA

TGAUAATGGT

143
RGRGTURGUUTUUUTGGAUUATTTUAUTUUTTUAAGTUAUTGGUTUUAURGRGTTTTUT

GTTUUUAUUURGTUAUAAUTTTGGGGUTTAAAGGUAUUAGGATUTGGUATUUURGGGA

TGUUAUUTGTUTTUUAGGATGTGTUUTGGUTUUXGATGUUUUAAGUTAAGUUURGGAU

AGGUTGGGAAUUTUTUUUTAGUAGUUUUTGUUUTGGTUUUTAGGGUUURGUAGGUUTT

GGGGTGGUAGTGGUUTTGTUUUATGTUATUUUAGAGGUUTRGGGAGGUAGATGGTTTT

GTUTAGGAA

144
TTUUTAGAUAAAAUUATUTGUUTUURGAGGUUTUTGGGATGAUATGGGAUAAGGUUAU

TGUUAUUUUAAGGUUTGRGGGGUUUTAGGGAUUAGGGUAGGGGUTGUTAGGGAGAGG

TTUUUAGUUTGTURGGGGUTTAGUTTGGGGUATXGGGAGUUAGGAUAUATUUTGGAAG

AUAGGTGGUATUURGGGGATGUUAGATUUTGGTGUUTTTAAGUUUUAAAGTTGTGARG

GGGTGGGAAUAGAAAARGRGGTGGAGUUAGTGAUTTGAAGGAGTGAAATGGTUUAGGG

AGGRGGARGRG

145
UTTTUTTTGTTATUTUURGTGAAAUUTTUAUTTAGUAGGTGGARGGAGUUURGRGAURG

GGUAGAGTURGGGUTRGUURGAGGAUAGGAGGAGGAGRGGGAGUURGRGRGTUURGG

GAGAGRGUUURGAGTGUAGGTUUURGUUURGUUXGGRGAGUUURGUTGGAGRGAGUU

UAGRGRGURGGGGUTGGGGGGRGGUUARGAUUUUUUUTGAAGGGGGTGGUUARGGAG

RGUAUUURGAGAAGRGAGUUUUUUTUUUUAGAGRGUTGUTUUTGRGGUTGUTGUTGUT

GUTGGTGAUUAA

146
TTGGTUAUUAGUAGUAGUAGUAGURGUAGGAGUAGRGUTUTGGGGAGGGGGGUTRGUT

TUTRGGGGTGRGUTURGTGGUUAUUUUUTTUAGGGGGGGTRGTGGURGUUUUUUAGUU

URGGRGRGUTGGGUTRGUTUUAGRGGGGUTRGUXGGGRGGGGRGGGGAUUTGUAUTRG

GGGRGUTUTUURGGGARGRGRGGGUTUURGUTUUTUUTUUTGTUUTRGGGRGAGUURG

GAUTUTGUURGGTRGRGGGGUTURGTUUAUUTGUTAAGTGAAGGTTTUARGGGAGATA

AUAAAGAAAG

147
GUUAGRGRGGGURGURGGRGATGARGGURGRGAAGUAGGAGURGUAGUUUAUUURGG

GGGUUAGGGRGAGUUAGGRGUAGURGGRGGAUUAGGTGAGAGTRGGUAGURGRGGUU

AGGUUUTUURGGGAGGGGTGGUTUUAGTGRGRGUTUXGUURGUUTUURGUTTUUUAGG

UTGGGUTUURGRGUUTUUUTUTTUTUAUUUTUUUURGUUURGUUUUAGTTUUAGGUTU

TUUTGUTTUTUUARGGAUTUTGRGGGAAGTTAGAGUUTUTGRGTGRGUTURGGGGUURG

GRGAGAGGATG

148
UATUUTUTRGURGGGUUURGGAGRGUARGUAGAGGUTUTAAUTTUURGUAGAGTURGT

GGAGAAGUAGGAGAGUUTGGAAUTGGGGRGGGGRGGGGGAGGGTGAGAAGAGGGAGG

RGRGGGAGUUUAGUUTGGGAAGRGGGAGGRGGGXGGAGRGRGUAUTGGAGUUAUUUU

TUURGGGAGGGUUTGGURGRGGUTGURGAUTUTUAUUTGGTURGURGGUTGRGUUTGG

UTRGUUUTGGUUUURGGGGTGGGUTGRGGUTUUTGUTTRGRGGURGTUATRGURGGRG

GUURGRGUTGGU

149
GGUTGAAGGTGUUURGGGGAAUUURGGRGGGRGGUUUAURGAGGGAGGGAGAGGRGG

URGGGAUUAAGGAATGGGGUUTUTTGGTTUUUUATTAARGUARGUTGAAGAAATUTGU

TGRGUTUUTGARGGURGUTUAURGGGTTRGAGUUUXGTUUTUUTATAGURGGGGRGUTR

GUTGGUUAAAGRGAUURGAGUAGGRGAATGAUUTTTAGGRGGARGGGGTTTTUUUTUT

GUTTTUTTGTTTUTTTTGAGGAGARGGGTGTGTGTTTGTGAGGTGGGGATGGGGGAAGAG

TGTUUUAG

150
UTGGGAUAUTUTTUUUUUATUUUUAUUTUAUAAAUAUAUAUURGTUTUUTUAAAAGAA

AUAAGAAAGUAGAGGGAAAAUUURGTURGUUTAAAGGTUATTRGUUTGUTRGGGTRGU

TTTGGUUAGRGAGRGUUURGGUTATAGGAGGAXGGGGUTRGAAUURGGTGAGRGGURG

TUAGGAGRGUAGUAGATTTUTTUAGRGTGRGTTAATGGGGAAUUAAGAGGUUUUATTU

UTTGGTUURGGURGUUTUTUUUTUUUTRGGTGGGURGUURGURGGGGTTUUURGGGGU

AUUTTUAGUU

151
GRGAGTTGTAAAUUTUAGAGAAAGGUAUTTGTUUUUAGUAAAARGUTTGGAGAGGAUR

GTGUARGUTGTGUTGUUUURGUUURGAGARGRGURGGGURGURGGGTUAURGGTTTTU

RGAAAGGGAUURGGUAGAGAUAAAGTGUUTTRGUXGUTGRGATAGGTTGGTTTTAUTTT

GUAATAAAUAGUUUUTAATGGGAURGGGRGURGGGRGGAGAGUTRGGUURGGGGRGR

GGUUTTTGURGUUTGGUTUTGRGGGURGUUURGURGGGRGUUAGGTTTTGGGGGGTGG

UURGGUUURG

152
RGGGGURGGGUUAUUUUUUAAAAUUTGGRGUURGGRGGGGRGGUURGUAGAGUUAGG

RGGUAAAGGURGRGUUURGGGURGAGUTUTURGUURGGRGUURGGTUUUATTAGGGGU

TGTTTATTGUAAAGTAAAAUUAAUUTATRGUAGXGGRGAAGGUAUTTTGTUTUTGURGG

GTUUUTTTRGGAAAAURGGTGAUURGGRGGUURGGRGRGTUTRGGGGRGGGGGUAGUA

UAGRGTGUARGGTUUTUTUUAAGRGTTTTGUTGGGGAUAAGTGUUTTTUTUTGAGGTTT

AUAAUTRGU

153
TTGRGGAGTURGURGRGGUUTRGGUTUURGUURGGRGUURGGUUTGGUUUUAURGURG

UTUAUTRGGTURGUATRGURGUUAUUTURGGAGUTGGTGGGGAGUURGGRGAGGGAGG

GUUTGGAAGGGGUUUTGGGRGURGAGTUUUURGUXGGRGURGAUAURGUUTGUUAGG

AGUAGURGGUUTGUTRGAGGTGAUTGUAGURGRGRGGTTUTGGRGRGGUTTUTTUAUU

AAUATGAGRGGGTUTUTGRGTURGAGAGTGAGRGUAGURGGGUAGRGUUTAGTGGATT

AUAGUAGARGU

154
GRGTUTGUTGTAATUUAUTAGGRGUTGUURGGUTGRGUTUAUTUTRGGARGUAGAGAU

URGUTUATGTTGGTGAAGAAGURGRGUUAGAAURGRGRGGUTGUAGTUAUUTRGAGUA

GGURGGUTGUTUUTGGUAGGRGGTGTRGGRGUXGGRGGGGGAUTRGGRGUUUAGGGUU

UUTTUUAGGUUUTUUUTRGURGGGUTUUUUAUUAGUTURGGAGGTGGRGGRGATGRGG

AURGAGTGAGRGGRGGTGGGGUUAGGURGGGRGURGGGRGGGAGURGAGGURGRGGR

GGAUTURGUAA

155
TAGGAGGUURGUTUTGUAUUTTUUTTAUTGTGGARGGGUUUTUTGAGUTUTGAGGUUTG

GRGGGAGAGRGRGTGUAGUTRGAGUTUUTUTTUUUAUUUAGUUUURGRGUUUTAUUTG

GTRGAUUTTURGUAGUUTUAGUURGGTUTTGUXGRGUTRGTUUTTGUAUAGGTGGATUT

RGRGUAUUURGGGUTTGATUTUAGUTRGURGUARGUUUAGGUTGTAUURGGTTAURGG

TGUUAUUATUTGGURGGGUURGGGGUURGAGAURGUTGTUTGUAGAUAUUAGGGGGU

AGGGGGTUA

156
TGAUUUUUTGUUUUUTGGTGTUTGUAGAUAGRGGTUTRGGGUUURGGGUURGGUUAGA

TGGTGGUAURGGTAAURGGGTAUAGUUTGGGRGTGRGGRGAGUTGAGATUAAGUURGG

GGTGRGRGAGATUUAUUTGTGUAAGGARGAGRGXGGUAAGAURGGGUTGAGGUTGRGG

AAGGTRGAUUAGGTAGGGRGRGGGGGUTGGGTGGGAAGAGGAGUTRGAGUTGUARGRG

UTUTUURGUUAGGUUTUAGAGUTUAGAGGGUURGTUUAUAGTAAGGAAGGTGUAGAGR

GGGUUTUUTA

157
UUTGTURGRGUTUUUAGUUUUUARGUAGUUAGAUAUURGRGGGTGUTGAGATGAAGAG

TGAAGTUAUUAGGAGAGAUAGGAAAUAGRGRGGGUUTRGGGGAUURGAGGGRGRGGA

AGUTRGGGGGURGGGGTRGAGUAUAGATTGGGAGGXGUAGRGUTGGGRGUARGTUUUR

GTRGGUURGGRGGUUUUTAATGAGGRGRGUTGTGRGUAGAUTGUTAAGAGUAGUATGA

GUARGGUTURGGRGGUUUUUAGGGUUURGRGGGTRGGRGUUAUUURGGAGRGRGGTG

UURGGAATUAUT

158
AGTGATTURGGGUAURGRGUTURGGGGTGGRGURGAUURGRGGGGUUUTGGGGGURGU

RGGAGURGTGUTUATGUTGUTUTTAGUAGTUTGRGUAUAGRGRGUUTUATTAGGGGURG

URGGGURGARGGGGARGTGRGUUUAGRGUTGXGUUTUUUAATUTGTGUTRGAUUURGG

UUUURGAGUTTURGRGUUUTRGGGTUUURGAGGUURGRGUTGTTTUUTGTUTUTUUTGG

TGAUTTUAUTUTTUATUTUAGUAUURGRGGGTGTUTGGUTGRGTGGGGGUTGGGAGRGR

GGAUAGG

159
UATUUTTUURGGAAAAGTAUAAAUAGTTUUTUAGARGAGGTUUUUUAUUTUUUARGRG

UTUUUUAGUUUTUUUTUUUTGRGGAGAGUUURGRGAUAGUUTUUUUAAUAUUTGTGAA

TUATURGGGAGGUTGUUAURGURGAGRGATURGXGUAUUAUUUUUTTUURGGGUURGG

GUARGGUUAGGGAGGAUAGTTAGGGTTGTTGUTTTATAATTATUAUTTTTAATUTUTAAT

ARGAUUAGUAUAAGTAGUUTTTGTUTUUURGUUUTGATTTGAGUATURGAGGGUUUUU

RGAGGUAG

160
UTGUUTRGGGGGGUUUTRGGATGUTUAAATUAGGGRGGGGAGAUAAAGGUTAUTTGTG

UTGGTRGTATTAGAGATTAAAAGTGATAATTATAAAGUAAUAAUUUTAAUTGTUUTUUU

TGGURGTGUURGGGUURGGGAAGGGGGTGGTGXGRGGATRGUTRGGRGGTGGUAGUUT

UURGGATGATTUAUAGGTGTTGGGGAGGUTGTRGRGGGGUTUTURGUAGGGAGGGAGG

GUTGGGGAGRGRGTGGGAGGTGGGGGAUUTRGTUTGAGGAAUTGTTTGTAUTTTTURGG

GAAGGATG

161
URGGAGARGAUTUUAGGGUTGGGTGAGGRGUTGAUUUUUAGGAGUTGGGGTGURGAGG

GGRGRGGGUUAUAGRGGTGRGAGUUAGTRGGGRGUURGRGRGGTGGGGRGRGRGGRGR

GGGGATTGGRGGGRGUTUUURGGTGUUXGUAGUTUTTUAGXGTAGURGGGAAGAGURG

RGRGTUTGRGRGUUAGUUUURGUUUTGGGUURGURGUURGAGUTUTUTGGRGUAGRGU

TAGUTURGURGRGUTUAGUTGUUUTGRGURGGUAUUUUTGGTUATGAGRGUUUUUTRG

ARGUTGUUUU

162
GGGGUAGRGTRGAGGGGGRGUTUATGAUUAGGGGTGURGGRGUAGGGUAGUTGAGRGR

GGRGGAGUTAGRGUTGRGUUAGAGAGUTRGGGRGGRGGGUUUAGGGRGGGGGUTGGR

GRGUAGARGRGRGGUTUTTUURGGUTAXGUTGAAGAGUTGXGGGUAURGGGGAGRGUU

RGUUAATUUURGRGURGRGRGUUUUAURGRGRGGGRGUURGAUTGGUTRGUAURGUTG

TGGUURGRGUUUUTRGGUAUUUUAGUTUUTGGGGGTUAGRGUUTUAUUUAGUUUTGGA

GTRGTUTURGG

163
RGTGUUARGUAAARGURGTRGTURGUAURGGTRGRGAUTRGGUAAGGGAGRGGGRGGA

GAGUTGAUTRGRGGRGGAGGGGGGTUAUTRGURGGARGGAARGUTGRGRGUAGUUATR

GUTRGUTGTUAGURGRGTUUUAAUURGUTAGGAXGUTGGGUUUTGURGGRGGGATUUT

UUUUTTAUTRGGAAAGGGGAGGRGURGGUUAUAGTAGGRGARGAGURGRGUTRGGUUA

UUAURGRGGTGGURGGRGGAAUUUTRGUTUTUUUUURGTUAUUURGTRGAGGGGGAAG

UTGAGGAGGG

164
UUUTUUTUAGUTTUUUUUTRGARGGGGTGARGGGGGGAGAGRGAGGGTTURGURGGUU

AURGRGGTGGTGGURGAGRGRGGUTRGTRGUUTAUTGTGGURGGRGUUTUUUUTTTURG

AGTAAGGGGAGGATUURGURGGUAGGGUUUAGXGTUUTAGRGGGTTGGGARGRGGUTG

AUAGRGAGRGATGGUTGRGRGUAGRGTTURGTURGGRGAGTGAUUUUUUTURGURGRG

AGTUAGUTUTURGUURGUTUUUTTGURGAGTRGRGAURGGTGRGGARGARGGRGTTTGR

GTGGUARG

165
UURGGRGAGARGAATGUAGUAUARGGURGUUTTTATGGGGUURGUAGAUAGRGRGTRG

UUAGGUTAAUUUTGRGTGGAAAATTRGGAGGTGGAAGGRGAGGRGUUTTATTGAGGGG

GURGGUAGRGGRGGRGGRGGRGGRGAGGGGGRGGXGGGGGUTGTGRGGUURGGGURG

GAAARGTGAGURGGGUTGGGGGRGGRGAUUAUUUURGRGUATTUUURGGUUUTUURGG

GAUUAURGRGRGUUURGGURGGUAGGTGGAGGAGUAGGAGGRGUUUTUAURGTUUUR

GUUUTGGGAGGA

166
TUUTUUUAGGGRGGGGARGGTGAGGGRGUUTUUTGUTUUTUUAUUTGURGGURGGGGR

GRGRGGTGGTUURGGGAGGGURGGGGAATGRGRGGGGGTGGTRGURGUUUUUAGUURG

GUTUARGTTTURGGUURGGGURGUAUAGUUUUXGURGUUUUUTRGURGURGURGURGU

RGUTGURGGUUUUUTUAATAAGGRGUUTRGUUTTUUAUUTURGAATTTTUUARGUAGG

GTTAGUUTGGRGARGRGUTGTUTGRGGGUUUUATAAAGGRGGURGTGTGUTGUATTRGT

UTRGURGGG

167
TUTGATGGTGGURGRGTGTTGGGUTTUAGTAGUUUUTGGAGUUUATUTAUTRGAAATAA

ATATTTUTRGGTAGUUATGGAAGTTGGAGUTGAGAAGUURGGGRGGGGGTGUUTGURG

GATGUUUURGGTTUUUUTUTTTTUUUAUTRGUXGUUAAUTTAAUTTTTURGGGGGATGG

ATRGGUTGGTTGAGAAAGAGGAUARGUATAUTRGGTGTGAARGAGGTUARGAAUUUTU

TGAUUUUAGGGAGAGATTTUTGUUAAGRGTAAGGTGTUTTTGGATUUUUUUAUUTUTGT

UUUAGGT

168
AUUTGGGAUAGAGGTGGGGGGATUUAAAGAUAUUTTARGUTTGGUAGAAATUTUTUUU

TGGGGTUAGAGGGTTRGTGAUUTRGTTUAUAURGAGTATGRGTGTUUTUTTTUTUAAUU

AGURGATUUATUUUURGGAAAAGTTAAGTTGGXGGRGAGTGGGAAAAGAGGGGAAURG

GGGGUATURGGUAGGUAUUUURGUURGGGUTTUTUAGUTUUAAUTTUUATGGUTAURG

AGAAATATTTATTTRGAGTAGATGGGUTUUAGGGGUTAUTGAAGUUUAAUARGRGGUU

AUUATUAGA

169
GGRGGRGATRGGAGRGGRGGRGGTGGTUTRGGRGGRGGRGGRGGRGGRGGRGGUAGGG

AGRGGGUTUURGGTGURGGGUAURGGGRGGGRGGRGGGGAAGATGAURGRGGGRGURG

GRGTGUTUUTTUTGUTGUTUTRGUTUTURGGRGXGUTURGGGTAAGTTGURGUUTUURG

UUUURGURGTTRGGAAGUUURGGGUAGRGGGAGGTRGTUUURGGATUURGRGGGGRGU

TUAUAUAUURGGRGGGGUTUTUURGGGUTUUUURGURGRGUTUUURGUTGUATUUAGU

URGGRGUUU

170
GGGRGURGGGUTGGATGUAGRGGGGAGRGRGGRGGGGGAGUURGGGAGAGUUURGUR

GGGTGTGTGAGRGUUURGRGGGATURGGGGARGAUUTUURGUTGUURGGGGUTTURGA

ARGGRGGGGGRGGGAGGRGGUAAUTTAUURGGAGXGRGURGGAGAGRGAGAGUAGUA

GAAGGAGUARGURGGRGUURGRGGTUATUTTUUURGURGUURGUURGGTGUURGGUAU

RGGGAGUURGUTUUUTGURGURGURGURGURGURGURGURGAGAUUAURGURGURGUT

URGATRGURGUU

171
URGRGUTGRGGGTGGGRGGRGGGGTAGGAUTGRGGGGUAUTUTUAUUUURGAGUUUTR

GGRGGGGUTGGAGUUTGRGTGTURGGRGGGGUUURGGGGGUTGGGAGUTGGGURGGGU

TTGGGURGGGAAGURGGARGURGGGRGGGRGRGGXGRGGGAGGGRGUUURGGGAGGG

RGAGRGGGTUTUURGUTGUUTUTGUAGAGUUURGRGGGURGGATTGUUAGUTTTGUTUT

GRGUUTGGRGAGGTGRGRGGUURGRGGGGGUAGAGAGRGRGGRGGUTURGGGGGRGUU

UUTGGGRGGA

172
TURGUUUAGGGGRGUUUURGGAGURGURGRGUTUTUTGUUUURGRGGGURGRGUAUUT

RGUUAGGRGUAGAGUAAAGUTGGUAATURGGUURGRGGGGUTUTGUAGAGGUAGRGG

GAGAUURGUTRGUUUTUURGGGGRGUUUTUURGXGURGRGUURGUURGGRGTURGGUT

TUURGGUUUAAGUURGGUUUAGUTUUUAGUUUURGGGGUUURGURGGAUARGUAGGU

TUUAGUUURGURGAGGGUTRGGGGGTGAGAGTGUUURGUAGTUUTAUUURGURGUUUA

UURGUAGRGRGG

173
UURGGRGTURGRGTUUUURGGRGRGRGUUTTGGGGAURGGGTTGGTGGRGUUURGRGT

GGGGUURGGTGGGUTTUURGGAGGGTTURGGGGGTRGGUUTGRGGRGRGTGRGGGGGA

GGAGARGGTTURGGGGGAURGGURGRGAUTGRGGXGGRGGTGGTGGGGGGAGURGRGG

GGATRGURGAGGGURGGTRGGURGUUURGGGTGURGRGRGGTGURGURGGRGGRGGTG

AGGUUURGRGRGTGTGTUURGGUTGRGGTRGGURGRGUTRGAGGGGTUUURGTGGRGT

UUUUTTUUUU

174
GGGGAAGGGGARGUUARGGGGAUUUUTRGAGRGRGGURGAURGUAGURGGGAUAUAR

GRGRGGGGUUTUAURGURGURGGRGGUAURGRGRGGUAUURGGGGRGGURGAURGGU

UUTRGGRGATUUURGRGGUTUUUUUUAUUAURGUXGURGUAGTRGRGGURGGTUUUUR

GGAAURGTUTUUTUUUURGUARGRGURGUAGGURGAUUUURGGAAUUUTURGGGAAG

UUUAURGGGUUUUARGRGGGGRGUUAUUAAUURGGTUUUUAAGGRGRGRGURGGGGG

ARGRGGARGURGGG

175
UUTTTUTRGRGUUTTUUURGTRGUUURGGUUTRGUURGTGGTUTUTRGTUTTUTUURGG

UURGUTUTTURGAAURGGGTRGGRGRGTUUUURGGGTGRGUUTRGUTTUURGGGUUTGU

RGRGGUUUTTUUURGAGGRGTURGTUURGGGXGTRGGRGTRGGGGAGAGUURGTUUTU

UURGRGTGGRGTRGUUURGTTRGGRGRGRGRGTGRGUURGAGRGRGGUURGGTGGTUU

UTUURGGAUAGGRGTTRGTGRGARGTGTGGRGTGGGTRGAUUTURGUUTTGURGGTRGU

TRGUUUT

176
AGGGRGAGRGAURGGUAAGGRGGAGGTRGAUUUARGUUAUARGTRGUARGAARGUUTG

TURGGGAGGGAUUAURGGGURGRGUTRGGGRGUARGRGRGRGURGAARGGGGRGARGU

UARGRGGGGAGGARGGGUTUTUUURGARGURGAXGUURGGGARGGARGUUTRGGGGA

AGGGURGRGGUAGGUURGGGAAGRGAGGRGUAUURGGGGGARGRGURGAUURGGTTR

GGAAGAGRGGGURGGGAGAAGARGAGAGAUUARGGGRGAGGURGGGGRGARGGGGAA

GGRGRGAGAAAGG

177
GGGRGGGTGGTTGGGGRGTURGGTTRGURGRGUUURGUUURGGUUUUAURGGTUURGG

URGURGUUUURGRGUURGUTRGUTUUUTUURGTURGUURGTURGRGGUURGTURGTUR

GTURGTURGTRGTUUTUUTRGUTTGRGGGGRGUXGGGUURGTUUTRGRGAGGUUUUURG

GURGGURGTURGGURGRGTRGGGGUUTRGURGRGUTUTAUUTAUUTAUUTGGTTGATUU

TGUUAGTAGUATATGUTTGTUTUAAAGATTAAGUUATGUATGTUTAAGTARGUARGGUR

GGTAUAG

178
UTGTAURGGURGTGRGTAUTTAGAUATGUATGGUTTAATUTTTGAGAUAAGUATATGUT

AUTGGUAGGATUAAUUAGGTAGGTAGGTAGAGRGRGGRGAGGUUURGARGRGGURGGA

RGGURGGURGGGGGGUUTRGRGAGGARGGGUUXGGRGUUURGUAAGRGAGGAGGARG

ARGGARGGARGGARGGARGGGURGRGGARGGGRGGARGGGAGGGAGRGAGRGGGRGR

GGGGGRGGRGGURGGGAURGGTGGGGURGGGGRGGGGRGRGGRGAAURGGARGUUUU

AAUUAUURGUUU

179
TATTUAGAUTGGGAGUTGGGGTUAGAGTGTUTURGTUTURGGAGGGGGAUUTTUUAGA

UUURGGGRGGGAGGGGUUUAUAGUAGGGTGUUURGGRGGUUUUAGGAGGGGGTURGG

GUUTRGGGRGAAGGUTGRGGRGUUURGGAAGGAGUXGRGUARGUUUUTGUARGRGGTG

UURGUTGRGGAGGUUARGGATURGGAGGTGAGAGRGUUUURGUUURGUTUUUTGUUUA

GUURGRGRGRGGUURGGTURGGGGUTGGGGGRGGGRGUUAAGAGGGGRGURGGGRGU

AGTGGGGRGGUU

180
GGURGUUUUAUTGRGUURGGRGUUUUTUTTGGRGUURGUUUUUAGUUURGGAURGGGU

RGRGRGRGGGUTGGGUAGGGAGRGGGGRGGGGGRGUTUTUAUUTURGGATURGTGGUU

TURGUAGRGGGUAURGRGTGUAGGGGRGTGRGXGGUTUUTTURGGGGRGURGUAGUUT

TRGUURGAGGUURGGAUUUUUTUUTGGGGURGURGGGGUAUUUTGUTGTGGGUUUUTU

URGUURGGGGTUTGGAAGGTUUUUUTURGGAGARGGAGAUAUTUTGAUUUUAGUTUUU

AGTUTGAATA

181
GRGAUTTRGTUTGGUUUUAAAAUUTTTGUUUTUUATTUUUUAGRGTUURGGAURGGTUU

TTGUTUATUTUTUAGGGGUAAUAUUTGAUUUARGGGGUURGTUURGGAGUTUTUTTRG

AUTURGGGAUUAGTTUUUAGUUUTTUAGTAUTXGGUATAURGGAAGGAGGATTUUTGU

TUUUTTUTUTUUTUUTAGAUUURGAGGUTTGGAGUTTAUTUTUUAGATGAGAUTAAAAA

GUUUTAAATTAATUUUUUTATAGRGUAUUUUTUUUTRGUAGRGUUUTGGTRGGGGGUU

TUUATTGT

182
AUAATGGAGGUUUURGAUUAGGGRGUTGRGAGGGAGGGGTGRGUTATAGGGGGATTAA

TTTAGGGUTTTTTAGTUTUATUTGGAGAGTAAGUTUUAAGUUTRGGGGTUTAGGAGGAG

AGAAGGGAGUAGGAATUUTUUTTURGGTATGUXGAGTAUTGAAGGGUTGGGAAUTGGT

UURGGAGTRGAAGAGAGUTURGGGARGGGUUURGTGGGTUAGGTGTTGUUUUTGAGAG

ATGAGUAAGGAURGGTURGGGARGUTGGGGAATGGAGGGUAAAGGTTTTGGGGUUAGA

RGAAGTRGU

183
UUARGGURGAGGGGURGGAGUTGUUTUTGTGRGAGGGAGUUUTGGURGRGGRGUAGGT

AGGAURGGGGAAUUUAAGGAGGGRGGGAGGAAAGRGUUUUTGGTGGGGGAGGGGURG

TGGRGGGGTUTGGGGGUUAAGGGUTURGGGURGAAXGAGGGGUAUARGURGGAGUAG

UTUUAGUUTGGGAGUATTGGGRGTGAARGTGGGGGAAAGTAGGGAUUAGATTAUAGGA

TUAAGGUTTTUUURGGUTUAGAAUTGGATTGTTUAGTGUUUUTAGAAGGGGUAGRGGG

TRGUUUARGRGA

184
TRGRGTGGGRGAUURGUTGUUUUTTUTAGGGGUAUTGAAUAATUUAGTTUTGAGURGG

GGAAAGUUTTGATUUTGTAATUTGGTUUUTAUTTTUUUUUARGTTUARGUUUAATGUTU

UUAGGUTGGAGUTGUTURGGRGTGTGUUUUTXGTTRGGUURGGAGUUUTTGGUUUUUA

GAUUURGUUARGGUUUUTUUUUUAUUAGGGGRGUTTTUUTUURGUUUTUUTTGGGTTU

UURGGTUUTAUUTGRGURGRGGUUAGGGUTUUUTRGUAUAGAGGUAGUTURGGUUUUT

RGGURGTGG

185
GAGAARGGAGUTGUURGTGRGGGGTGRGRGUUAAGGRGGGGGAUAGGARGGGUURGG

RGTUTGURGAGAUUTAGGUTGUURGUATTURGUTRGRGGUTUUUUTTUTGUUTUURGUT

UTAGRGGRGURGGAGURGXGUTGGUUUURGUUURGUURGGUAGUTUUXGXGAGTUAG

AGUURGGTGUUUUUAGAURGRGGGUAGTRGGGGUUTUATTURGGGUUAGAGUAGGAA

AGAGUURGAUUUAUUTUUTRGGTTUTTUUAGGGAAUUUUTTUTRGGAGGGRGUUUTGG

GUUTURGRGUUA

186
TGGRGRGGAGGUUUAGGGRGUUUTURGAGAAGGGGTTUUUTGGAAGAAURGAGGAGGT

GGGTRGGGUTUTTTUUTGUTUTGGUURGGAATGAGGUUURGAUTGUURGRGGTUTGGG

GGUAURGGGUTUTGAUTXGXGGGAGUTGURGGGRGGGGRGGGGGUUAGXGRGGUTUR

GGRGURGUTAGAGRGGGAGGUAGAAGGGGAGURGRGAGRGGAATGRGGGUAGUUTAG

GTUTRGGUAGARGURGGGUURGTUUTGTUUUURGUUTTGGRGRGUAUUURGUARGGGU

AGUTURGTTUTU

187
AGTAGGTGAAUTTGAGGTAGAAGRGGGARGTGTAUTTUTURGGUTGURGGTRGGGGTRG

RGGUTUTRGTTGAGRGTGTUUURGAAGAUUTUUTTGGUUAGRGRGATGRGUURGUAUA

UUATGATGRGRGUUAUARGURGGAATTTGGRGTXGGUUTGGTTGTRGRGUARGGTGGTG

TAGGAGUUURGGTAGUUUAGRGTGAGGAAGUURGAGRGUTTGTUUTGRGRGURGURGU

RGURGUUAUUAURGTGRGUTUURGGGUURGAGGGUARGGRGGURGURGUUURGRGUA

GUAGUAGRG

188
RGUTGUTGUTGRGRGGGGRGGRGGURGURGTGUUUTRGGGUURGGGAGRGUARGGTGG

TGGRGGRGGRGGRGGRGRGUAGGAUAAGRGUTRGGGUTTUUTUARGUTGGGUTAURGG

GGUTUUTAUAUUAURGTGRGRGAUAAUUAGGUXGARGUUAAATTURGGRGTGTGGRGR

GUATUATGGTGTGRGGGRGUATRGRGUTGGUUAAGGAGGTUTTRGGGGAUARGUTUAA

RGAGAGURGRGAUUURGAURGGUAGURGGAGAAGTAUARGTUURGUTTUTAUUTUAAG

TTUAUUTAUT

189
GURGRGAUUUUTGRGUTUUUAGUAGGGUUAAGAGGAUUTRGRGGGUAUUAGRGTURG

GGRGGGAAGGGARGTGTGUUUAAGUUTRGUUTUUTGGUUUTUAGTGGGUTGGGARGUU

UTTGATUAURGGRGUAGGAAAGAGGUTUUUUAGUUXGTGAGUTTRGTURGGGRGUUAG

GGUAGGGATGGUTGGTGGTGTGUAUTGGAGAGUARGARGGTGARGUTGRGTGGGAAAG

AGARGTGGGAAGGGUATAGURGGATTATUUAUTUAGUTUUAATTTTUTUUAAGRGUUA

UTUAUUUUAUA

190
TGTGGGGTGAGTGGRGUTTGGAGAAAATTGGAGUTGAGTGGATAATURGGUTATGUUUT

TUUUARGTUTUTTTUUUARGUAGRGTUAURGTRGTGUTUTUUAGTGUAUAUUAUUAGU

UATUUUTGUUUTGGRGUURGGARGAAGUTUAXGGGUTGGGGAGUUTUTTTUUTGRGUR

GGTGATUAAGGGRGTUUUAGUUUAUTGAGGGUUAGGAGGRGAGGUTTGGGUAUARGTU

UUTTUURGUURGGARGUTGGTGUURGRGAGGTUUTUTTGGUUUTGUTGGGAGRGUAGG

GGTRGRGGU

191
CTGGGTTTGGAGTTGGGTAGRGRGGCCAGGTGARGGTCTCCTTCCCTGGGCRGTCAGGGC

TGCAGGRGGCTGTGACACTCRGGAGGCCTRGACAGAGGGGTCCCAGGRGCAGRGTACCR

GGGCTCCTCTTGCCTCAGRGGAGRGTCCCAXGGCAGTCCRGGACCRGRGCCTCTCCRGCC

RGGCTCCTGCARGCCCAGRGAGCCTGCAGCCTGGGATCCTCRGCTCCTGCATCCRGGCTR

GGGATGGGTCCRGGCTCCCCACCTCARGCRGGRGGGGCCACCCTTTCACTCRGCCTCCAT

C

192
GATGGAGGRGGAGTGAAAGGGTGGCCCRGCRGGRGTGAGGTGGGGAGCRGGGACCCAT

CCRGAGCRGGGATGCAGGAGRGGAGGATCCCAGGCTGCAGGCTRGCTGGGRGTGCAGG

AGCRGGGRGGGAGAGGRGRGGGTCRGGGACTGCXGTGGGARGCTCRGCTGAGGCAAGA

GGAGCCRGGGTARGCTGRGCCTGGGACCCCTCTGTRGAGGCCTCRGGAGTGTCACAGCR

GCCTGCAGCCCTGARGGCCCAGGGAAGGAGACRGTCACCTGGCRGRGCTACCCAACTCC

AAACCCAG

193
UUUTUAUUTUTAGUUUUTRGGUTTUAGUTUAGGUUUUTRGGGGAGUATUUUTTGURGT

GAGAUTGAUAGUUTTTGGGGGRGUAGGGTUUTGTTUTUTGRGUTUTAGUUUATUTGTGR

GUAGAGUUTRGTTUUUAGGRGUUTGGAAUURGGXGGGUATTGARGTUAAGRGURGGRG

GAGRGUTGUUTAUAGARGGTTGAUURGGGUUUTUUTUUAUAUUUUUTTUUTTUTTRGU

UTUUTUUUTUTTTUUTGUARGGGGGUTRGGGUTUAUTATAAAAGGTGGGAGRGRGTGGT

GUUUUAGU

194
GUTGGGGUAUUARGRGUTUUUAUUTTTTATAGTGAGUURGAGUUUURGTGUAGGAAAG

AGGGAGGAGGRGAAGAAGGAAGGGGGTGTGGAGGAGGGUURGGGTUAAURGTUTGTA

GGUAGRGUTURGURGGRGUTTGARGTUAATGUUXGURGGGTTUUAGGRGUUTGGGAAR

GAGGUTUTGRGUAUAGATGGGUTAGAGRGUAGAGAAUAGGAUUUTGRGUUUUUAAAG

GUTGTUAGTUTUARGGUAAGGGATGUTUUURGAGGGGUUTGAGUTGAAGURGAGGGGU

TAGAGGTGAGGG

195
TTGUTGUTAAATGTUAUAAAAGTUAUUTAAAGGUAUAGAGGAGGURGUTUTGTTTTTGR

GAAAUTTGUTAAAATTAATUTGRGUTGGGUUAUTTGUAGAAAGUAGAAUUAUUTUURG

UUUUUAUUTRGUUTUUAGURGURGGGGTTUAGGXGTTTGTGAAAGAUAGAAUUTTTGG

GUTAGGGAUURGGGUAUTGGTGUTTRGAAGTURGAATURGURGGURGAGAAAARGAUA

AGAGAAAGAAAATUUAGRGGGRGUTUTUTUUAGRGUUAGGURGGTGTAGGAGGGRGUT

GGGGUTRGG

196
URGAGUUUUAGRGUUUTUUTAUAURGGUUTGGRGUTGGAGAGAGRGUURGUTGGATTT

TUTTTUTUTTGTRGTTTTUTRGGURGGRGGATTRGGAUTTRGAAGUAUUAGTGUURGGGT

UUUTAGUUUAAAGGTTUTGTUTTTUAUAAAXGUUTGAAUUURGGRGGUTGGAGGRGAG

GTGGGGGRGGGAGGTGGTTUTGUTTTUTGUAAGTGGUUUAGRGUAGATTAATTTTAGUA

AGTTTRGUAAAAAUAGAGRGGUUTUUTUTGTGUUTTTAGGTGAUTTTTGTGAUATTTAG

UAGUAA

197
AURGAGRGUTGGAGGURGUTUUARGRGRGAGUTRGAAUUARGGAGGGUTUTUAGUTRG

GAUAAGRGTRGUTGTUTAAGAGUTUUAAGUTUUARGGAAUTTTGATTTTATGUURGGGA

GUURGGTUATUUAUTTUTGUATUTUAGAUAAGXGURGGAGGUTRGTGUAGUTTUUTRG

RGTUUTUTTUUTGGARGRGGGGRGUUUAUUTTAGTUATAGUUUUTGGTUURGGUAUTG

UTUARGURGTRGGTTTUUUUAUTGUUUAGGUTUUUTUAGUURGAAUUUTGRGTUTUTGT

TUAUATTG

198
UAATGTGAAUAGAGARGUAGGGTTRGGGUTGAGGGAGUUTGGGUAGTGGGGAAAURGA

RGGRGTGAGUAGTGURGGGAUUAGGGGUTATGAUTAAGGTGGGRGUUURGRGTUUAGG

AAGAGGARGRGAGGAAGUTGUARGAGUUTURGGXGUTTGTUTGAGATGUAGAAGTGGA

TGAURGGGUTUURGGGUATAAAATUAAAGTTURGTGGAGUTTGGAGUTUTTAGAUAGR

GARGUTTGTURGAGUTGAGAGUUUTURGTGGTTRGAGUTRGRGRGTGGAGRGGUUTUUA

GRGUTRGGT

199
AAUAGUAGGUTGAAUUAGAAUTAAGAGAAAATTGGGUAGAGAGAAGGUAATGGRGAG

TUUAUUTAGGGGUTGGGGUTGRGGAGAGUTGUTGUTGUUUTTUATGUTUUTGGGGARG

UTGTGRGAGUUAGGATURGGGUAGATURGUTAUTXGATGURGGAGGAGUTGGAUAAAG

GUTUUTTRGTRGGUAAUATAGUUAAGGAUUTTGGGUTGGAGUUUUAGGAGUTGGRGGA

GRGRGGAGTURGUATRGTUTUUAGAGGTAGGARGUAGUTTTTTGUUUTGAAUURGRGA

AGRGGUAGUTT

200
AAGUTGURGUTTRGRGGGTTUAGGGUAAAAAGUTGRGTUUTAUUTUTGGAGARGATGR

GGAUTURGRGUTURGUUAGUTUUTGGGGUTUUAGUUUAAGGTUUTTGGUTATGTTGUR

GARGAAGGAGUUTTTGTUUAGUTUUTURGGUATXGAGTAGRGGATUTGUURGGATUUT

GGUTRGUAUAGRGTUUUUAGGAGUATGAAGGGUAGUAGUAGUTUTURGUAGUUUUAG

UUUUTAGGTGGAUTRGUUATTGUUTTUTUTUTGUUUAATTTTUTUTTAGTTUTGGTTUAG

UUTGUTGTT

201
GGTGTUURGRGAGUAGGTGTUUAGUAGURGRGRGUUUAGGRGUARGURGGGUAGUAGU

TRGGGGTRGGRGTTGARGRGGTUUAGRGRGTAUAGUATGGUUTUUAGURGGTGUARGU

UUTGUTUUTTUTTUAGUTGUURGUARGUURGGUUXGURGRGUUURGRGRGTGUAURGG

GAAUAGGURGUUUAGRGTUAGGURGUURGUUAGGRGUAUAGAGUURGURGRGRGRGU

UAGGUURGUUTGRGUUAGUUARGUUAGRGGUAGUAGRGUUARGAGUAGRGGUTUURG

GGUTUTURGGGG

202
UUURGGAGAGUURGGGAGURGUTGUTRGTGGRGUTGUTGURGUTGGRGTGGUTGGRGU

AGGRGGGUUTGGRGRGRGRGGRGGGUTUTGTGRGUUTGGRGGGRGGUUTGARGUTGGG

RGGUUTGTTUURGGTGUARGRGRGGGGRGRGGXGGGURGGGRGTGRGGGUAGUTGAAG

AAGGAGUAGGGRGTGUAURGGUTGGAGGUUATGUTGTARGRGUTGGAURGRGTUAARG

URGAUUURGAGUTGUTGUURGGRGTGRGUUTGGGRGRGRGGUTGUTGGAUAUUTGUTR

GRGGGAUAUU

203
GAAUUAGGGTAGGUATAGUAAAGGUAAUAGGTGGGARGGGGAGUUAGGGAGTGGAAA

RGURGGAGGTUUTUAGGUTGGGGATUURGGAGTUUTRGUTUAGATTURGATTURGGAG

AAGGUUAATTURGGGAUUUTATUUATGTUUUATTUXGGGUAUUTTUUTUAAAAGTAGG

GGTGGGAGGGGUATAAGURGGTUAAGTATTTUURGGGUUTTURGGAGUAATUTTGUUT

AAGRGTTUATATTTAARGUATTGGAUTGGGAAAUATTTGGAGATUATTGATTAAGAAAT

AUTUAAAUUT

204
AGGTTTGAGTATTTUTTAATUAATGATUTUUAAATGTTTUUUAGTUUAATGRGTTAAATA

TGAARGUTTAGGUAAGATTGUTURGGAAGGUURGGGAAATAUTTGAURGGUTTATGUU

UUTUUUAUUUUTAUTTTTGAGGAAGGTGUUXGGAATGGGAUATGGATAGGGTUURGGA

ATTGGUUTTUTURGGAATRGGAATUTGAGRGAGGAUTURGGGATUUUUAGUUTGAGGA

UUTURGGRGTTTUUAUTUUUTGGUTUUURGTUUUAUUTGTTGUUTTTGUTATGUUTAUU

UTGGTTU

205
TTUTRGTRGGTAGRGUTUTGGUUTTGGGRGUURGURGUTTTGGUTATGGAGGTAUAGTG

GURGRGTUURGGGGAGUUTGGGUUTUUTTGRGGUTURGAGUUTURGRGGRGUURGRGU

UURGAUTUTURGGTUURGGUTUTUTUUAGGUUXGGUTGTUTURGGTUUUTUAUTGAUTT

RGUUAGURGUAUUAUUUAUUTUTTTUTUURGUAGARGTAAAUURGAATARGUUURGGU

UTTGGGUUTAGGGUUTRGGUUTRGUUTGGUTUTAUAGGATTRGGGGGRGGGGTAGGUR

GGUAGAGU

206
GUTUTGURGGUUTAUUURGUUUURGAATUUTGTAGAGUUAGGRGAGGURGAGGUUUTA

GGUUUAAGGURGGGGRGTATTRGGGTTTARGTUTGRGGGAGAAAGAGGTGGGTGGTGR

GGUTGGRGAAGTUAGTGAGGGAURGGAGAUAGUXGGGUUTGGAGAGAGURGGGAURG

GAGAGTRGGGGRGRGGGRGURGRGGAGGUTRGGAGURGUAAGGAGGUUUAGGUTUUU

RGGGARGRGGUUAUTGTAUUTUUATAGUUAAAGRGGRGGGRGUUUAAGGUUAGAGRG

UTAURGARGAGAA

207
RGUAUAURGARGGUUTUURGGGTTRGGRGGGGARGAUURGAGUTAGGAGUURGRGGGG

GURGTGGGAGUTGUTURGURGAGGTGGAUURGGGGRGUURGUAUUUUTUAUUTTUTTT

URGTGGGGTURGGGGUUAGGGARGUAGRGGGGGAXGGRGGGUATUAGURGGGUAGRG

UURGAGURGTUUUTGGURGGGTUUUUAUAUUTGRGTGUTRGGGUATUUUUAGGGTRGG

GGGRGRGGTGAATRGTGGTUUURGUAGUURGGGGUURGUUAGGUURGRGGAGTUAGUU

UAUUUTGGRGA

208
TRGUUAGGGTGGGUTGAUTURGRGGGUUTGGRGGGUUURGGGUTGRGGGGAUUARGAT

TUAURGRGUUUURGAUUUTGGGGATGUURGAGUARGUAGGTGTGGGGAUURGGUUAG

GGARGGUTRGGGRGUTGUURGGUTGATGUURGUXGTUUUURGUTGRGTUUUTGGUUUR

GGAUUUUARGGAAAGAAGGTGAGGGGTGRGGGRGUUURGGGTUUAUUTRGGRGGAGU

AGUTUUUARGGUUUURGRGGGUTUUTAGUTRGGGTRGTUUURGURGAAUURGGGAGGU

RGTRGGTGTGRG

209
GAUUAAAAGGAGUTURGRGUUAGGURGGAGGUTGRGUURGTUAUTRGRGGTUURGGUR

GGGTUUUTGGRGRGTAGTUAGGURGUAURGUURGAGUUUUAURGRGRGUUUAUUURGG

URGGGTGRGTURGGUTRGRGGURGTUUUTUURGXGAUUTGTGGUURGGGGUTGUTGRG

GGRGUURGGGGAAGAGAGGRGGGGGURGRGGGGGGUAGGAGGAGRGGUTGRGGURGG

UAUAGRGUUAGGGRGAGTGAGGRGGGTGGRGRGGGGGAGGRGGRGGAGTAAAGAGAG

GURGURGGUTGG

210
UUAGURGGRGGUUTUTUTTTAUTURGURGUUTUUUURGRGUUAUURGUUTUAUTRGUU

UTGGRGUTGTGURGGURGUAGURGUTUUTUUTGUUUUURGRGGUUUURGUUTUTUTTU

UURGGGRGUURGUAGUAGUUURGGGUUAUAGGTXGRGGGAGGGARGGURGRGAGURG

GARGUAUURGGURGGGGTGGGRGRGRGGTGGGGUTRGGGRGGTGRGGUUTGAUTARGR

GUUAGGGAUURGGURGGGAURGRGAGTGARGGGRGUAGUUTURGGUUTGGRGRGGAG

UTUUTTTTGGTU

211
GURGGUAUUTGURGUTURGUURGGGATTAGGGAGUTUURGGUTTGTGGGGGGTGRGGG

RGGURGGTGGTUUAGUTRGUURGUUURGGURGAGARGUTGGGUURGGUUUUUAUAGG

UTGRGUUTGARGGGAURGGRGGGAGGUUTTTGTTXGUAGUURGGAGGUUUUAGGUTUU

AAAUUAUAUTRGGGUTGRGGGGAGRGAGRGRGGGRGTTGURGUTAATTGUTGUTAATTT

TGTTUUATTAAUTTTATTTAUTUTTGAAUUUUTTAATTGTTTTUUAUTTATTTTTAGTAAT

TTTATAA

212
TTATAAAATTAUTAAAAATAAGTGGAAAAUAATTAAGGGGTTUAAGAGTAAATAAAGTT

AATGGAAUAAAATTAGUAGUAATTAGRGGUAARGUURGRGUTRGUTUUURGUAGUURG

AGTGTGGTTTGGAGUUTGGGGUUTURGGGUTGXGAAUAAAGGUUTUURGURGGTUURG

TUAGGRGUAGUUTGTGGGGGURGGGUUUAGRGTUTRGGURGGGGRGGGRGAGUTGGAU

UAURGGURGUURGUAUUUUUUAUAAGURGGGAGUTUUUTAATUURGGGRGGAGRGGU

AGGTGURGGU

213
TUAGUAURGGGGAUAGUTUUTGUUTRGUUAAUTTRGGUAGUTUTTUTRGATGTUTRGUR

GRGGGUTGTGUUTUAUAGGUTARGGGAGGGGAGAGTGTUTUUUUARGURGGGGTUURG

GUUTUTGGGTTTTAGGGAGRGRGAATGGGTUTURGAUAGUAARGGGAGUAGURGGTGG

RGUUTUAGGUTGRGGTGGUAARGAGUURGAUTGUAUTARGGUTTTGRGGGAURGRGUU

AGRGRGGAGGAGAURGAGUUUATUTUAGRGGGURGGGURGGAUUUAGGTGAGRGGUU

UTGGUTUUUU

214
GGGGAGUUAGGGURGUTUAUUTGGGTURGGUURGGUURGUTGAGATGGGUTRGGTUTU

UTURGRGUTGGRGRGGTUURGUAAAGURGTAGTGUAGTRGGGUTRGTTGUUAURGUAG

UUTGAGGRGUUAURGGUTGUTUURGTTGUTGTRGGAGAUUUATTRGRGUTUUUTAAAA

UUUAGAGGURGGGAUUURGGRGTGGGGAGAUAUTUTUUUUTUURGTAGUUTGTGAGGU

AUAGUURGRGGRGAGAUATRGAGAAGAGUTGURGAAGTTGGRGAGGUAGGAGUTGTUU

URGGTGUTGA

215
TTUUTURGAGGUURGUAGGGAGGGGGRGGTGRGGAATGGATGRGURGAGRGGGUAGRG

UTUAGUUTUTRGUTUAUAUUUUUAGUAGGUAGURGRGTUUURGTGURGGUATUUTRGU

TGURGURGGUTUUUTRGGRGUUUURGGGUXGUTUUUUAXGRGRGRGURGGGAUUTGUA

RGAGUUUUUTRGTRGAUTRGGAGRGRGATUTGGGRGRGTGUUTUUUTGTUUTTGTUUTU

TGUTUTRGTUTGGGGARGTGTGUUURGUAUUUUUTGUAURGRGRGUTUUTUTAUUUUTU

URGUTUUU

216
GGGAGRGGGAGGGGTAGAGGAGRGRGRGGTGUAGGGGGTGRGGGGUAUARGTUUUUA

GARGAGAGUAGAGGAUAAGGAUAGGGAGGUARGRGUUUAGATRGRGUTURGAGTRGA

RGAGGGGGUTRGTGUAGGTUURGGRGRGRGXGTGGGGAGXGGUURGGGGGRGURGAGG

GAGURGGRGGUAGRGAGGATGURGGUARGGGGARGRGGUTGUUTGUTGGGGGTGTGAG

RGAGAGGUTGAGRGUTGUURGUTRGGRGUATUUATTURGUAURGUUUUUTUUUTGRGG

GUUTRGGAGGAA

217
AAGGGGAGGGRGGGGURGAUURGGAUUUTUUAGGGAARGUUUTTGAGTAAUTRGRGUA

UTTGGGAUUUATTUUUAUUTAGGAUUATAGGUTUAAGATGGUUTGGTGGATGURGURG

GUARGRGUUTUTUUTUTGGRGGGAAUXGAAGGRGUXGGTAGGTTTTUAUAUTTGUAGU

RGATRGGUTAAGAGAARGRGGGATTUAGURGAGAAGUUAUTGGGAGUURGAGGAGRGG

AGUAGAGGUAUUUAGGUAGUUTGRGRGGAGAAATRGGATRGGUTAGGARGGUUTGUA

GUUUUTGRGRG

218
RGRGUAGGGGUTGUAGGURGTUUTAGURGATURGATTTUTURGRGUAGGUTGUUTGGGT

GUUTUTGUTURGUTUUTRGGGUTUUUAGTGGUTTUTRGGUTGAATUURGRGTTUTUTTA

GURGATRGGUTGUAAGTGTGAAAAUUTAUXGGRGUUTTXGGTTUURGUUAGAGGAGAG

GRGRGTGURGGRGGUATUUAUUAGGUUATUTTGAGUUTATGGTUUTAGGTGGGAATGG

GTUUUAAGTGRGRGAGTTAUTUAAGGGRGTTUUUTGGAGGGTURGGGTRGGUUURGUU

UTUUUUTT

219
TGTGGRGGGGGUTTGGAGUTGUTGAGAGURGAGAGGRGUAGAGRGUAAGUTGGUAGGU

TGGGUTGUTATUURGGRGRGUAGATGUUURGURGUUAGTRGAGRGRGAAUATUTUTUR

GGAAUATRGATUTATUAUUTUUUTTTAAGGAUUXGGAURGGGAAATTTUUATTTTUTGT

TTTGGGAATAAGAAATAAAAGRGAUUAAGUTUTTGUUUTAATTTUUUUURGRGGGUUU

TTUUARGRGGGUTGGRGGGATUAGAAGGARGGGTURGAGUTRGGGGGRGRGGGGTTUU

TGTGAAUTU

220
GAGTTUAUAGGAAUUURGRGUUUURGAGUTRGGAUURGTUUTTUTGATUURGUUAGUU

RGRGTGGAAGGGUURGRGGGGGGAAATTAGGGUAAGAGUTTGGTRGUTTTTATTTUTTA

TTUUUAAAAUAGAAAATGGAAATTTUURGGTUXGGGTUUTTAAAGGGAGGTGATAGAT

RGATGTTURGGAGAGATGTTRGRGUTRGAUTGGRGGRGGGGUATUTGRGRGURGGGATA

GUAGUUUAGUUTGUUAGUTTGRGUTUTGRGUUTUTRGGUTUTUAGUAGUTUUAAGUUU

URGUUAUA

221
UAGUUUTTGGUTTTUURGUTTUAGGUAAAATUTTUUUTUUTTUUTUTTTTTTUTGGGGUU

TTUUUUARGAUUUUTUTTUUTAGUUUTTUTGATURGTUUUUTGATGUATGAUTGGGGUU

AURGGAGGGGUTGAUUUTUURGGAGAGUUUXGTRGGTUUTGGTGGTUTTGGGAURGGA

GAGRGAUAGATGTGGAAAURGAGGUUUUTUAGTGAAGAGUTGUUAGGGTGGTRGUUTT

AGAGUAAAGGRGTTUUTUATTURGUUTGRGUAGUTGUUUUTURGUURGGUTGURGUUU

UAGUURG

222
RGGGUTGGGGRGGUAGURGGGRGGAGGGGUAGUTGRGUAGGRGGAATGAGGAARGUU

TTTGUTUTAAGGRGAUUAUUUTGGUAGUTUTTUAUTGAGGGGUUTRGGTTTUUAUATUT

GTRGUTUTURGGTUUUAAGAUUAUUAGGAURGAXGGGGUTUTURGGGAGGGTUAGUUU

UTURGGTGGUUUUAGTUATGUATUAGGGGARGGATUAGAAGGGUTAGGAAGAGGGGTR

GTGGGGAAGGUUUUAGAAAAAAGAGGAAGGAGGGAAGATTTTGUUTGAAGRGGGAAA

GUUAAGGGUTG

223
GGGAAAGGGGGGAGAGGGAGAGGAGGRGRGGGGTGGGGGAGGGGAGTGAGUAGGGAG

URGGGAGAGGGAGGAGGGGRGGGAAUUAGGGGAGRGGUURGAAUUURGTTTGGTURG

GAUUURGUAGUUAURGUTGGGTUTRGURGURGGGTXGUUUTTRGRGTGGAGATURGGT

URGRGUUUUUTUUURGGTUTUUTUUUUTUUUUTUUUUTURGUUUUUUTUUUTURGGUU

UAUAUAGUUTUTTUUAGAAAGAAGTUAUTUTAGAGURGRGRGAUUUAGUUUUAGAGTU

RGURGGGGTURG

224
RGGAUUURGGRGGAUTUTGGGGUTGGGTRGRGRGGUTUTAGAGTGAUTTUTTTUTGGAA

GAGGUTGTGTGGGURGGAGGGAGGGGGGRGGAGGGGAGGGGAGGGGAGGAGAURGGG

GAGGGGGRGRGGAURGGATUTUUARGRGAAGGGXGAUURGGRGGRGAGAUUUAGRGG

TGGUTGRGGGGTURGGAUUAAARGGGGTTRGGGURGUTUUUUTGGTTUURGUUUUTUU

TUUUTUTUURGGUTUUUTGUTUAUTUUUUTUUUUUAUUURGRGUUTUUTUTUUUTUTU

UUUUUTTTUUU

225
RGGTURGRGUUUUUTUUURGGTUTUUTUUUUTUUUUTUUUUTURGUUUUUUTUUUTUR

GGUUUAUAUAGUUTUTTUUAGAAAGAAGTUAUTUTAGAGURGRGRGAUUUAGUUUUA

GAGTURGURGGGGTURGUUUAURGGGTUTUUTGXGXGUUUUTUUURGUUUUTUUURGG

GUAUAGUURGTTUARGAAAUUTAAGGRGURGGUUARGRGUUAUUTUUUURGGGURGG

GGTUTUUTRGGTUUURGRGRGGGRGUTGGTTUTUURGGGTGGGRGGUAGUUURGUUUT

GTGUUUTUUTGG

226
UUAGGAGGGUAUAGGGRGGGGUTGURGUUUAUURGGGAGAAUUAGRGUURGRGRGGG

GAURGAGGAGAUUURGGUURGGGGGAGGTGGRGRGTGGURGGRGUUTTAGGTTTRGTG

AARGGGUTGTGUURGGGGAGGGGRGGGGAGGGGXGXGUAGGAGAUURGGTGGGRGGA

UUURGGRGGAUTUTGGGGUTGGGTRGRGRGGUTUTAGAGTGAUTTUTTTUTGGAAGAGG

UTGTGTGGGURGGAGGGAGGGGGGRGGAGGGGAGGGGAGGGGAGGAGAURGGGGAGG

GGGRGRGGAURG

227
GRGGTRGGRGUUUUARGUTUUUTGAARGUUUUUUAGGRGGUAUUAGTGURGGGUAGA

GTUUUUTRGGRGGURGRGGGRGTUAAATRGAUAUUTGAUTUUAUAGUTUUUTTUUUTU

TUUTUTTUTTUUAUUTUUURGGGGGTUUAGGUAUXGURGTGRGTUUAUTUURGGTUTUU

ARGGUTTAGGUAGARGGAGTGGGGGAUTURGGGGAUURGRGRGTUUTUTUUTUUTRGG

UUUTGGRGGGAGGAGURGGGUTGGGGTTTURGRGGGURGRGGRGRGTTTTAGGGUTGU

RGGGGAUTGU

228
GUAGTUUURGGUAGUUUTAAAARGRGURGRGGUURGRGGAAAUUUUAGUURGGUTUUT

UURGUUAGGGURGAGGAGGAGAGGARGRGRGGGTUUURGGAGTUUUUUAUTURGTUTG

UUTAAGURGTGGAGAURGGGAGTGGARGUARGGXGGTGUUTGGAUUUURGGGGAGGTG

GAAGAAGAGGAGAGGGAAGGGAGUTGTGGAGTUAGGTGTRGATTTGARGUURGRGGUR

GURGAGGGGAUTUTGUURGGUAUTGGTGURGUUTGGGGGGRGTTUAGGGAGRGTGGGG

RGURGAURGU

229
GUUUTUUTURGTGUTGGGUUTGTUUTAUUTUUAGGGRGGAGGRGRGGGUTUTGRGTUR

GGAGGRGUUTRGGGRGGUAGUTURGGTGGGGURGRGTUTGGTGRGGGGUURGGGAUUU

AGUAGGGUAGUUXGGGATGGAGUUAGGRGGGAGUXGARGGAGURGUTTAUAUUURGU

XGURGGTGTRGURGRGUTTUTUUTTUURGGGGAUUAURGGGTUUUTGGRGGURGURGU

RGURGUTGURGRGGUURGGGAAGUTGRGGUUTAUAGUAGTGGRGGRGGAGRGGRGGGT

GRGGGUUTGGU

230
GUUAGGUURGUAUURGURGUTURGURGUUAUTGUTGTAGGURGUAGUTTUURGGGURG

RGGUAGRGGRGGRGGRGGURGUUAGGGAUURGGTGGTUUURGGGAAGGAGAAGRGRG

GRGAUAURGGXGGXGGGGTGTAAGRGGUTURGTXGGUTUURGUUTGGUTUUATUUXGG

GUTGUUUTGUTGGGTUURGGGUUURGUAUUAGARGRGGUUUUAURGGAGUTGURGUUR

GAGGRGUUTURGGARGUAGAGUURGRGUUTURGUUUTGGAGGTAGGAUAGGUUUAGU

ARGGAGGAGGGU

231
AGAAAAGUAGRGARGTGGRGTTUAUUURGUTGUAGAAUTRGGAUUAUTRGGGUTRGGT

GUAGGGATTGGUTUUAGGUTTGURGTRGGGGTRGGGAGURGAGGARGAGGAGGRGGUR

GGGGGRGGUTGUTGUURGGARGGRGGRGGUTGUTXGRGUTGUTGUTGUTGUTGRGURG

GGAGTGGRGGUTURGRGGGUTRGGGRGGUTURGGRGGRGTRGURGGUURGGGRGGRGG

RGGGGRGGGUTRGGUTGRGUTGTGUUTGRGUUTGGGUAGGGAGUAGRGGRGUTAUTUA

UTGTGGGAUT

232
AGTUUUAUAGTGAGTAGRGURGUTGUTUUUTGUUUAGGRGUAGGUAUAGRGUAGURGA

GUURGUUURGURGURGUURGGGURGGRGARGURGURGGAGURGUURGAGUURGRGGA

GURGUUAUTUURGGRGUAGUAGUAGUAGUAGRGXGAGUAGURGURGURGTURGGGUA

GUAGURGUUUURGGURGUUTUUTRGTUUTRGGUTUURGAUUURGARGGUAAGUUTGGA

GUUAATUUUTGUAURGAGUURGAGTGGTURGAGTTUTGUAGRGGGGTGAARGUUARGT

RGUTGUTTTTUT

233
GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAATGUATTTAGAG

GARGGGAGUTGUUTUTAGGGAGAAAGGGGAGGGGGGAGGGGAGAAGAAAAAGGAAAG

AGRGRGTTTUUTRGURGGGGTTAURGGGGTGGXGRGGGUTTRGRGGURGAGGGGUTGUR

GGUURGGUURGUAGUTUUTRGRGGURGGUTGGGAAGTGGRGGGTURGGUTAGTURGRG

GGRGGGRGGAGRGRGURGTRGUTGUUAGTGTUUUUUTTTAAUATTUUTTUUURGAGUUT

TTUUUT

234
AGGGAAAGGUTRGGGGAAGGAATGTTAAAGGGGGAUAUTGGUAGRGARGGRGRGUTUR

GUURGUURGRGGAUTAGURGGAUURGUUAUTTUUUAGURGGURGRGAGGAGUTGRGGG

URGGGURGGUAGUUUUTRGGURGRGAAGUURGXGUUAUUURGGTAAUUURGGRGAGG

AAARGRGUTUTTTUUTTTTTUTTUTUUUUTUUUUUUTUUUUTTTUTUUUTAGAGGUAGU

TUURGTUUTUTAAATGUATTAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUAUA

UAUAUAUAUAU

235
UTUUTGGGRGAUUTAAAUTTRGATAAUTGUTGGAAAGGTUUAGUAGGAGAGGAGGGRG

AGGAGGGGRGGAGUUAGGAGGUURGGUUURGUUUAURGRGRGUURGUUTUTUTUTGU

AGAUUAURGGUTAGAGUAGGAGUARGAGUTUTTUXGUTGTTTUUAGGATUUUTGURGG

UTGGGUARGRGUUAAAAGUAGUUUTGGGUUUTGGGTATRGRGUTTGGGGGGAGGGTAU

UUURGURGGUTGGGUARGRGUUAAGAGUAGUUUTGGGUUUTGGGTATRGTGUTTAGGG

GGAGGGTATRG

236
RGATAUUUTUUUUUTAAGUARGATAUUUAGGGUUUAGGGUTGUTUTTGGRGRGTGUUU

AGURGGRGGGGGTAUUUTUUUUUUAAGRGRGATAUUUAGGGUUUAGGGUTGUTTTTGG

RGRGTGUUUAGURGGUAGGGATUUTGGAAAUAGXGGAAGAGUTRGTGUTUUTGUTUTA

GURGGTGGTUTGUAGAGAGAGGRGGGRGRGRGGTGGGRGGGGURGGGUUTUUTGGUTU

RGUUUUTUUTRGUUUTUUTUTUUTGUTGGAUUTTTUUAGUAGTTATRGAAGTTTAGGTR

GUUUAGGAG

237
GGRGURGGGRGUTGGGUTTAUAGUAGAGURGRGGGURGRGGGGTRGGAAAGTUUTTUR

GGGGRGGGGURGUAGRGGUUTUTTUURGUAGUUUUTRGGGUURGGGUUURGGTGGAAR

GGAAAUUTUUUUUTAUUURGGGAGGGGUTGUUAGXGGGUTGGGGGTGRGAAAARGGR

GGUAGGAGRGGGRGAGGGGUURGGGURGRGUAUTTTGRGUUTGGGTTTGRGRGURGRG

GURGRGGGAGTUURGRGRGGAURGGURGGARGUURGGUUTUUUUUAGUUUUAGUTTTT

TGTGTGTGTGT

238
AUAUAUAUAUAAAAAGUTGGGGUTGGGGGAGGURGGGRGTURGGURGGTURGRGRGG

GAUTUURGRGGURGRGGRGRGUAAAUUUAGGRGUAAAGTGRGRGGUURGGGUUUUTRG

UURGUTUUTGURGURGTTTTRGUAUUUUUAGUUXGUTGGUAGUUUUTUURGGGGTAGG

GGGAGGTTTURGTTUUAURGGGGUURGGGUURGAGGGGUTGRGGGAAGAGGURGUTGR

GGUUURGUUURGGAAGGAUTTTURGAUUURGRGGUURGRGGUTUTGUTGTAAGUUUAG

RGUURGGRGUU

239
AGGUAUAGAAAGGRGUAGURGUTAGUUAGAGUURGUAUAGAGUUAGAGUUAGGTUUU

UAUTGGRGUAGATGGGGGAUTGUAGGUAUAGUAGTAGUUARGRGGGTGTAAARGTAGA

GURGRGTGAAUURGGGTTGTGGGATUUUAGGUUUXGAUUAGUUUUUATUUURGGUTGG

UATUTRGGTUURGGGGAGTUTUAGUTTUUUTTTUTGUAGAATGGGUTGGAGGRGUUUU

UUAUAGGUURGUUUAGGRGUUUUURGGGGUUAGRGUUUUTUUUUUAUUTGUUURGUU

UUUAUURGRGGG

240
UURGRGGGTGGGGGRGGGGUAGGTGGGGGAGGGGRGUTGGUUURGGGGGGRGUUTGG

GRGGGUUTGTGGGGGGRGUUTUUAGUUUATTUTGUAGAAAGGGAAGUTGAGAUTUUUR

GGGAURGAGATGUUAGURGGGGATGGGGGUTGGTXGGGGUUTGGGATUUUAUAAUUR

GGGTTUARGRGGUTUTARGTTTAUAUURGRGTGGUTAUTGUTGTGUUTGUAGTUUUUUA

TUTGRGUUAGTGGGGAUUTGGUTUTGGUTUTGTGRGGGUTUTGGUTAGRGGUTGRGUUT

TTUTGTGUUT

241
GGGGURGRGGAGTRGGGTGAGGRGGRGGRGGUTGRGGRGGTGGGGURGGGRGAGGTUR

GUTGRGGTUURGGRGGUTURGTGGUTGUTURGUTUTGAGRGUUTGGRGRGUUURGRGU

UUTUUUTGURGGGGURGUTGGGURGGGGATGUAXGRGGGGUURGGGAGUUATGGTURG

UTTRGGGGARGAGUTGGGRGGURGUTATGGGGGUUURGGRGGRGGAGAGRGGGUURGG

GGRGGRGGGGURGGRGGGGRGGGGGGUURGGGTUURGGGGGGUTGUAGUURGGUUAG

RGGGTUUTUTA

242
TAGAGGAUURGUTGGURGGGUTGUAGUUUUURGGGAUURGGGUUUUURGUUURGURG

GUUURGURGUUURGGGUURGUTUTURGURGURGGGGUUUUUATAGRGGURGUUUAGUT

RGTUUURGAAGRGGAUUATGGUTUURGGGUUURGXGTGUATUUURGGUUUAGRGGUUU

RGGUAGGGAGGGRGRGGGGRGRGUUAGGRGUTUAGAGRGGAGUAGUUARGGAGURGU

RGGGAURGUAGRGGAUUTRGUURGGUUUUAURGURGUAGURGURGURGUUTUAUURG

AUTURGRGGUUUU

Compositions for Detecting Methylation

Also provided herein are probes and primers that are complementary to one or more of SEQ ID NOS: 1-242. In embodiments, pairs of primers complementary to nucleotide sequences on either side of a methylation site of interest listed in Table 1 are provided. In embodiments, a plurality of probes and/or primers are provided to detect and/or amplify a polynucleotide (e.g., a polynucleotide obtained by bisulfate treatment of DNA) comprising a methylation site of interest. In embodiments, a probe or primer is complementary to a polynucleotide sequence that encompasses the methylation site of interest. In embodiments, the probe or primer is complementary to a sequence that is proximal to the methylation site of interest (e.g., within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, or 25 nucleotides of the methylation site of interest in a genomic or bisulfate-treatment-derived polynucleotide).

In embodiments, a deoxyribonucleic acid selected from SEQ ID NO:243 to SEQ ID NO:356 is included. In embodiments, the deoxyribonucleic acid selected from SEQ ID NO:243 to SEQ ID NO:356 is hybridized to a complementary DNA sequence having uridine or cytosine. In embodiments, each of the nucleic acids is different. In embodiments, each of the nucleic acids does not simultaneously have the same sequence selected from SEQ ID NO:243 to SEQ ID NO:356. SEQ ID NOS: 243 to 356 are listed in Table 5 below.

TABLE 5

SEQ ID NOS: 243 to 356

SEQ ID NO:
Sequence

243
GTTTTTTTTTTAAAGTAGTTTTT

244
CTCCTACAACCCCCTTCC

245
TTTTGAGTAGTTGGAGTTATAG

246
TAAATTCATCCTCTACCTATTA

247
ATGGTATGAATTAATTAATTTGA

248
CTCCCTAAAAAAAAAAATAAA

249
TATTTGTTTGATTTTAATTATATTT

250
AAACTCTACTCTCTAAACCTTTC

251
ATTTTATTGATTATGTTTAGTTGATTA

252
AAAAATACCCCAAAAACAA

253
GTGGAAGGGAAAAAAAAAGAG

254
ACAAACTCCCCTATACCTCAAATA

255
TTTTTTAAATGGTGAAATAT

256
AATTTTACTTCTTCTTCTTATC

257
TGGTAATAATTGGAGGAATTG

258
ACAAATAAAATCATAAAAAATAACAAAC

259
GATTTTTTGATTTGAAAATAGTT

260
AATCTCAACCCCAAACTC

261
TTAGGTAAAGATTTGGTTTTAGAA

262
AACTTATTATATAAATTATAAAAAAATAAA

263
GGAGGTGTTTTTTAGTAAGTTTG

264
CCTCATACCTATAACCTACACTCA

265
AAGGGTTGTTATGTTAGTGTAGT

266
CCCAAAAATAAATAATTTAACTA

267
ATTTTTGTTTTAATATGGAGTTG

268
AAACCTAAATCTACACTTAAACATC

269
AGGGTTAGGGTTTTTTTG

270
AAATCTCTTTATTACTCATTTTCTATA

271
GTTATTTATATTTTTGAGTATTAAGAGTT

272
ACCAACAAATACAACACCTTCT

273
GGTTTTTTAGTTTTATGAATTATTTA

274
AACCCCTCTACAACCTACTAC

275
GATGAGGAAGTTGAGGTATAG

276
CTCCAACCCATTCTACAA

277
GAGGGGATTGAGTAGGTGAATAG

278
CAAAACAAAAAAAAAATAAAAAAAAAACT

279
AAGGAGAGGAGAAGAGGTATAG

280
AATAATAAAAAAAAACTAAATTCAAAC

281
TTGGGGTTTAGGGATTTAG

282
AATAAAATATACCTCCTTTCAAACTAA

283
AAAGGTTAAATTAAAAATTTTTTTAT

284
CTCCCAACTATACTTCTTAATCTC

285
GGTTTAGAGTTATTGAATAAATGAAGTG

286
CAAAATCAAATTCTCCAACAAA

287
TGATGGAGGAAGTTTTTG

288
AACCCTAAACTAAACAACCC

289
TTAAGGATTTAGATATTTTGTAAT

290
AATAAATTTATAAATTTACTCTCTTAC

291
AATGTTTTGGAGATTAGTAATAT

292
TCTTCCTAAAAAAAAAATAAA

293
GGGAATAATGAGGAGGAGA

294
AAAAATACAATAAAAAATCTTAAAATAAA

295
GGTAGGGGATTGGGATAG

296
AACAAAACAAAACTATAAAATTAAACTAA

297
GGTTAGGAGATTAGGGATTG

298
AAAAAAAAAAACAACTTAAAAAAC

299
TGTTTATTTTAGAGGTGTTTATTT

300
ACCCTAATCCAATATCCC

301
GGTTGGTTAGGTTGTTATTT

302
CAAACACCACATACTTATTC

303
TTTGGGTTTAGAAAGTTG

304
CAAAAACTAAAAAAAAAATAAC

305
TTTTGAGTTTGGTTTAGTT

306
CCTAAAAAAAATAAAAATCC

307
GGAATTTGGAGGGTAAAA

308
CTAATCTCTATCCTATATATTTCTTTATATT

309
TATTATATTGTTATGTTGATT

310
ATATTAATTTCTTCCAACTAA

311
TGGGATTGGTAAGTAGGTATT

312
AAAAAAAAAAACCTATAACCTATAAA

313
ATGGTTTTTGTTTGTAGAC

314
ATTATTATTATTTATTTATTTATTATCA

315
TTTTTTTGAAAGTTAGTGAATTTATTTATT

316
ACCCATCTCCCCACACAC

317
TTGGGGTTTAAAGGTATTAG

318
AAAACAAAAACTACTAAAAAAAAAT

319
TTGGTTAAGGAAGAAAGGAGTAG

320
CACCCCCTTCAAAAAAAA

321
TGGGGTTTTTTGGTTTTTT

322
CCACCTCACAAACACACAC

323
AGAAAGGTATTTGTTTTTAGTAAA

324
TTACAAAATAAAACCAACCTAT

325
GAGTTTTTTTTTTTATTTAGTTTT

326
CCTACCCCCTAATATCTACA

327
TTTTTTTAATATTTGTGAATTAT

328
AAACAACAACCCTAACTATC

329
ATGGAAGTTGGAGTTGAGAAG

330
ACACCTAAAACAAAAATAAAAAAATC

331
ATTAGTTTTTAGTTTTTTAGTATT

332
TCCCCTTAACATTAAATC

333
TTGAGATTAGATTAGAGTTTATTT

334
AAAAAAACCTTAATCCTATAAT

335
TTTTTAGTAGGGTTAAGAGGATTT

336
TCCAATACACACCACCAA

337
TTTGAATGGTAGAGGAAATAGTT

338
ACCCAAAAATTCTATCTTTCAC

339
TTTATGGGTTTTTATTTTAGTA

340
AAAAATATTCCAATATAAACAAA

341
AGAGTTGTTGTTGTTTTTTATGT

342
CAACCCAAAATCCTTAACTATA

343
AAAAGAATTAGGGTAGGTATAGT

344
CTTAATCAATAATCTCCAAATAT

345
TTTGGTTATGGAGGTATAGT

346
AAAAAAAAAATAAATAATAC

347
ATTTTATTTGGGGATTTTTAATA

348
TAAAACAAAATTAACAACAATTAAC

349
TAAGATGGTTTGGTGGATGT

350
AAATCTCTCACTCACCCTTTC

351
GGTTTGGAGTTGTTGAGAG

352
AAAAAAATTAAAACAAAAACTTAATC

353
ATTTTTTTTTTTAGTTTTTTTGAT

354
CACCCTAACAACTCTTCACT

355
GGGGAGAAGAAAAAGGAAAG

356
AAAAAAAATATTAAAAAAAAACACTAACA

In embodiments, aspects include a deoxyribonucleic acid selected from SEQ ID NO:243 to SEQ ID NO:356, hybridized to corresponding a complementary DNA sequence having uridine or cytosine, and in a complex with an enzyme, e.g., a thermostable DNA polymerase. In embodiments, the thermostable DNA polymerase is Taq DNA polymerase.

In some aspects, the method includes deoxyribonucleic acid that has a sequence that is at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to a nucleic acid having a sequence of at least one of SEQ ID NO:243 to SEQ ID NO:356.

Kit for Detecting Methylation Level of a DCIS Cell Mass

Also provided is a kit including a plurality (e.g., at least about 10, 20, 40, 50, 100, 110 or 118) nucleic acids each independently comprising one sequence selected from SEQ ID NO:243 to SEQ ID NO:356, in which the nucleic acids do not simultaneously include the same sequence.

In some aspects, the kit includes deoxyribonucleic acid that has a sequence that is at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to a nucleic acid having a sequence of at least one of SEQ ID NO:243 to SEQ ID NO:356.

The kit provided herein may include enzymes, reagents for deamination of cytosine, buffers, vials, control DNA, devices for collecting DCIS tissue samples, reagents for isolating DNA, reagents for labeling DNA, labels, or any combinations thereof.

The kit provided herein may include enzymes such as thermostable DNA polymerase enzymes, restriction enzymes, and combination thereof.

In embodiments, the kit(s) may further include enzymes, reagents for deamination of cytosine, buffers, vials, control DNA, devices for tissue samples, or reagents for labeling DNA, or any combinations thereof.

In embodiments, a kit provided herein may include a solid carrier capable of adsorbing the nucleic acids containing in a sample of a body fluid, for example blood (whole blood, plasma, or serum). The kit may also contain other components for example, reagents, in concentrated or final dilution form, chromatographic materials for the separation of the nucleic acids, aqueous solutions (buffers, optionally also in concentrated form for final adjusting by the user) or chromatographic materials for desalting nucleic acids which have been eluted with sodium chloride.

In embodiments, a kit provided herein includes materials for purifying nucleic acids, for example, inorganic and/or organic carriers and optionally solutions, excipients and/or accessories. Such agents are known and are commercially available. For solid phase nucleic acid isolation methods, many solid supports have been used including membrane filters, magnetic beads, metal oxides, and latex particles.

In addition, a kit can also contain excipients such as, for example, a protease such as proteinase K, or enzymes and other agents for manipulating nucleic acids, e.g., at least one amplification primer, nucleic acid bases (A, T, G, C, and/or U), and enzymes suitable for amplifying nucleic acids, e.g., DNase, a nucleic acid polymerase and/or at least one restriction endonuclease. Alternatively, a commercial polymerase chain reaction kit may be used to amplify the DNA samples.

Exemplary Techniques for Detecting Specific Sequences

Specific sequences, such as the sequences listed in Table 1 (or portions thereof containing a methylation site of interest), can be detected by numerous methods that are well-established in the art (e.g., PCR-based sequence specific amplification, isozyme markers, northern analysis, sequence specific hybridization, and array based hybridization). In embodiments, the presence or absence of methylation is determined through nucleotide sequencing of the site of interest (e.g., the site in bisulfite-treated DNA or an amplicon thereof). Any of these methods are readily adapted to high throughput analysis.

Some techniques for detecting specific sequences utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the methylation site of interest (e.g., amplified nucleic acids produced using bisulfite-treated DNA as a template or the bisulfite-treated DNA itself). Hybridization formats, including, but not limited to: solution phase, solid phase, mixed phase, or in situ hybridization assays are useful for sequence detection. A non-limiting guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Elsevier, N.Y., as well as in Sambrook, Berger and Ausubel.

Nucleic acid probes complementary to a methylation site can be cloned and/or synthesized. Any suitable label can be used with a probe. Detectable labels suitable for use with nucleic acid probes include, for example, any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. A probe can also constitute radiolabelled PCR primers that are used to generate a radiolabelled amplicon. Labeling strategies for labeling nucleic acids and corresponding detection strategies can be found, e.g., in Haugland (2003) Handbook of Probes and Research Chemicals Ninth Edition by Molecular Probes, Inc. (Eugene Oreg.). Additional non-limiting details regarding sequence detection strategies are found below.

PCR, RT-PCR and LCR are in particularly broad use as amplification and amplification-detection methods for amplifying nucleic acids (e.g., those comprising a methylation site), facilitating detection of the nucleic acids of interest.

In embodiments, real time PCR or LCR is performed on the amplification mixtures described herein, e.g., using molecular beacons or TaqMan™ probes. A molecular beacon (MB) is an oligonucleotide or peptide nucleic acid (PNA) which, under appropriate hybridization conditions, self-hybridizes to form a stem and loop structure. The MB has a label and a quencher at the termini of the oligonucleotide or PNA; thus, under conditions that permit intra-molecular hybridization, the label is typically quenched (or at least altered in its fluorescence) by the quencher. Under conditions where the MB does not display intra-molecular hybridization (e.g., when bound to a target nucleic acid, e.g., to a region of an amplicon during amplification), the MB label is unquenched. Details regarding standard methods of making and using MBs are well established in the literature and MBs are available from a number of commercial reagent sources. See also, e.g., Leone et al. (1995) “Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.” Nucleic Acids Res. 26:2150-2155; Tyagi and Kramer (1996) “Molecular beacons: probes that fluoresce upon hybridization” Nature Biotechnology 14:303-308; Blok and Kramer (1997) “Amplifiable hybridization probes containing a molecular switch” Mol Cell Probes 11:187-194; Hsuih et al. (1997) “Novel, ligation-dependent PCR assay for detection of hepatitis C in serum” J Clin Microbiol 34:501-507; Kostrikis et al. (1998) “Molecular beacons: spectral genotyping of human alleles” Science 279:1228-1229; Sokol et al. (1998) “Real time detection of DNA:RNA hybridization in living cells” Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons for allele discrimination” Nature Biotechnology 16:49-53; Bonnet et al. (1999) “Thermodynamic basis of the chemical specificity of structured DNA probes” Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang et al. (1999) “Designing a novel molecular beacon for surface-immobilized DNA hybridization studies” J. Am. Chem. Soc. 121:2921-2922; Marras et al. (1999) “Multiplex detection of single-nucleotide variation using molecular beacons” Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al. (1999) “Multiplex detection of four pathogenic retroviruses using molecular beacons” Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399. Additional details regarding MB construction and use is found in the patent literature, e.g., U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al. entitled “Detectably labeled dual conformation oligonucleotide probes, assays and kits;” U.S. Pat. No. 6,150,097 to Tyagi et al (Nov. 21, 2000) entitled “Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes” and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000), entitled “Wavelength-shifting probes and primers and their use in assays and kits.”

PCR detection and quantification using dual-labeled fluorogenic oligonucleotide probes, commonly referred to as “TaqMan™” probes, can also be performed. These probes are composed of short (e.g., 20-25 base) oligodeoxynucleotides that are labeled with two different fluorescent dyes. On the 5′ terminus of each probe is a reporter dye, and on the 3′ terminus of each probe a quenching dye is found. The oligonucleotide probe sequence is complementary to an internal target sequence present in a PCR amplicon. When the probe is intact, energy transfer occurs between the two fluorophores and emission from the reporter is quenched by the quencher by FRET. During the extension phase of PCR, the probe is cleaved by 5′ nuclease activity of the polymerase used in the reaction, thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity. Accordingly, TaqMan™ probes are oligonucleotides that have a label and a quencher, where the label is released during amplification by the exonuclease action of the polymerase used in amplification. This provides a real time measure of amplification during synthesis. A variety of TaqMan™ reagents are commercially available, e.g., from Applied Biosystems (Division Headquarters in Foster City, Calif.) as well as from a variety of specialty vendors such as Biosearch Technologies (e.g., black hole quencher probes). Further details regarding dual-label probe strategies can be found, e.g., in WO92/02638.

Other similar methods include e.g. fluorescence resonance energy transfer between two adjacently hybridized probes, e.g., using the “LightCycler™” format described in U.S. Pat. No. 6,174,670.

Amplification and Sequencing Primers

In embodiments, methylation sites are detected using primers, e.g., to amplify and/or sequence polynucleotides comprising the methylation sites.

Suitable primers can be designed and is not intended that the present subject matter be limited to any particular primer or primer pair. For example, primers can be designed using any suitable software program, such as LASERGENE™, e.g., taking account of publicly available sequence information. Flanking sequences for the methylation sites identified herein are publicly available; accordingly, suitable amplification primers can be constructed based on well understood base-pairing rules. The sequence of any amplicon can be detected as has already been discussed above, e.g., by sequencing, hybridization, array hybridization, PCR, LCR, or the like.

In embodiments, the primers are radiolabelled, or labeled by any suitable means (e.g., using a non-radioactive fluorescent tag), to allow for rapid visualization of differently sized amplicons following an amplification reaction without any additional labeling step or visualization step. In embodiments, the primers are not labeled, and the amplicons are visualized following their size resolution, e.g., following agarose or acrylamide gel electrophoresis. In embodiments, ethidium bromide staining of the PCR amplicons following size resolution allows visualization of the different size amplicons.

It is not intended that the primers be limited to generating an amplicon of any particular size. The primers can generate an amplicon of any suitable length for detection (e.g., by sequencing or hybridization). In embodiments, amplification produces an amplicon at least 20 nucleotides in length, or alternatively, at least 50 nucleotides in length, or alternatively, at least 100 nucleotides in length, or alternatively, at least 200 nucleotides in length. Amplicons of any size can be detected and/or sequenced using various technologies described herein and known in the art.

Detection of Methylation Levels Using Sequencing

Sequencing is the process of determining the precise order of nucleotides within a DNA molecule. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Non-limiting examples and descriptions are provided below. However, embodiments are not limited to the use of a particular sequencing assay, technology, or approach.

Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication (Sanger F; Coulson A R (May 1975) J. Mol. Biol. 94 (3): 441-8; Sanger et al. (December 1977) Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5463-7).

In embodiments, next-generation sequencing is used. Non-limiting examples of next-generation sequencing methods include massively parallel signature sequencing (MPSS), single-molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by ligation, chain termination, DNA nanoball sequencing, helicos single molecule sequencing, single molecule real time sequencing, nanopore DNA sequencing, tunnelling currents DNA sequencing, and sequencing by hybridization.

Many commercially available sequencing technologies, devices, and services are available. In embodiments, an Illumina sequencer is used. In embodiments, PCR products are ligated with a linker and sequenced using a high throughput sequencer, such as an Illumina sequencer. In embodiments, the ligation step can be avoided, omitted, or eliminated by adding a linker to amplification primers.

Array-Based Sequence Detection

Array-based detection can be performed using commercially available arrays, e.g., from Affymetrix (Santa Clara, Calif.) or other manufacturers. Reviews regarding the operation of nucleic acid arrays include Sapolsky et al. (1999) “High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays.” Genetic Analysis: Biomolecular Engineering 14:187-192; Lockhart (1998) “Mutant yeast on drugs” Nature Medicine 4:1235-1236; Fodor (1997) “Genes, Chips and the Human Genome.” FASEB Journal 11:A879; Fodor (1997) “Massively Parallel Genomics.” Science 277: 393-395; and Chee et al. (1996) “Accessing Genetic Information with High-Density DNA Arrays.” Science 274:610-614.

A variety of probe arrays have been described in the literature and can be used for detection of methylation. For example, DNA probe array chips or larger DNA probe array wafers (from which individual chips would otherwise be obtained by breaking up the wafer) may be used in embodiments described herein. DNA probe array wafers generally comprise glass wafers on which high density arrays of DNA probes (short segments of DNA) have been placed. Each of these wafers can hold, for example, approximately 60 million DNA probes that are used to recognize longer sample DNA sequences (e.g., from individuals or populations, e.g., that comprise methylation sites of interest). The recognition of sample DNA by the set of DNA probes on the glass wafer takes place through DNA hybridization. When a DNA sample hybridizes with an array of DNA probes, the sample binds to those probes that are complementary to the sample DNA sequence. By evaluating to which probes the sample DNA for an individual hybridizes more strongly, it is possible to determine whether a known sequence of nucleic acid is present or not in the sample, thereby determining whether a uracil, thymine, or cytosine is present at a polynucleotide site corresponding to a genomic methylation site. One can also use this approach to control the hybridization conditions to permit single nucleotide discrimination, e.g., for the identification of methylation at a site of interest. Arrays provide one convenient embodiment for detecting multiple methylation sites simultaneously (or in series). Of course, any detection technology (PCR, LCR, and/or sequencing etc.) can similarly be used, e.g., with multiplex amplification/detection/sequencing reactions, or simply by running several separate reactions, e.g., simultaneously or in series.

In embodiments, the use of DNA probe arrays to obtain methylation information involves the following general steps: design and manufacture of DNA probe arrays, preparation of the sample, bisulfite treatment, hybridization of sample DNA to the array, detection of hybridization events and data analysis to determine sequence. In embodiments, an array is used to capture polynucleotides containing a methylation site of interest, and the captured polynucleotides are subsequently amplified and/or sequenced. Preferred wafers are manufactured using a process adapted from semiconductor manufacturing to achieve cost effectiveness and high quality, and are available, e.g., from Affymetrix, Inc. of Santa Clara, Calif.

For example, probe arrays can be manufactured by light-directed chemical synthesis processes, which combine solid-phase chemical synthesis with photolithographic fabrication techniques as employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density arrays of oligonucleotides, with each probe in a predefined position in the array. Multiple probe arrays can be synthesized simultaneously on a large glass wafer. This parallel process enhances reproducibility and helps achieve economies of scale.

In embodiments, DNA probe arrays can be used to obtain data regarding presence of sequences (e.g., corresponding to methylated or unmethylated DNA) of interest. The DNA samples may be tagged with biotin and/or a fluorescent reporter group by standard biochemical methods. The labeled samples are incubated with an array, and segments of the samples bind, or hybridize, with complementary sequences on the array. The array can be washed and/or stained to produce a hybridization pattern. The array is then scanned and the patterns of hybridization are detected by emission of light from the fluorescent reporter groups. Because the identity and position of each probe on the array is known, the nature of the DNA sequences in the sample applied to the array can be determined.

In embodiments, the nucleic acid sample to be analyzed is isolated, bisulfite-treated, amplified and, optionally, labeled with biotin and/or a fluorescent reporter group. The labeled nucleic acid sample may then be incubated with the array using a fluidics station and hybridization oven. The array can be washed and or stained or counter-stained, as appropriate to the detection method. After hybridization, washing and staining, the array is inserted into a scanner, where patterns of hybridization are detected. The hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the labeled nucleic acid, which is now bound to the probe array. Probes that most clearly match the labeled nucleic acid produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, by complementarity, the identity of the nucleic acid sample applied to the probe array can be identified. In embodiments, hybridization techniques and conditions that allow only fully complementary nucleotide sequences to hybridize with probes in an array are used.

Prior to amplification and/or detection of a nucleic acid comprising a sequence of interest, the nucleic acid is optionally purified from the samples by any available method, e.g., those taught in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 (“Sambrook”); and/or Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) (“Ausubel”)). A plethora of kits are also commercially available for the purification of nucleic acids from cells or other samples (see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Alternately, samples can simply be directly subjected to amplification or detection, e.g., following aliquotting and/or dilution.

Breast Cancer Diagnostic System and Processes

In embodiments, included herein is a system for detecting methylation or unmethylation of a DCIS cancer cell proliferation DNA molecule of a subject. In embodiments, the system provides at least one processor; and at least one memory including program code which when executed by the at least one processor provides operations comprising: contacting an isolated DCIS cancer cell proliferation DNA molecule from the subject with a bisulfite salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule; detecting the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at a methylation site set forth in Table 1, thereby detecting methylation or unmethylation of the DCIS cancer cell proliferation DNA molecule of the subject; generating a diagnosis for the subject based at least in part on the presence or absence of uracil in the reacted DCIS cancer cell proliferation DNA molecule at the methylation site set forth in Table 1; and providing, via a user interface, the diagnosis or prognosis for the subject. In embodiments, the system provides at least one processor; and at least one memory including program code which when executed by the at least one processor provides operations comprising: contacting the plurality of isolated DCIS cancer cell proliferation DNA molecules with a bisulfite salt thereby forming a plurality of reacted DCIS cancer cell proliferation DNA molecules; detecting the level of reacted DCIS cancer cell proliferation DNA molecules in the plurality of reacted DCIS cancer cell proliferation DNA molecules having a uracil at a methylation site set forth in Table 1 thereby detecting the level of methylation or unmethylation in the plurality of DCIS cancer cell proliferation DNA molecules of the subject; generating a diagnosis for the subject based at least in part on the level of methylation or unmethylation at the plurality of methylation sites set forth in Table 1; and providing, via a user interface, the diagnosis or prognosis for the subject.

FIG. 2 depicts a block diagram illustrating an exemplary breast cancer diagnostic system 600. Referring to FIG. 2, the breast cancer diagnostic system 600 can include an input module 610, an isolation module 612, a conversion module 614, a detection module 616, a diagnosis module 618, a treatment module 620, and a user interface (UI) module 622. The breast cancer diagnostic system 600 can be configured to provide a diagnosis indicative of a presence of IDC and/or a risk of developing IDC. Moreover, the breast cancer diagnostic system 600 can be further configured to generate a treatment plan for a subject based on the diagnosis. For instance, when the diagnosis indicates a presence and/or risk of IDC in a subject, the breast cancer diagnostic system 600 can recommend one or more treatments including, for example, surgery (e.g., lumpectomy or mastectomy), radiation therapy, chemotherapy, hormone therapy, targeted therapy, and/or administration of an active agent.

One or more modules of the breast cancer diagnostic system 600 can be realized in digital electronic circuitry, integrated circuitry, specially designed application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs) computer hardware, firmware, software, and/or combinations thereof. The breast cancer diagnostic system 600 can further be communicatively coupled with one or more devices including, for example, a device 630. The breast cancer diagnostic system 600 can communicate with the device 620 via a wired and/or wireless network 640 (e.g., a wide area network (WAN), a local area network (LAN), and/or the Internet). As shown in FIG. 2, the breast cancer diagnostic system 600 can be further coupled with a data store 650.

The input module 610 can be adapted to receive and/or collect a sample of a DCIS cancer cell proliferation obtained from a subject. The isolation module 612 can be configured to isolate DNA from the DCIS cancer cell proliferation sample received by the input module 610 thereby forming isolated DCIS cancer cell proliferation DNA. The conversion module 614 can be configured to treat the isolated DCIS cancer cell proliferation DNA including by contacting the isolated DCIS cancer cell proliferation DNA with one or more bisulfite reagents including, for example, a bisulfite salt. Exposing the isolated DCIS cancer cell proliferation DNA to one or more bisulfite reagents can convert cytosine to uracil while 5-mC is left unmodified. Thus, the 5-mC present in the isolated DCIS cancer cell proliferation DNA will remain in the reacted DCIS cancer cell proliferation DNA. Meanwhile, any cytosine in the isolated DCIS cancer cell proliferation DNA will be replaced by uracil in the reacted DCIS cancer cell proliferation DNA. In embodiments, the treatment of the isolated DCIS cancer cell proliferation DNA can be performed by applying one or more kits (e.g., the Bisulflash DNA Modification Kit (Epigentek) or Imprint DNA Modification Kit (Sigma)).

In embodiments, the conversion module 614 can be further adapted to ensure optimal bisulfite conversion (e.g., with desired DNA fragment size for post-bisulfite ligation) by controlling one or more of a concentration of the bisulfite reagents, temperature, and reaction time period. It should be appreciated that the conversion module 614 can be adapted to use a different and/or additional type of reagent without departing from the scope of the present subject matter. For example, the conversion module 614 can treat the isolated DCIS cancer cell proliferation DNA with potassium chloride, which may reduce the thermophilic DNA degradation associated with the conversion of cytosine to uracil. Moreover, the conversion module 614 can be configured to perform additional processing of the reacted DCIS cancer cell proliferation DNA including, for example, desulphonation (e.g., with an alkalized solution), cleansing (e.g., by elution), and amplification (e.g., using the PCR method).

The detection module 616 can be configured to detect a methylation and/or unmethylation of the DCIS cancer cell proliferation DNA. For instance, the detection module 616 can detect methylation by detecting a presence of uracil in the reacted DCIS cancer cell proliferation DNA generated by the conversion module 614. Alternately and/or additionally, the detection module 616 can detect unmethylation by detecting an absence of uracil in the reacted DCIS cancer cell proliferation DNA. In embodiments, the detection module 616 can be configured detect the presence and/or absence of uracil at specific methylation sites. That is, the detection module 616 can be configured to detect the presence and/or absence of uracil at specific chromosomal positions of certain chromosomes. For example, the breast cancer diagnostic system 600 can store a plurality of specific methylation sites (e.g., Table 1) in the data store 650. As such, to detect methylation, the detection module 616 can be configured to obtain, from the data store 650, one or more specific methylation sites at which to test for the presence and/or absence of uracil. Moreover, in embodiments, the detection module 616 can be configured to determine a level of methylation and/or unmethylation at the specific methylation sites. The level of methylation at a particular site can correspond to a proportion of the reacted DCIS cancer cell proliferation DNA that has a cytosine rather than a uracil at that site. By contrast, the level of unmethylation at a particular site can correspond to a proportion of reacted DCIS cancer cell proliferation DNA that has a uracil rather than a cytosine at that site.

In embodiments, the conversion module 614 may amplify the reacted DCIS cancer cell proliferation DNA such as by using a PCR method. The detection of methylation and/or unmethylation in amplified reacted DCIS cancer cell proliferation DNA may require detection of a presence and/or absence of thymidine at a site of interest in amplicons amplified from the reacted DCIS cancer cell proliferation DNA. That is, instead of detecting the presence and/or absence of uracil, the detection module 616 can be configured to detect methylation and/or unmethylation of amplified reacted DCIS cancer cell proliferation DNA by detecting a presence and/or absence of thymidine at specific methylation sites (e.g., as set forth in Table 1).

The diagnosis module 618 can be configured to generate a diagnosis for the subject based on whether the detection module 616 detects methylation and/or unmethylation at the plurality of specific methylation sites (e.g., Table 1). Alternately or additionally, the diagnosis module 618 can be configured to generate a diagnosis for the subject based on a level of methylation and/or unmethylation detected by the detection module 616 at the plurality of specific methylation sites. For instance, diagnosis module 618 can determine that the DCIS cancer cell proliferation is invasive competent when the unmethylation level (e.g., proportion of uracil) at different methylation sites exceeds the corresponding thresholds (e.g., as set forth in Table 2).

The treatment module 620 can be configured to formulate a treatment plan for the subject based on the diagnosis generated by the diagnosis module 618. For instance, when the diagnosis generated by the diagnosis module 618 indicates a presence and/or risk of IDC, the treatment module 620 can prescribe or suggest one or more treatments including, for example, surgery, radiation therapy, chemotherapy, hormone therapy, and/or administration of an active agent. In embodiments, the treatment module 620 can be configured to provide the treatment plan to the device 630 via the network 640. Alternately or additionally, the treatment module 620 can store the treatment plan in the data store 650.

The UI module 622 can be configured to generate a UI through which a user (e.g., a physician) can interface with the breast cancer diagnostic system 600. For example, the UI module 622 can provide one or more graphic user interfaces (GUIs) configured to display the diagnosis and/or treatment plan for the subject.

FIG. 5 depicts a flowchart illustrating an exemplary process 700 for diagnosing invasive or invasive competent breast cancer. Referring to FIGS. 3 and 4, the process 700 can be performed by the breast cancer diagnostic system 600.

The breast cancer diagnostic system 600 (e.g., the input module 610) can receive a sample of a DCIS cancer cell proliferation from a subject (702). The breast cancer diagnostic system 600 (e.g., the isolation module 612) can isolate DCIS cancer cell proliferation DNA from the DCIS cancer cell proliferation sample (704). The breast cancer diagnostic system 600 (e.g., the conversion module 614) can treat the isolated DCIS cancer cell proliferation DNA with a bisulfite salt to generate reacted DCIS cancer cell proliferation DNA (706). Treating the isolated DCIS cancer cell proliferation DNA with the bisulfite salt can form a reacted DCIS cancer cell proliferation DNA by converting the cytosine present in the isolated DCIS cancer cell proliferation DNA to uracil. In embodiments, the breast cancer diagnostic system 600 can further process the reacted DCIS cancer cell proliferation DNA by desulphonating, cleansing, and/or amplifying the reacted DCIS cancer cell proliferation DNA.

The breast cancer diagnostic system 600 (e.g., the detection module 616) can detect methylation and/or unmethylation of the isolated DCIS cancer cell proliferation DNA by at least detecting a presence and/or absence of uracil in the reacted DCIS cancer cell proliferation DNA (708). In embodiments, the breast cancer diagnostic system 600 can be configured to detect a presence and/or absence of uracil at specific methylation sites (e.g., as set forth in Table 1). Moreover, the breast cancer diagnostic system 600 can be configured to detect a level of methylation and/or unmethylation at the methylation sites.

The breast cancer diagnostic system 600 (e.g., the diagnostics module 618) can generate a diagnosis for the subject based on the methylation and/or unmethylation of the isolated DCIS cancer cell proliferation DNA (710). For example, the breast cancer diagnostic system 600 can generate a diagnosis based on a level of methylation and/or unmethylation at a plurality of specific methylation sites. Each methylation site may be associated with a certain threshold unmethylation level (e.g., as set forth in Table 2). As such, the breast cancer diagnostic system 600 can determine that the DCIS cancer cell proliferation from the subject is invasive if the level of unmethylation at the plurality of methylation sites exceeds (e.g., as set forth in Table 2) or is below (e.g., as set forth in Table 3) the corresponding thresholds.

The breast cancer diagnostic system 600 (e.g., the treatment module 620) can formulate, based on the diagnosis, a treatment plan for the subject (712). For example, when the diagnosis indicates that a presence and/or risk of IDC in the subject, the breast cancer diagnostic system 600 can prescribe or suggest surgery, radiation therapy, chemotherapy, hormone therapy, and/or administration of an active agent. The breast cancer diagnostic system 600 (e.g., the UI module 622) can provide, via a UI (e.g., GUI at the device 630), the diagnosis and/or the treatment plan for the subject (714).

FIG. 6 depicts a flowchart illustrating an exemplary process 800 for diagnosing IDC. Referring to FIGS. 2 and 4, the process 700 can be performed by the breast cancer diagnostic system 600.

The breast cancer diagnostic system 600 (e.g., the input module 610) can receive a sample of a DCIS cancer cell proliferation from a subject (802). The breast cancer diagnostic system 600 (e.g., the isolation module 612) can isolate DCIS cancer cell proliferation DNA from the DCIS cancer cell proliferation sample (804). The breast cancer diagnostic system 600 (e.g., the conversion module 614) can treat the isolated DCIS cancer cell proliferation DNA with a bisulfite salt to generate reacted DCIS cancer cell proliferation DNA (806).

As shown in FIG. 4, the breast cancer diagnostic system 600 (e.g., the conversion module 614) can amplify the reacted DCIS cancer cell proliferation DNA (808). For instance, the breast cancer diagnostic system 600 can amplify the reacted DCIS cancer cell proliferation DNA subsequent to treating the isolated DCIS cancer cell proliferation DNA with the bisulfite salt to generate amplicons of the reacted DCIS cancer cell proliferation DNA. The breast cancer diagnostic system 600 can detect methylation and/or unmethylation of the isolated DCIS cancer cell proliferation DNA by detecting a presence and/or absence of thymidine in the amplified reacted DCIS cancer cell proliferation DNA (810).

It should be appreciated that the process 700 and/or 800 can include different and/or additional operations without departing from the scope of the present subject matter. Moreover, one or more operations of the process 700 and/or 800 can be omitted and/or repeated without departing from the scope of the present subject matter.

Implementations of the present subject matter can include, but are not limited to, methods consistent with the descriptions provided above as well as articles that comprise a tangibly embodied machine-readable medium operable to cause one or more machines (e.g., computers, etc.) to result in operations implementing one or more of the described features. Similarly, computer systems are also described that can include one or more processors and one or more memories coupled to the one or more processors. A memory, which can include a computer-readable storage medium, can include, encode, store, or the like one or more programs that cause one or more processors to perform one or more of the operations described herein. Computer implemented methods consistent with one or more implementations of the current subject matter can be implemented by one or more data processors residing in a single computing system or multiple computing systems. Such multiple computing systems can be connected and can exchange data and/or commands or other instructions or the like via one or more connections, including but not limited to a connection over a network (e.g. the Internet, a wireless wide area network, a local area network, a wide area network, a wired network, or the like), via a direct connection between one or more of the multiple computing systems, etc.

One or more aspects or features of the subject matter described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs, FPGAs, computer hardware, firmware, software, and/or combinations thereof. These various aspects or features can include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which can be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device. The programmable system or computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

These computer programs, which can also be referred to programs, software, software applications, applications, components, or code, include machine instructions for a programmable processor, and can be implemented in a high-level procedural language, an object-oriented programming language, a functional programming language, a logical programming language, and/or in assembly/machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus and/or device, such as for example magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and/or data to a programmable processor. The machine-readable medium can store such machine instructions non-transitorily, such as for example as would a non-transient solid-state memory or a magnetic hard drive or any equivalent storage medium. The machine-readable medium can alternatively or additionally store such machine instructions in a transient manner, such as for example as would a processor cache or other random access memory associated with one or more physical processor cores.

To provide for interaction with a user, one or more aspects or features of the subject matter described herein can be implemented on a computer having a display device, such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) or a light emitting diode (LED) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback; and input from the user can be received in any form, including, but not limited to, acoustic, speech, or tactile input. Other possible input devices include, but are not limited to, touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive trackpads, voice recognition hardware and software, optical scanners, optical pointers, digital MRI image capture devices and associated interpretation software, and the like.

EXAMPLES

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1. Development of an Epigenetic Biomarker Panel for DCIS Invasiveness

DNA methylation patterns were evaluated in 7 DCIS samples from patients without reported invasion (pure DCIS), and 3 DCIS samples from patients with IDC. In order to identify DNA methylation changes exclusive to cancer cells, malignant cells located within the involved ducts of DICS were isolated by laser capture microdissection (FIG. 1A). Genome-wide profiling of DNA methylation was performed. Reduced representation bisulfite sequencing (RRBS) was used to evaluate DNA methylation patterns at gene promoters in captured cells (FIG. 1C). Clustering analysis clearly separated DCIS samples into two epigenetic groups. The first DCIS epigenetic group was associated with almost no DNA methylation at promoters and obtained from patients lacking invasion (pure DCIS). At the same time, specimens from the second epigenetic group of DCIS were characterized by extensive accumulation of DNA methylation at promoters. This group contained all analyzed DCIS from patients with IDC and 3 pure DCIS, according to a clinical pathology report.

In general, DNA methylation patterns strikingly separate normal from cancer cells and reflect genome-wide alterations of chromatin. While normal cells are missing DNA methylation at promoters, cancer is characterized by aberrant accumulation of promoter DNA methylation, which is frequently associated with gene silencing. Therefore, the first DCIS epigenetic group has a DNA methylation pattern very similar to normal cells. At the same time, the second DCIS epigenetic group exhibited a clear accumulation of cancer-associated DNA methylation and was highly enriched in DCIS associated with invasion.

Not to be bound by scientific theory, it was hypothesized that there are two different epigenetic programs driving DCIS progression, and patients with the “invasion incompetent” signature are unlikely to develop invasion while patients with pure DCIS from the second epigenetic group having “invasion competent” signature will have a very high chance to develop invasion. This hypothesis on the predictive power of epigenetic profiling is supported by the fact that on independent blinded pathology review of the “pure DCIS” cases, DCIS sample number 6 (DCIS_6) was reclassified as invasive (by a pathologist) that confirms the “invasion competent” signature identified by DNA methylation analysis. Thus, based on the DNA methylation pattern, the potential for invasiveness of DCIS can be predicted.

140 cytosines with DNA methylation patterns that strictly distinguish these two DCIS groups (“invasion incompetent” and “invasion competent”) were identified (FIG. 1D). These epigenetic signatures can be used in diagnostic and prognostic DCIS tests for invasiveness that guide clinical management and potentially de-escalate therapy for DCIS with no potential for invasion.

DNA Methylation Profiling

By using LDM 7000 (Leica Microsystems), cancer cells were isolated from breast tissues by using laser capture procedure. Genomic DNA was purified by using a standard phenol/chloroform extraction approach followed by ethanol precipitation. Further genomic DNA underwent RRBS procedure. RRBS DNA amplicons were paired-end sequenced by using HighSeq (Illumina). For each sample, at least 15 million aligned reads were obtained. Specific methylation signatures for “invasion incompetent” and “invasion competent” cells were determined based on cytosines which are characterized by at least 5 sequencing reads in each sample.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

	Number	Date	Country
Parent	16235968	Dec 2018	US
Child	17561434		US

DNA METHYLATION DIAGNOSTIC TEST FOR BREAST CANCER

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)

Continuations (1)