Mitochondrial DNA deletion between about residues 12317-16254 for use in the detection of cancer

Abstract

The present invention relates to methods for predicting, diagnosing and monitoring cancer. The methods comprise obtaining biological samples, extracting mitochondrial DNA (mtDNA) from the samples, quantifying a mtDNA mutation in the sample and comparing the level of the mtDNA mutation present in the sample with a reference value The methods of the invention may also be effective in screening for new therapeutic agents and treatment regimes Further, said methods may be also be useful for monitoring the response of a subject to a preventative or therapeutic treatment.

Description

FIELD OF THE INVENTION

[0001] The present invention pertains to the field of mitochondrial genomics. In particular it is related to the detection of human mitochondrial genome mutations and their utility as an indicators of cancer.

BACKGROUND OF THE INVENTION

Mitochondrial DNA as a Diagnostic Tool

[0002] Mitochondrial DNA (mtDNA) sequence dynamics are important diagnostic tools. Mutations in mtDNA are often preliminary indicators of developing disease, often associated with nuclear mutations, and act as biomarkers specifically related to: disease, such as but not limited to, tissue damage and cancer from smoking and exposure to second hand tobacco smoke (Lee et al., 1998; Wei, 1998); longevity, based on accumulation of mitochondrial genome mutations beginning around 20 years of age and increasing thereafter (von Wurmb, 1998); metastatic disease caused by mutation or exposure to carcinogens, mutagens, ultraviolet radiation (Birch-Machin, 2000); osteoarthritis; cardiovascular, Alzheimer, Parkinson disease (Shoffner et al., 1993; Sherratt et al., 1997; Zhang et al, 1998); age associated hearing loss (Seidman et al., 1997); optic nerve degeneration and cardiac dysrhythmia (Brown et al., 1997; Wallace et al., 1988); chronic progressive external exophthalmoplegia (Taniike et al., 1992); atherosclerosis (Bogliolo et al., 1999); papillary thyroid carcinomas and thyroid tumours (Yeh et al., 2000); as well as others (e.g. Naviaux, 1997; Chinnery and Turnbull, 1999).

[0003] Mutations at specific sites of the mitochondrial genome can be associated with certain diseases. For example, mutations at positions 4216, 4217 and 4917 are associated with Leber's Hereditary Optic Neuropathy (LHON) (Mitochondrial Research Society; Huoponen (2001); MitoMap). A mutation at 15452 was found in 5/5 patients to be associated with ubiquinol cytochrome c reductase (complex III) deficiency (Valnot et al. 1999).

[0004] Specifically, these mutations or alterations include point mutations (transitions, transversions), deletions (one base to thousands of bases), inversions, duplications, (one base to thousands of bases), recombinations and insertions (one base to thousands of bases). In addition, specific base pair alterations, deletions, or combinations thereof have been found to be associated with early onset of prostate, skin, and lung cancer, as well as aging (e.g. Polyak et al., 1998), premature aging, exposure to carcinogens (Lee et al., 1998), etc.

Prostate Cancer

[0005] Prostate cancer is a frequently diagnosed solid tumour that most likely originates in the prostate epithelium (Huang et al. 1999). In 1997, nearly 10 million American men were screened for prostate specific antigen (PSA), the presence of which suggests prostate cancer (Woodwell, 1999). Indeed, this indicates an even higher number of men screened by an initial digital rectal exam (DRE). In the same year, 31 million men had a DRE (Woodwell, 1999). Moreover, the annual number of newly diagnosed cases of prostate cancer in the United States is estimated at 179,000 (Landis et al., 1999). It is the second most commonly diagnosed cancer and second leading cause of cancer mortality in Canadian men. In 1997 prostate cancer accounted for 19,800 of newly diagnosed cancers in Canadian men (28%) (National Cancer Institute of Canada). It is estimated that 30% to 40% of all men over the age of forty-nine (49) have some cancerous prostate cells, yet only 20% to 25% of these men have a clinically significant form of prostate cancer (SpringNet--CE Connection, internet, www.springnet.com/ce/j803a.htm). Prostate cancer exhibits a wide variety of histological behaviour involving both endogenous and exogenous factors, i.e. socio-economic situations, diet, geography, hormonal imbalance, family history and genetic constitution (Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNA alterations have been previously associated with prostate cancer, the need exists for further markers for the detection of prostate cancer.

Breast Cancer

[0006] Breast cancer is a cancer of the glandular breast tissue and is the fifth most common cause of cancer death. In 2005, breast cancer caused 502,000 deaths (7% of cancer deaths; almost 1% of all deaths) worldwide (World Health Organization Cancer Fact Sheet No. 297). Among women worldwide, breast cancer is the most common cancer and the most common cause of cancer death (World Health Organization Cancer Fact Sheet No. 297). Although certain mtDNA alterations have been previously associated with breast cancer, for example in Parrella et al. (Cancer Research: 61, 2001), the need exists for further markers for the detection of breast cancer.

[0007] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[0008] The present invention pertains to mitochondrial DNA mutations for use in the detection of cancer. In accordance with an aspect of the present invention, there is provided a method of detecting a cancer in an individual comprising: [0009] a) obtaining a biological sample from the individual; [0010] b) extracting mitochondrial DNA (mtDNA) from the sample; [0011] c) quantifying the amount of mtDNA in the sample having a deletion in the mtDNA sequence between about residue 12317 and about residue 16254 of the human mtDNA genome; and [0012] d) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value.

[0013] In accordance with another aspect of the present invention, there is provided a method of monitoring an individual for the development of a cancer comprising: [0014] a) obtaining a biological sample; [0015] b) extracting mitochondrial DNA (mtDNA) from the sample; [0016] c) quantifying the amount of mtDNA in the sample having a deletion in the mtDNA sequence between about residue 12317 and about residue 16254 of the human mtDNA genome; and [0017] d) repeating steps a) to c) over a duration of time;

[0018] wherein an increasing level of the deletion over the duration of time is indicative of cancer.

[0019] In accordance with another aspect of the present invention, there is provided a method of detecting a cancer in an individual comprising: [0020] a) obtaining a biological sample from the individual; [0021] b) extracting mitochondrial DNA (mtDNA) from the sample; [0022] c) quantifying the amount of mtDNA in the sample having a sequence corresponding to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and [0023] d) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 to at least one known reference value.

[0024] In accordance with another aspect of the present invention, there is provided a diagnostic kit for carrying out the method of the invention comprising:

[0025] (a) material for collecting one or more biological samples; and

[0026] (b) suitable primers and reagents for detecting the mtDNA deletion.

BRIEF DESCRIPTION OF THE FIGURES

[0027] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[0028] FIG. 1 is a graph showing cycle threshold as related to Example 1.

[0029] FIG. 2 shows a ROC curve illustrating the specificity and sensitivity of one embodiment of the present invention.

[0030] FIG. 3 is a graph showing cycle threshold as related to Example 2.

[0031] FIG. 4 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.

[0032] FIG. 5 is a schematic diagram showing the design and sequence of a primer useful for the detection of the 4 kb deletion.

[0033] FIG. 6 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides methods of predicting, diagnosing and monitoring cancer. The methods comprise obtaining one or more biological samples, extracting mitochondrial DNA (mtDNA) from the samples, quantifying the amount of a mitochondrial mutation in the samples and comparing the quantity of the mutation in a sample with a reference value. In this regard, the methods provide a comprehensive tool for determining disease onset and for assessing the predisposition of an individual to cancer. The methods also allow for the monitoring of an individual's risk factors over time and/or for monitoring a patient's response to therapeutic agents and treatment regimes.

Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] As used herein, the term "about" refers to an understood variation from the stated value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0037] As defined herein, "biological sample" refers to a tissue or bodily fluid containing cells from which mtDNA can be obtained. For example, the biological sample can be derived from tissue such as breast or prostate tissue, or from blood, saliva, cerebral spinal fluid, sputa, urine, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like. The biological sample may be a surgical specimen or a biopsy specimen. The biological sample can be used either directly as obtained from the source or following a pre-treatment to modify the character of the sample. Thus, the biological sample can be pre-treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, and the like.

[0038] As used herein, "cycle threshold" (C.sub.T) is the point at which target amplification using real-time PCR rises above background, as indicated by a signal such as a fluorescence signal. The C.sub.T is inversely related to the quantity of the sequence being investigated.

[0039] As used herein, "diagnostic" or "diagnosing" means using the presence or absence of a mutation or combination of mutations as a factor in disease diagnosis or management. The detection of the mutation(s) can be a step in the diagnosis of a disease.

[0040] As used herein, "deletion" means removal of a region of mtDNA from a contiguous sequence of mtDNA. Deletions can range in size from one base to thousands of bases or larger.

[0041] As used herein, "mitochondrial DNA" or "mtDNA" is DNA present in mitochondria.

[0042] As used herein, "mutation" encompasses any modification or change in mitochondrial DNA from the wild type sequence, including without limitation point mutations, transitions, insertions, transversions, translocations, deletions, inversions, duplications, recombinations or combinations thereof. The modification or change of the sequence can extend from a single base change to the addition or elimination of an entire DNA fragment.

[0043] As defined herein, "sensitivity" refers to the fraction of true positives (true positive rate) results obtained using the method of the present invention.

[0044] As defined herein, "specificity" refers to the fraction of false positives (false positive rate) results obtained using the method of the present invention.

[0045] The terms "therapy" and "treatment," as used interchangeably herein, refer to an intervention performed with the intention of improving a subject's status. The improvement can be subjective or objective and is related to ameliorating the symptoms associated with, preventing the development of, or altering the pathology of a disease. Thus, the terms therapy and treatment are used in the broadest sense, and include the prevention (prophylaxis), moderation, reduction, and curing of a disease, at various stages. Preventing deterioration of a subject's status is also encompassed by the term. Subjects in need of therapy/treatment thus include those already having the disease, as well as those prone to, or at risk of developing, the disease, and those in whom the disease is to be prevented.

Assays for Predicting, Diagnosing and Monitoring Cancer

Assay for Detection of Mitochondrial Mutation

[0046] Mitochondrial DNA (mtDNA) dynamics are an important diagnostic tool. Mutations in mtDNA are often preliminary indicators of developing disease and may act as biomarkers indicative of risk factors associated with disease onset. As discussed herein, measuring the level of mitochondrial DNA aberration in a biological sample can determine the presence of one or more cancers and identify the potential risk or predisposition of a patient to one or more cancers. Furthermore, measurement of mtDNA at regular intervals can provide health care professionals with a real-time, quantitative monitoring tool for measuring the progression of a patient over time and/or as an assessment for treatment recommendations in order to determine their effectiveness in preventing or treating cancer.

[0047] The present invention, therefore, provides methods for predicting, diagnosing or monitoring cancer, comprising obtaining one or more biological samples, extracting mitochondrial DNA (mtDNA) from the samples, and assaying the samples for mitochondrial mutation by: quantifying the amount of an mtDNA aberration in the sample and comparing the level of the aberration with a reference value. As would be understood by those of skill in the art, the reference value is based on whether the method seeks to predict, diagnose or monitor cancer. Accordingly, the reference value may relate to mtDNA data collected from one or more known non-cancerous biological samples, from one or more known cancerous biological samples, and/or from one or more biological samples taken over time. These reference values are used for comparison with the mtDNA data collected from the one or more biological samples wherein, for example, a similar or elevated amount of deletion in the biological sample compared to the reference sample is indicative of a predisposition to or the onset of cancer, or wherein an increasing level of the deletion over time is indicative of cancer onset.

[0048] In accordance with an aspect of the invention, the methods for predicting, monitoring and diagnosing cancer comprise an assay for detecting and quantifying one or more mitochondrial mutations. In accordance with one embodiment of the invention, the mutation is an mtDNA deletion. In accordance with another embodiment, the mutation is an mtDNA deletion of 3926 bp of mtDNA (referred to herein as "the 4 kb deletion" or "4 kb sequence"). In accordance with yet another embodiment, the mutation is an mtDNA deletion having the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, there being no difference between SEQ ID NO: 1 and SEQ ID NO: 2 when in circular form.

[0049] The 4 kb deletion spans approximately nucleotides 12317 and 16254 of the human mtDNA genome. The human mtDNA genome is listed herein as SEQ ID NO:3 (Genbank accession no. AC.sub.--000021). The 4 kb deletion is characterized by direct flanking repeats 12 bp in size, with the repeats located at positions 12317-12328 and 16243 to 16254. The repeat sequence is 5'-TGCAACTCCAAA-3'. Thus, in accordance with one embodiment of the invention, the mutation is an mtDNA deletion of between about residue 12317 and about residue 16254 of the human mtDNA genome.

[0050] The inventors have determined, as provided by way of example below, that this deletion is associated with cancer and in particular prostate and breast cancer. Therefore, such deletion provides an accurate biomarker and, therefore, a valuable tool for the detection, diagnosis, or monitoring of cancer in at least these tissues.

[0051] The deletion results in the creation of two deletion monomers, one of 4 kb in size (small sublimon) and one of approximately 12.5 kb in size (large sublimon). The occurrence of the deletion may be detected by either identifying the presence of the small sublimon or the large sublimon, the 4 kb or 12.5 kb sequence respectively.

[0052] Exemplary methods for assaying the mitochondrial mutation are provided in the Example section. Extraction of mtDNA from a sample may be undertaken using any suitable known method. MtDNA extraction is followed by amplification of all or a region of the mitochondrial genome, and may include sequencing of the mitochondrial genome, as is known in the art and described, for example, in Current Protocols in Molecular Biology (Ausubel et al., John Wiley & Sons, New York, 2007). Likewise, methods for detecting the presence of mutations in the mtDNA can be selected from suitable techniques known to those skilled in the art. For example, analyzing mtDNA can comprise sequencing the mtDNA, amplifying mtDNA by PCR, Southern, Northern, Western South-Western blot hybridizations, denaturing HPLC, hybridization to microarrays, biochips or gene chips, molecular marker analysis, biosensors, melting temperature profiling or a combination of any of the above.

[0053] Any suitable means to sequence mitochondrial DNA may be used. Preferably, mtDNA is amplified by PCR prior to sequencing. The method of PCR is well known in the art and may be performed as described in Mullis and Faloona, 1987, Methods Enzymol., 155: 335. PCR products can be sequenced directly or cloned into a vector which is then placed into a bacterial host. Examples of DNA sequencing methods are found in Brumley, R. L. Jr. and Smith, L. M., 1991, Rapid DNA sequencing by horizontal ultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 and Luckey, J. A., et al, 1993, High speed DNA sequencing by capillary gel electrophoresis, Methods Enzymol. 218: 154-172. The combined use of PCR and sequencing of mtDNA is described in Hopgood, R., et al, 1992, Strategies for automated sequencing of human mtDNA directly from PCR products, Biotechniques 13:82-92 and Tanaka, M. et al, 1996, Automated sequencing of mtDNA, Methods Enzymol. 264: 407-421.

[0054] Although real-time quantitative PCR methods, as described in the examples below, represent the preferred means for detecting and quantifying the presence or absence of the 4 kb deletion, other methods would be well known to an individual of skill in the art and could be utilized as indicated above. In addition, quantification of the deletion could be made using Bio-Rad's Bioplex.TM. System and Suspension Array technology. Generally, the method requires amplification and quantification of sequences using any known methods.

[0055] The following primer sequences are examples of primers that may be used for the detection of the 4 kb deletion:

TABLE-US-00001 4 forward (binds to bases 12313-12328/16255-16267 of the human mtDNA genome) (SEQ ID NO: 4) 5'-TTGGTGCAACTCCAAAGCCACCCCTCACC-3'; 4 reverse (binds to bases 16391-16409 of the human mtDNA genome) (SEQ ID NO: 5) 5'-AGGATGGTGGTCAAGGGAC-3'.

[0056] In one embodiment of the present invention, a pair of amplification primers are used to amplify a target region indicative of the presence of the 4 kb deletion. In this embodiment, one of the pair of amplification primers overlaps a spliced region of mtDNA after deletion of the 4 kb sequence has occurred and the mtDNA has reformed as a circular mtDNA molecule (eg. a splice at a position between 12328 and 16255 of the mtDNA genome). Therefore, extension of the overlapping primer can only occur if the 4 kb section is deleted. FIG. 5 is a schematic diagram showing the design and sequence of the primer (ie. SEQ ID NO: 4).

[0057] In another embodiment of the present invention, a pair of amplification primers are used to amplify a target region associated with the deleted 4 kb sequence. The deleted 4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. In this embodiment, one of the pair of amplification primers overlaps the rejoining site of the ends of the 4 kb sequence. Thus, an increase in the amount of the 4 kb molecule detected in a sample is indicative of cancer.

[0058] In still another embodiment of the present invention, the breakpoint of the deletion is unknown thereby resulting in two possibilities for primer location. In this embodiment, two separate forward primers may be designed to amplify the target region associated with the deleted 4 kb sequence. The following primer sequences are examples of those that may be used for the detection of the 4 kb deletion in this scenario:

TABLE-US-00002 Forward Primers: Primer A (binds to bases 12313-12328/16255-16267 of the human mtDNA genome) (SEQ ID NO: 4) 5'-TTGGTGCAACTCCAAAGCCACCCCTCACC-3'; Primer B (binds to bases 12302-12316 of the human mtDNA genome) (SEQ ID NO: 6) 5'-CCCAAAAATTTTGGTGCAACTCCAAAGCCAC-3'. Reverse Primer: Primer C (binds to bases 16391-16409 of the human mtDNA genome) (SEQ ID NO: 5) 5'-AGGATGGTGGTCAAGGGAC-3'.

[0059] As would be understood by a person of skill in the art, the forward primers A or B can be used with reverse primer C to create PCR products that are useful in qPCR assays.

Biological Sample

[0060] The present invention provides for diagnostic tests which involve obtaining or collecting one or more biological samples. In the context of the present invention, "biological sample" refers to a tissue or bodily fluid containing cells from which mtDNA can be obtained. For example, the biological sample can be derived from tissue including, but not limited to, breast, prostate, nervous, muscle, heart, stomach, colon tissue and the like; or from blood, saliva, cerebral spinal fluid, sputa, urine, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like. The biological sample may be obtained from a cancerous or non-cancerous tissue and may be a surgical specimen or a biopsy specimen.

[0061] The biological sample can be used either directly as obtained from the source or following a pre-treatment to modify the character of the sample. Thus, the biological sample can be pre-treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, and the like.

[0062] One skilled in the art will understand that more than one sample type may be assayed at a single time (i.e. for the detection of more than one cancer). Furthermore, where a course of collections are required, for example, for the monitoring of risk factors or cancer over time, a given sample may be diagnosed alone or together with other sample taken throughout the test period. In this regard, biological samples may be taken once only, or at regular intervals such as biweekly, monthly, semi-annually or annually.

[0063] One of skill will also appreciate that mitochondrial DNA targets are in much greater abundance (approximately 1000 fold greater) than nucleic acid targets and as such sample sizes comprising extremely low yields of nucleic acids would be suitable for use with the present invention.

Applications for Predicating, Diagnosing and Monitoring Cancer

Diagnosing and Monitoring Cancer

[0064] The prevalence of cancer in most tissue types and age groups necessitates the availability of a tool to not only detect the presence of cancer, but also to monitor the success and appropriateness of preventative measures and therapies being advised to prevent onset, progression and spread of the disease. Measuring the level of mitochondrial DNA deletions in one or more biological samples of an individual can provide initial diagnosis of risk factors, cancer and/or stages of the disease.

[0065] The system and method of the present invention, for example, may be used to detect cancer at an early stage, and before any histological abnormalities. Furthermore, sample testing at regular intervals such as biweekly, monthly, semi-annually or annually (or any other suitable interval) can provide health care professionals with a real-time, quantitative monitoring tool to compare against treatment recommendations to determine their effectiveness in preventing or treating the disease.

[0066] Turning now to the examples, in one embodiment the present invention may be used for detecting the presence of pre-neoplasia, neoplasia and progression towards potential malignancy of prostate cancer and breast cancer. In one aspect, the present invention involves the detection and quantification of the 4 kb mtDNA deletion for the detection, diagnosis, and/or monitoring of cancer. In this method, mtDNA is extracted from a biological sample (for example body tissue, or body fluids such as urine, prostate massage fluid). The extracted mtDNA is then tested in order to determine the levels (ie. quantity) of the 4 kb deletion in the sample. In tests conducted by the present inventors, the levels of the deletion were found to be elevated in samples obtained from subjects with cancer when compared to samples obtained from subjects without cancer. Based on the information and data supplied below, the inventors have concluded that elevated levels of the 4 kb deletion in human mtDNA is indicative of cancer.

[0067] In another embodiment, samples of, for instance prostate tissue, prostate massage fluid, urine or breast tissue, are obtained from an individual and tested over a period of time (eg. years) in order to monitor the genesis or progression of cancer. Increasing levels of the 4 kb deletion over time could be indicative of the beginning or progression of cancer.

[0068] One of ordinary skill in the art will appreciate that analysing one or more biological samples from an individual for quantification of a mitochondrial DNA target provides a means for a health care worker to monitor the effectiveness of treatment regimes. One of ordinary skill will also appreciate the utility of mtDNA analysis for use by health care providers in identifying (and providing recommendations for) lifestyle habits, such as poor diet and exercise, or activities that cause over exposure of an individual to known carcinogens (eg. tobacco, pollutants).

[0069] Another aspect of the invention provides methods for confirming or refuting the results of a cancer biopsy test from a biopsy sample (eg. prostate or breast cancer), comprising: obtaining non-cancerous tissue from a biopsy sample; and detecting and quantifying the amount of the 4 kb mtDNA deletion in the non-diseased tissue.

Determining Genetic Predisposition to Cancer

[0070] In order to fully evaluate an individual's risk of one or more cancers it is imperative that health care providers are provided with as much information as possible to understand and communicate their patient's risk factors. The utilization of the present invention to determine the level of mtDNA aberration will not only prove helpful in assessing an individual's susceptibility to one or more cancers, it provides a valuable tool to identify patients with greater risk who are potentially in need of more aggressive monitoring and treatment measures.

[0071] In this regard, the various examples provided below illustrate a difference in the amount of mtDNA having the 4 kb deletion between samples obtained from subjects having cancer, and subjects without cancer. The amount of the 4 kb deletion was found to be higher in the samples obtained from subjects having cancer. This determination was made by comparing the amount of the 4 kb deletion in the samples from known cancer cells and/or known non-cancer cells.

[0072] As such, the inventors determined that screening of biological samples would prove useful in identifying an individual's predisposition to one or more cancers. Thus, in accordance with one embodiment of the present invention there is provided a method for screening individuals for cancer from one or more biological samples comprising: obtaining the one or more samples, and detecting and quantifying the level of the 4 kb mtDNA deletion in the samples. In a specific embodiment of the invention, there is provided a method for screening individuals for prostate or breast cancer from a body fluid or tissue sample comprising; obtaining the body fluid or tissue sample, and detecting and quantifying the level of the 4 kb mtDNA deletion in the body fluid or tissue sample.

[0073] Age related accumulation of the 4 kb mtDNA deletion may also predispose an individual to, for example, prostate cancer or breast cancer, which is prevalent in middle aged and older men, and middle aged and older women, respectively. Similarly, an accumulation of the 4 kb mtDNA deletion may be associated with a particular lifestyle based on an individual's diet, exercise habits, and exposure to known carcinogens. Thus, in accordance with one aspect of the invention, a method is provided wherein regular cancer screening may take place by monitoring over time the amount of the 4 kb deletion in one or more biological samples, non-limiting examples of which include breast and prostate tissues or body fluids such as prostate massage fluid, or urine.

Evaluation of Therapeutic Agents

[0074] The method of the present invention may also be used for screening potential therapeutic agents for use in cancer treatment or for monitoring the therapeutic effect of such agents. The method of the present invention may be used to measure various biomarkers associated with the cancers identified herein. The ability to assess the level of DNA damage in any biological sample at any time point provides the foundation for a unique and informative screening test for an individual's health and to assess the safety and efficacy of existing and new therapeutic agents and treatment regimes. Furthermore, by identifying the specific genetic changes underlying a subject's state of health, it may be readily determined whether and to what extent a patient will respond to a particular therapeutic agent or regime.

Kits

[0075] The present invention provides diagnostic/screening kits for use in a clinical environment. Such kits could not only include one or more sampling means, but other materials necessary for the identification of mtDNA mutations.

[0076] The kits can optionally include reagents required to conduct a diagnostic assay, such as buffers, salts, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a biological sample, may also be included in the kit. One or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised components.

[0077] Where appropriate, the kit may also contain reaction vessels, mixing vessels and other components that facilitate the preparation of the test sample. The kit may also optionally include instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.

[0078] In one aspect of the invention there is provided a kit for diagnosing cancer comprising means for extraction of mtDNA, primers, reagents and instructions.

[0079] In another aspect of the invention there is provided a kit for diagnosing cancer, for example prostate or breast cancer, comprising means for extraction of mtDNA, primers having the nucleic acid sequences recited in SEQ ID NOs: 4 and 5, reagents and instructions.

[0080] In another aspect of the invention there is provided a kit for diagnosing cancer, for example prostate or breast cancer, comprising means for extraction of mtDNA, primers having the nucleic acid sequences recited in SEQ ID NOs: 6 and 5, reagents and instructions.

[0081] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1

Association of Prostate Cancer with 4 kb Deletion in Human mtDNA

[0082] Urine samples were collected from five patients who had been diagnosed with prostate cancer and five who had a needle biopsy procedure which was unable to detect prostate malignancy. These samples were collected following a digital rectal exam (DRE) to facilitate the collection of prostate cells.

[0083] Upon receipt of the samples a 5 ml aliquot was removed and then 2 mls were centrifuged at 14,000.times.g to form a pellet. The supernatant was removed and discarded.

[0084] Pellets were resuspended in 200 ul phosphate buffered saline solution. Both the resuspended pellet and the whole urine sample were subjected to a DNA extraction procedure using the QiaAMP DNA Mini Kit (Qiagen P/N 51304) according to the manufacturer's directions. The resulting DNA extracts were then quantified using a NanoDrop ND-1000 Spectrophotometer and normalized to a concentration of 0.1 ng/ul.

[0085] Samples were analyzed by quantitative real-time PCR with the 4 kb deletion specific primers according to the following:

[0086] 1.times. iQ SYBR Green Supermix (Bio-Rad product no. 170-8880)

[0087] 100 nmol forward primer (5'-TTGGTGCAACTCCAAAGCCACCCCTCACC-3') (SEQ ID NO: 4)

[0088] 100 nmol reverse primer (5'-AGGATGGTGGTCAAGGGAC-3') (SEQ ID NO: 5)

[0089] 1 ng template DNA in a 25 ul reaction

[0090] Reactions were cycled on an Opticon 2 DNA Engine (Bio-Rad Canada) according to the following protocol: [0091] 1. 95.degree. C. for 3 minutes [0092] 2. 95.degree. C. for 30 seconds [0093] 3. 69.degree. C. for 30 seconds [0094] 4. 72.degree. C. for 30 seconds [0095] 5. Plate Read [0096] 6. Repeat steps 2-5 44 times [0097] 7. 72.degree. C. for 10 minutes [0098] 8. Melting Curve from 50.degree. C. to 105.degree. C., read every 1.degree. C., hold for 3 seconds [0099] 9. 10.degree. C. Hold

Results

[0100] Results from the urine pellet did not yield significant differences in the mean cycle threshold observed or a useful cutoff point. However, the results from the whole urine sample did yield significant differences as provided below.

[0101] Tables 1 and 2, and FIG. 1 show the difference in the mean C.sub.T scores for urine samples from subjects having prostate malignant tissue and benign tissue at the 0.04 significance level.

TABLE-US-00003 TABLE 1 Mean Values for C.sub.T scores: Urine Samples Std. Error N Mean Std. Deviation Mean Benign 7 38.0357 3.40974 1.288876 Malignant 7 31.9300 6.12583 2.31534

TABLE-US-00004 TABLE 2 Significance Test for Mean C.sub.T scores Independent Samples Test Levene's Test for Test for Equality Means Equality 95% Confidence of Sig. Std. Interval of the CTt40 Variances (2- Mean Error Difference fluid F Sig. t df tailed) Diff. Diff. Lower Upper Equal 1.707 .216 2304 12 .040 610571 264985 .33218 11.87925 variances assumed Equal 2304 9392 .046 610571 264985 .14927 12.06215 variances not assumed

[0102] Tables 3 and 4, and FIG. 2 illustrate that when using a cut-off cycle threshold of 36.255 the sensitivity of the assay for prostate cancer is 86% and the specificity is 86%.

[0103] FIG. 2 is a Receiver Operating Characteristic (ROC) curve illustrating the specificity and sensitivity of the 4 kb mtDNA deletion as a marker for prostate cancer when testing urine. These results were obtained using a cutoff C.sub.T of 36.255. The sensitivity of the marker at this C.sub.T is 86%, while the specificity is 86%.

[0104] The determination of the cutoff C.sub.T of 36.255 is shown in Table 3. The results listed in Table 3 show that a cutoff C.sub.T of 36.255 provided the highest sensitivity and specificity.

[0105] The accuracy of the test depends on how well the test separates the group being tested into those with and without the prostate cancer. Accuracy is measured by the area under the ROC curve. Table 4 shows the calculation of the area under the curve for the present example.

TABLE-US-00005 TABLE 3 Determination of Specificity and Sensitivity Positive if .ltoreq..sup.a Sensitivity 1 - specificity 19.86 .000 .000 24.87 .143 .000 29.48 .286 .000 30.54 .429 .000 32.235 .429 .143 33.77 .571 .143 35.11 .714 .143 36.255 .857 .143 37.415 .857 .286 39.23 .857 .429 39.995 1.000 .429 40.21 1.000 .857 41.42 1.000 1.000 .sup.athe smallest cutoff value is the minimum observed test value minus 1 and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.

TABLE-US-00006 TABLE 4 Results Showing Area Under the ROC Curve Asymptotic 95% Confidence Interval Area Std. Error.sup.a Asymptotic Sig..sup.b Lower bound Upper bound .878 .096 .018 .689 1.066 Notes: .sup.aunder the non-parametric assumption .sup.bnull hypothesis: true area = 0.5

Example 2

Association of Breast Cancer with 4 kb Deletion in Human mtDNA

[0106] Twenty breast tissue samples were collected, ten of which were malignant and ten of which had benign breast disease or no abnormalities. These samples were formalin-fixed paraffin embedded and 20 micron sections of each were cut into individual sample tubes for extraction according to the manufacturer's protocol for the QiaAMP DNA Mini Kit (Qiagen P/N 51304). DNA was then quantified using a Nanodrop ND-1000 and normalized to a concentration of 2 ng/ul.

[0107] Samples were then assayed for the levels of the 4 kb deletion by quantitative real-time PCR using the following protocol:

TABLE-US-00007 X iQ SYBR Green Supermix (Bio-Rad product no. 170-8880) 175 nmol forward primer (SEQ ID NO: 4) (5'-TTGGTGCAACTCCAAAGCCACCCCTCACC-3') 175 nmol reverse primer (SEQ ID NO: 5) (5'-AGGATGGTGGTCAAGGGAC-3')

[0108] 20 ng template DNA in a 25 ul reaction

[0109] Reactions were cycled on an Opticon 2 DNA Engine (Bio-Rad Canada) according to the following protocol: [0110] 1. 95.degree. C. for 3 minutes [0111] 2. 95.degree. C. for 30 seconds [0112] 3. 70.degree. C. for 30 seconds [0113] 4. 72.degree. C. for 30 seconds [0114] 5. Plate Read [0115] 6. Repeat steps 2-5 44 times [0116] 7. 72.degree. C. for 10 minutes [0117] 8. Melting Curve from 50.degree. C. to 105.degree. C., read every 1.degree. C., hold for 3 seconds [0118] 9. 10.degree. C. Hold

[0119] Tables 5 and 6, and FIG. 3 show the difference in the mean C.sub.T scores for breast tissue samples from subjects having malignant breast tissue and benign breast tissue at the 0.065 level.

TABLE-US-00008 TABLE 5 Mean Values for C.sub.T scores: Breast Tissue Samples Group N Mean Std. Dev. Std. Error Mean Normal 9 21.5278 2.71939 .90646 Malignant 9 18.9089 2.89126 .96375

TABLE-US-00009 TABLE 6 Significance Test for Mean C.sub.T scores Levene's Test for Equality Means Test for 95% Equality Confidence of Sig. Std. Interval of the Variances (2- Mean Error Difference CTt40 fluid F Sig. t df tailed) Diff. Diff. Lower Upper Equal .007 .934 1.979 16 .065 2.61889 1.32306 -.18588 5.42366 variances assumed Equal 1.979 15.94 .065 2.61889 1.32306 -.18674 5.42452 variances not assumed

[0120] Tables 7 and 8, and FIG. 4 illustrate that when using a cut-off cycle threshold of 19.845 the sensitivity of the assay for breast cancer is 78% and the specificity is 78%.

[0121] FIG. 4 is an ROC curve illustrating the specificity and sensitivity of the 4 kb mtDNA deletion as a marker for breast cancer when testing breast tissue. These results were obtained using a cutoff C.sub.T of 19.845. The sensitivity of the marker at this C.sub.T is 78%, while the specificity is 78%.

[0122] The determination of the cutoff C.sub.T of 19.845 is shown in Table 7. The results listed in Table 7 show that a cutoff C.sub.T of 19.845 provided the highest sensitivity and specificity.

[0123] The accuracy of the test depends on how well the test separates the group being tested into those with and without the breast cancer. Accuracy is measured by the area under the ROC curve. Table 8 shows the calculation of the area under the curve for the present example.

TABLE-US-00010 TABLE 7 Determination of Specificity and Sensitivity Positive if .ltoreq..sup.a Sensitivity 1 - specificity 15.28 .000 .000 16.305 .111 .000 16.69 .222 .000 17.075 .333 .000 17.4 .444 .000 17.71 .556 .000 18.0 .556 .111 18.835 .556 .222 19.415 .667 .222 19.845 .778 .222 20.475 .778 .333 10.79 .778 .444 21.38 .778 .556 22.005 .778 .667 23.145 .889 .667 24.19 .889 .778 24.49 .889 .889 25.21 1.00 .889 26.66 1.00 1.00 .sup.athe smallest cutoff value is the minimum observed test value minus 1 and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.

TABLE-US-00011 TABLE 8 Results Showing Area Under the ROC Curve Asymptotic 95% Confidence Interval Area Std. Error.sup.a Asymptotic Sig..sup.b Lower bound Upper bound .778 .117 .047 .548 1.008

Example 3

Association of Prostate Cancer with 4 kb Deletion in Human mtDNA Using Needle Biopsy Samples

[0124] Prostate needle biopsy specimens were obtained from 19 individuals, 9 without prostate cancer and 10 with prostate cancer. Needle biopsy tissues were formalin-fixed paraffin embedded (FFPE) as is standard in the clinical diagnostic setting. 10 micron sections of each biopsy were deposited directly into centrifuge tubes and the DNA was extracted using the QiaAMP DNA Mini Kit (Qiagen, p/n 51306). DNA extracts were quantified by absorbance at 260 nm using a NanoDrop ND-1000 Spectrophotometer. Yields ranged from 347 ng to 750 ng. These samples were diluted to 2 ng/ul and amplification reactions setup according to Table 9 and the following:

TABLE-US-00012 TABLE 9 Reagents and Concentrations for Amplification Reaction Final Concen- Reagent tration iQ SYBR Green Supermix 1X (Bio-Rad Laboratories, p/n 170-8882) Forward Primer 12303-12316/16243-16259F 175 nmol 5'-CCCAAAAATTTTGGTGCAACTCCAAAGCCAC-3' (SEQ ID NO: 6) Reverse Primer 16410R 175 nmol 5'-AGGATGGTGGTCAAGGGAC-3' (SEQ ID NO: 5) DNA extract 0.8 ng/ul

[0125] Nuclease-free water was added to a final reaction volume of 25 ul. Amplifications were carried out on a DNA Engine Chromo4 Real Time PCR Instrument (Bio-Rad Laboratories) according the following cycling conditions:

[0126] 1) 95.degree. C. for 3 minutes

[0127] 2) Followed by 45 cycles of

[0128] 3) 95.degree. C. for 30 seconds

[0129] 4) 69.degree. C. for 30 seconds

[0130] 5) 72.degree. C. for 30 seconds

[0131] 6) Plate Read

Then

[0132] 7) 72.degree. C. for 10 minutes

[0133] 8) Melting Curve 50.degree. C.-105.degree. C. reading every 1.degree. C., hold for 3 seconds

[0134] 9) 4.degree. C. Hold

[0135] Results, shown in Table 10, demonstrate that those individuals with prostate cancer have a lower C.sub.T value and therefore higher levels of the 4 kb deletion in prostate tissue than do those without prostate cancer. Patients with prostate cancer have an average C.sub.T value of 30.7 while the patients without prostate cancer have an average C.sub.T value of 36.4. This difference of 5.7 C.sub.T corresponds to nearly 100 fold greater 4 kb deletion levels in the group with prostate malignancy than in the group without.

TABLE-US-00013 TABLE 10 Patient Diagnosis and Associated C.sub.T Score Patient Number and Diagnosis C(t) CUG 1301 Malignant 25.7 CUG 1268 Malignant 27.7 CUG RN 345 Normal 28.3 CUG 1272 Malignant 28.8 CUG 1375 Malignant 29.1 CUG 1259 Malignant 29.1 CUG 1381 Malignant 30.2 CUG RN 82 Normal 30.5 CUG 1372 Malignant 30.9 CUG 1085 C T1 Normal 31.5 CUG 1317 Malignant 31.7 CUG 1377 F Normal 33.6 CUG 1365 B Normal 34.6 CUG 1370 Malignant 35.9 CUG RN 405 Normal 37.5 CUG 1366 Malignant 37.9 CUG RN 701 Normal 41.7 CUG RN 420 Normal 45 CUG RN 373 Normal 45

[0136] Tables 11 and 12 show the difference in the mean C.sub.T scores for prostate tissue samples from subjects having normal and malignant prostate tissue.

TABLE-US-00014 TABLE 11 Mean Values for C.sub.T Score: Prostate Needle Biopsy Tissue Group N Mean Std. Dev. Std. Error Mean Normal 9 36.4111 6.25229 2.08410 Malignant 10 30.7 3.69534 1.16857

TABLE-US-00015 TABLE 12 Significance Test for C.sub.T Scores Levene's Test for Equality Means Test for 95% Equality Confidence of Sig. Std. Interval of the Variances (2- Mean Error Difference CTt40 fluid F Sig. t df tailed) Diff. Diff. Lower Upper Equal 4.426 .051 2.455 17 .025 5.71111 2.32589 .80391 10.61831 variances assumed Equal 2.390 12.705 .033 5.71111 2.38935 .53701 10.88522 variances not assumed

[0137] Table 13 and FIG. 6 illustrate that when using a cutoff of C.sub.T 32.65 the sensitivity and specificity of correctly diagnosing these patients is 80% and 67% respectively.

TABLE-US-00016 TABLE 13 Determination of Specificity and Sensitivity Positive if .ltoreq..sup.a Sensitivity 1 - specificity 24.7 .000 .000 26.7 .100 .000 28.0 .200 .000 28.55 .200 .111 28.95 .300 .111 29.65 .500 .111 30.35 .600 .111 30.7 .600 .222 31.2 .700 .222 31.6 .700 .333 32.65 .800 .333 34.1 .800 .444 32.25 .800 .556 36.7 .900 .556 37.7 .900 .667 39.8 1.000 .667 43.35 1.000 .778 46.0 1.000 1.000

[0138] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims. All documents recited in the present application are incorporated herein by reference.

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[0141] Birch-Machin, M. A. (2000). Mitochondria and skin disease. Clin Exp Dermatol, 25, 141-6.

[0142] Brown, M. D., et al., Am J. Humn Genet, 60: 381-387, 1997

[0143] Bogliolo, M, et al., Mutagenesis, 14: 77-82, 1999

[0144] Chinnery P F and Turnbull D M., Lancet 354 (supplement 1): 17-21, 1999

[0145] Huoponen, Kirsi, Leber hereditary optic neuropathy: clinical and molecular genetic findings, Neurogenetics (2001) 3: 119-125.

[0146] Hayward S W, Grossfeld G D, Tlsty T D, Cunha G R., Int J Oncol 13:35-47, 1998

[0147] Huang G M, Ng W L, Farkas J, He L, Liang H A, Gordon D, Hood R., Genomics 59(2):178-86,1999

[0148] Konishi N, Cho M, Yamamoto K, Hiasa Y. Pathol. Int. 47:735-747,1997

[0149] Landis S H, Murray T, Bolden S, Wingo P A. Cancer J. Clin. 49:8-31

[0150] Lee H C, Lu C Y, Fahn H J, Wei Y Hu. Federation of European Biochemical Societies, 441:292-296,1998

[0151] Mitochondrial Research Society http:www.mitoresearch.org/diseases.html.

[0152] MITOMAP: A human mt genome database (www.gen.emory.edu/mitomap.html)

[0153] Naviaux, R K., Mitochondrial Disease--Primary Care Physican's Guide. Psy-Ed. Corp D/B/A Exceptional Parents Guide: 3-10, 1997

[0154] Parrella P, Xiao Y, Fliss M, Sanchez-Cespedes M, Mazzarelli P, Rinaldi M, Nicol T, Gabrielson E, Cuomo C, Cohen D, Pandit S, Spencer M, Rabitti C, Fazio V M, Sidransky D: Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates. Cancer Res 2001, 61:7623-7626

[0155] Polyak Y, et al., Nature Genet. 20 (3):291-293, 1998

[0156] Seidman, M. D. et al., Arch. Otolaryngol Head Neck Surg., 123: 1039-1045, 1997

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[0158] Shoffner J M, Brown M D, Torroni A, Lott M T, Cabell M F, Mirra S S, Beal M F, Yang C, Gearing M, Salvo R, Watts R L, Juncos J L, Hansen L A, Crain B J, Fayad M, Reckford C L, and Wallace D C., Genomics 17: 171-184, 1993

[0159] SpringNet--CE Connection: Screening, Diagnosis: Improving Primary Care Outcomes. Website: http://www.springnet.com/ce/j803a.htm

[0160] Taniike, M. et al., BioChem BioPhys Res Comun, 186: 47-53, 1992

[0161] Valnot, Isabelle, et al., A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency, Human Genetics (1999) 104: 460-466

[0162] von Wurmb, N, Oehmichen, M, Meissner, C., Mutat Res. 422:247-254, 1998

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[0168] Zhang, C., et al., BioChem. BioPhys. Res. Comun., 195: 1104-1110, 1993

Sequence CWU 1

1

613926DNAHomo sapiens 1taaaagtaat aaccatgcac actactataa ccaccctaac cctgacttcc ctaattcccc 60ccatccttac caccctcgtt aaccctaaca aaaaaaactc atacccccat tatgtaaaat 120ccattgtcgc atccaccttt attatcagtc tcttccccac aacaatattc atgtgcctag 180accaagaagt tattatctcg aactgacact gagccacaac ccaaacaacc cagctctccc 240taagcttcaa actagactac ttctccataa tattcatccc tgtagcattg ttcgttacat 300ggtccatcat agaattctca ctgtgatata taaactcaga cccaaacatt aatcagttct 360tcaaatatct actcatcttc ctaattacca tactaatctt agttaccgct aacaacctat 420tccaactgtt catcggctga gagggcgtag gaattatatc cttcttgctc atcagttgat 480gatacgcccg agcagatgcc aacacagcag ccattcaagc aatcctatac aaccgtatcg 540gcgatatcgg tttcatcctc gccttagcat gatttatcct acactccaac tcatgagacc 600cacaacaaat agcccttcta aacgctaatc caagcctcac cccactacta ggcctcctcc 660tagcagcagc aggcaaatca gcccaattag gtctccaccc ctgactcccc tcagccatag 720aaggccccac cccagtctca gccctactcc actcaagcac tatagttgta gcaggaatct 780tcttactcat ccgcttccac cccctagcag aaaatagccc actaatccaa actctaacac 840tatgcttagg cgctatcacc actctgttcg cagcagtctg cgcccttaca caaaatgaca 900tcaaaaaaat cgtagccttc tccacttcaa gtcaactagg actcataata gttacaatcg 960gcatcaacca accacaccta gcattcctgc acatctgtac ccacgccttc ttcaaagcca 1020tactatttat gtgctccggg tccatcatcc acaaccttaa caatgaacaa gatattcgaa 1080aaataggagg actactcaaa accatacctc tcacttcaac ctccctcacc attggcagcc 1140tagcattagc aggaatacct ttcctcacag gtttctactc caaagaccac atcatcgaaa 1200ccgcaaacat atcatacaca aacgcctgag ccctatctat tactctcatc gctacctccc 1260tgacaagcgc ctatagcact cgaataattc ttctcaccct aacaggtcaa cctcgcttcc 1320ccacccttac taacattaac gaaaataacc ccaccctact aaaccccatt aaacgcctgg 1380cagccggaag cctattcgca ggatttctca ttactaacaa catttccccc gcatccccct 1440tccaaacaac aatccccctc tacctaaaac tcacagccct cgctgtcact ttcctaggac 1500ttctaacagc cctagacctc aactacctaa ccaacaaact taaaataaaa tccccactat 1560gcacatttta tttctccaac atactcggat tctaccctag catcacacac cgcacaatcc 1620cctatctagg ccttcttacg agccaaaacc tgcccctact cctcctagac ctaacctgac 1680tagaaaagct attacctaaa acaatttcac agcaccaaat ctccacctcc atcatcacct 1740caacccaaaa aggcataatt aaactttact tcctctcttt cttcttccca ctcatcctaa 1800ccctactcct aatcacataa cctattcccc cgagcaatct caattacaat atatacacca 1860acaaacaatg ttcaaccagt aactactact aatcaacgcc cataatcata caaagccccc 1920gcaccaatag gatcctcccg aatcaaccct gacccctctc cttcataaat tattcagctt 1980cctacactat taaagtttac cacaaccacc accccatcat actctttcac ccacagcacc 2040aatcctacct ccatcgctaa ccccactaaa acactcacca agacctcaac ccctgacccc 2100catgcctcag gatactcctc aatagccatc gctgtagtat atccaaagac aaccatcatt 2160ccccctaaat aaattaaaaa aactattaaa cccatataac ctcccccaaa attcagaata 2220ataacacacc cgaccacacc gctaacaatc aatactaaac ccccataaat aggagaaggc 2280ttagaagaaa accccacaaa ccccattact aaacccacac tcaacagaaa caaagcatac 2340atcattattc tcgcacggac tacaaccacg accaatgata tgaaaaacca tcgttgtatt 2400tcaactacaa gaacaccaat gaccccaata cgcaaaacta accccctaat aaaattaatt 2460aaccactcat tcatcgacct ccccacccca tccaacatct ccgcatgatg aaacttcggc 2520tcactccttg gcgcctgcct gatcctccaa atcaccacag gactattcct agccatgcac 2580tactcaccag acgcctcaac cgccttttca tcaatcgccc acatcactcg agacgtaaat 2640tatggctgaa tcatccgcta ccttcacgcc aatggcgcct caatattctt tatctgcctc 2700ttcctacaca tcgggcgagg cctatattac ggatcatttc tctactcaga aacctgaaac 2760atcggcatta tcctcctgct tgcaactata gcaacagcct tcataggcta tgtcctcccg 2820tgaggccaaa tatcattctg aggggccaca gtaattacaa acttactatc cgccatccca 2880tacattggga cagacctagt tcaatgaatc tgaggaggct actcagtaga cagtcccacc 2940ctcacacgat tctttacctt tcacttcatc ttgcccttca ttattgcagc cctagcaaca 3000ctccacctcc tattcttgca cgaaacggga tcaaacaacc ccctaggaat cacctcccat 3060tccgataaaa tcaccttcca cccttactac acaatcaaag acgccctcgg cttacttctc 3120ttccttctct ccttaatgac attaacacta ttctcaccag acctcctagg cgacccagac 3180aattataccc tagccaaccc cttaaacacc cctccccaca tcaagcccga atgatatttc 3240ctattcgcct acacaattct ccgatccgtc cctaacaaac taggaggcgt ccttgcccta 3300ttactatcca tcctcatcct agcaataatc cccatcctcc atatatccaa acaacaaagc 3360ataatatttc gcccactaag ccaatcactt tattgactcc tagccgcaga cctcctcatt 3420ctaacctgaa tcggaggaca accagtaagc taccctttta ccatcattgg acaagtagca 3480tccgtactat acttcacaac aatcctaatc ctaataccaa ctatctccct aattgaaaac 3540aaaatactca aatgggcctg tccttgtagt ataaactaat acaccagtct tgtaaaccgg 3600agatgaaaac ctttttccaa ggacaaatca gagaaaaagt ctttaactcc accattagca 3660cccaaagcta agattctaat ttaaactatt ctctgttctt tcatggggaa gcagatttgg 3720gtaccaccca agtattgact cacccatcaa caaccgctat gtatttcgta cattactgcc 3780agccaccatg aatattgtac ggtaccataa atacttgacc acctgtagta cataaaaacc 3840caatccacat caaaaccccc tccccatgct tacaagcaag tacagcaatc aaccctcaac 3900tatcacacat caactgcaac tccaaa 392623926DNAHomo sapiens 2tgcaactcca aataaaagta ataaccatgc acactactat aaccacccta accctgactt 60ccctaattcc ccccatcctt accaccctcg ttaaccctaa caaaaaaaac tcataccccc 120attatgtaaa atccattgtc gcatccacct ttattatcag tctcttcccc acaacaatat 180tcatgtgcct agaccaagaa gttattatct cgaactgaca ctgagccaca acccaaacaa 240cccagctctc cctaagcttc aaactagact acttctccat aatattcatc cctgtagcat 300tgttcgttac atggtccatc atagaattct cactgtgata tataaactca gacccaaaca 360ttaatcagtt cttcaaatat ctactcatct tcctaattac catactaatc ttagttaccg 420ctaacaacct attccaactg ttcatcggct gagagggcgt aggaattata tccttcttgc 480tcatcagttg atgatacgcc cgagcagatg ccaacacagc agccattcaa gcaatcctat 540acaaccgtat cggcgatatc ggtttcatcc tcgccttagc atgatttatc ctacactcca 600actcatgaga cccacaacaa atagcccttc taaacgctaa tccaagcctc accccactac 660taggcctcct cctagcagca gcaggcaaat cagcccaatt aggtctccac ccctgactcc 720cctcagccat agaaggcccc accccagtct cagccctact ccactcaagc actatagttg 780tagcaggaat cttcttactc atccgcttcc accccctagc agaaaatagc ccactaatcc 840aaactctaac actatgctta ggcgctatca ccactctgtt cgcagcagtc tgcgccctta 900cacaaaatga catcaaaaaa atcgtagcct tctccacttc aagtcaacta ggactcataa 960tagttacaat cggcatcaac caaccacacc tagcattcct gcacatctgt acccacgcct 1020tcttcaaagc catactattt atgtgctccg ggtccatcat ccacaacctt aacaatgaac 1080aagatattcg aaaaatagga ggactactca aaaccatacc tctcacttca acctccctca 1140ccattggcag cctagcatta gcaggaatac ctttcctcac aggtttctac tccaaagacc 1200acatcatcga aaccgcaaac atatcataca caaacgcctg agccctatct attactctca 1260tcgctacctc cctgacaagc gcctatagca ctcgaataat tcttctcacc ctaacaggtc 1320aacctcgctt ccccaccctt actaacatta acgaaaataa ccccacccta ctaaacccca 1380ttaaacgcct ggcagccgga agcctattcg caggatttct cattactaac aacatttccc 1440ccgcatcccc cttccaaaca acaatccccc tctacctaaa actcacagcc ctcgctgtca 1500ctttcctagg acttctaaca gccctagacc tcaactacct aaccaacaaa cttaaaataa 1560aatccccact atgcacattt tatttctcca acatactcgg attctaccct agcatcacac 1620accgcacaat cccctatcta ggccttctta cgagccaaaa cctgccccta ctcctcctag 1680acctaacctg actagaaaag ctattaccta aaacaatttc acagcaccaa atctccacct 1740ccatcatcac ctcaacccaa aaaggcataa ttaaacttta cttcctctct ttcttcttcc 1800cactcatcct aaccctactc ctaatcacat aacctattcc cccgagcaat ctcaattaca 1860atatatacac caacaaacaa tgttcaacca gtaactacta ctaatcaacg cccataatca 1920tacaaagccc ccgcaccaat aggatcctcc cgaatcaacc ctgacccctc tccttcataa 1980attattcagc ttcctacact attaaagttt accacaacca ccaccccatc atactctttc 2040acccacagca ccaatcctac ctccatcgct aaccccacta aaacactcac caagacctca 2100acccctgacc cccatgcctc aggatactcc tcaatagcca tcgctgtagt atatccaaag 2160acaaccatca ttccccctaa ataaattaaa aaaactatta aacccatata acctccccca 2220aaattcagaa taataacaca cccgaccaca ccgctaacaa tcaatactaa acccccataa 2280ataggagaag gcttagaaga aaaccccaca aaccccatta ctaaacccac actcaacaga 2340aacaaagcat acatcattat tctcgcacgg actacaacca cgaccaatga tatgaaaaac 2400catcgttgta tttcaactac aagaacacca atgaccccaa tacgcaaaac taacccccta 2460ataaaattaa ttaaccactc attcatcgac ctccccaccc catccaacat ctccgcatga 2520tgaaacttcg gctcactcct tggcgcctgc ctgatcctcc aaatcaccac aggactattc 2580ctagccatgc actactcacc agacgcctca accgcctttt catcaatcgc ccacatcact 2640cgagacgtaa attatggctg aatcatccgc taccttcacg ccaatggcgc ctcaatattc 2700tttatctgcc tcttcctaca catcgggcga ggcctatatt acggatcatt tctctactca 2760gaaacctgaa acatcggcat tatcctcctg cttgcaacta tagcaacagc cttcataggc 2820tatgtcctcc cgtgaggcca aatatcattc tgaggggcca cagtaattac aaacttacta 2880tccgccatcc catacattgg gacagaccta gttcaatgaa tctgaggagg ctactcagta 2940gacagtccca ccctcacacg attctttacc tttcacttca tcttgccctt cattattgca 3000gccctagcaa cactccacct cctattcttg cacgaaacgg gatcaaacaa ccccctagga 3060atcacctccc attccgataa aatcaccttc cacccttact acacaatcaa agacgccctc 3120ggcttacttc tcttccttct ctccttaatg acattaacac tattctcacc agacctccta 3180ggcgacccag acaattatac cctagccaac cccttaaaca cccctcccca catcaagccc 3240gaatgatatt tcctattcgc ctacacaatt ctccgatccg tccctaacaa actaggaggc 3300gtccttgccc tattactatc catcctcatc ctagcaataa tccccatcct ccatatatcc 3360aaacaacaaa gcataatatt tcgcccacta agccaatcac tttattgact cctagccgca 3420gacctcctca ttctaacctg aatcggagga caaccagtaa gctacccttt taccatcatt 3480ggacaagtag catccgtact atacttcaca acaatcctaa tcctaatacc aactatctcc 3540ctaattgaaa acaaaatact caaatgggcc tgtccttgta gtataaacta atacaccagt 3600cttgtaaacc ggagatgaaa acctttttcc aaggacaaat cagagaaaaa gtctttaact 3660ccaccattag cacccaaagc taagattcta atttaaacta ttctctgttc tttcatgggg 3720aagcagattt gggtaccacc caagtattga ctcacccatc aacaaccgct atgtatttcg 3780tacattactg ccagccacca tgaatattgt acggtaccat aaatacttga ccacctgtag 3840tacataaaaa cccaatccac atcaaaaccc cctccccatg cttacaagca agtacagcaa 3900tcaaccctca actatcacac atcaac 3926316569DNAHomo sapiensmisc_feature(3107)..(3107)n is a, c, g, or t 3gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720tcaccctcta aatcaccacg atcaaaagga acaagcatca agcacgcagc aatgcagctc 780aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacnttc aaattcctcc 3120ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc ccgtaaatga 3180tatcatctca acttagtatt atacccacac ccacccaaga acagggtttg ttaagatggc 3240agagcccggt aatcgcataa aacttaaaac tttacagtca gaggttcaat tcctcttctt 3300aacaacatac ccatggccaa cctcctactc ctcattgtac ccattctaat cgcaatggca 3360ttcctaatgc ttaccgaacg aaaaattcta ggctatatac aactacgcaa aggccccaac 3420gttgtaggcc cctacgggct actacaaccc ttcgctgacg ccataaaact cttcaccaaa 3480gagcccctaa aacccgccac atctaccatc accctctaca tcaccgcccc gaccttagct 3540ctcaccatcg ctcttctact atgaaccccc ctccccatac ccaaccccct ggtcaacctc 3600aacctaggcc tcctatttat tctagccacc tctagcctag ccgtttactc aatcctctga 3660tcagggtgag catcaaactc aaactacgcc ctgatcggcg cactgcgagc agtagcccaa 3720acaatctcat atgaagtcac cctagccatc attctactat caacattact aataagtggc 3780tcctttaacc tctccaccct tatcacaaca caagaacacc tctgattact cctgccatca 3840tgacccttgg ccataatatg atttatctcc acactagcag agaccaaccg aacccccttc 3900gaccttgccg aaggggagtc cgaactagtc tcaggcttca acatcgaata cgccgcaggc 3960cccttcgccc tattcttcat agccgaatac acaaacatta ttataataaa caccctcacc 4020actacaatct tcctaggaac aacatatgac gcactctccc ctgaactcta cacaacatat 4080tttgtcacca agaccctact tctaacctcc ctgttcttat gaattcgaac agcatacccc 4140cgattccgct acgaccaact catacacctc ctatgaaaaa acttcctacc actcacccta 4200gcattactta tatgatatgt ctccataccc attacaatct ccagcattcc ccctcaaacc 4260taagaaatat gtctgataaa agagttactt tgatagagta aataatagga gcttaaaccc 4320ccttatttct aggactatga gaatcgaacc catccctgag aatccaaaat tctccgtgcc 4380acctatcaca ccccatccta aagtaaggtc agctaaataa gctatcgggc ccataccccg 4440aaaatgttgg ttataccctt cccgtactaa ttaatcccct ggcccaaccc gtcatctact 4500ctaccatctt tgcaggcaca ctcatcacag cgctaagctc gcactgattt tttacctgag 4560taggcctaga aataaacatg ctagctttta ttccagttct aaccaaaaaa ataaaccctc 4620gttccacaga agctgccatc aagtatttcc tcacgcaagc aaccgcatcc ataatccttc 4680taatagctat cctcttcaac aatatactct ccggacaatg aaccataacc aatactacca 4740atcaatactc atcattaata atcataatag ctatagcaat aaaactagga atagccccct 4800ttcacttctg agtcccagag gttacccaag gcacccctct gacatccggc ctgcttcttc 4860tcacatgaca aaaactagcc cccatctcaa tcatatacca aatctctccc tcactaaacg 4920taagccttct cctcactctc tcaatcttat ccatcatagc aggcagttga ggtggattaa 4980accaaaccca gctacgcaaa atcttagcat actcctcaat tacccacata ggatgaataa 5040tagcagttct accgtacaac cctaacataa ccattcttaa tttaactatt tatattatcc 5100taactactac cgcattccta ctactcaact taaactccag caccacgacc ctactactat 5160ctcgcacctg aaacaagcta acatgactaa cacccttaat tccatccacc ctcctctccc 5220taggaggcct gcccccgcta accggctttt tgcccaaatg ggccattatc gaagaattca 5280caaaaaacaa tagcctcatc atccccacca tcatagccac catcaccctc cttaacctct 5340acttctacct acgcctaatc tactccacct caatcacact actccccata tctaacaacg 5400taaaaataaa atgacagttt gaacatacaa aacccacccc attcctcccc acactcatcg 5460cccttaccac gctactccta cctatctccc cttttatact aataatctta tagaaattta 5520ggttaaatac agaccaagag ccttcaaagc cctcagtaag ttgcaatact taatttctgt 5580aacagctaag gactgcaaaa ccccactctg catcaactga acgcaaatca gccactttaa 5640ttaagctaag cccttactag accaatggga cttaaaccca caaacactta gttaacagct 5700aagcacccta atcaactggc ttcaatctac ttctcccgcc gccgggaaaa aaggcgggag 5760aagccccggc aggtttgaag ctgcttcttc gaatttgcaa ttcaatatga aaatcacctc 5820ggagctggta aaaagaggcc taacccctgt ctttagattt acagtccaat gcttcactca 5880gccattttac ctcaccccca ctgatgttcg ccgaccgttg actattctct acaaaccaca 5940aagacattgg aacactatac ctattattcg gcgcatgagc tggagtccta ggcacagctc 6000taagcctcct tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca 6060tctacaacgt tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca 6120tcataatcgg aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg 6180cgtttccccg cataaacaac ataagcttct gactcttacc tccctctctc ctactcctgc 6240tcgcatctgc tatagtggag gccggagcag gaacaggttg aacagtctac cctcccttag 6300cagggaacta ctcccaccct ggagcctccg tagacctaac catcttctcc ttacacctag 6360caggtgtctc ctctatctta ggggccatca atttcatcac aacaattatc aatataaaac 6420cccctgccat aacccaatac caaacgcccc tcttcgtctg atccgtccta atcacagcag 6480tcctacttct cctatctctc ccagtcctag ctgctggcat cactatacta ctaacagacc 6540gcaacctcaa caccaccttc ttcgaccccg ccggaggagg agaccccatt ctataccaac 6600acctattctg atttttcggt caccctgaag tttatattct tatcctacca ggcttcggaa 6660taatctccca tattgtaact tactactccg gaaaaaaaga accatttgga tacataggta 6720tggtctgagc tatgatatca attggcttcc tagggtttat cgtgtgagca caccatatat 6780ttacagtagg aatagacgta gacacacgag catatttcac ctccgctacc ataatcatcg 6840ctatccccac cggcgtcaaa gtatttagct gactcgccac actccacgga agcaatatga 6900aatgatctgc tgcagtgctc tgagccctag gattcatctt tcttttcacc gtaggtggcc 6960tgactggcat tgtattagca aactcatcac tagacatcgt actacacgac acgtactacg 7020ttgtagccca

cttccactat gtcctatcaa taggagctgt atttgccatc ataggaggct 7080tcattcactg atttccccta ttctcaggct acaccctaga ccaaacctac gccaaaatcc 7140atttcactat catattcatc ggcgtaaatc taactttctt cccacaacac tttctcggcc 7200tatccggaat gccccgacgt tactcggact accccgatgc atacaccaca tgaaacatcc 7260tatcatctgt aggctcattc atttctctaa cagcagtaat attaataatt ttcatgattt 7320gagaagcctt cgcttcgaag cgaaaagtcc taatagtaga agaaccctcc ataaacctgg 7380agtgactata tggatgcccc ccaccctacc acacattcga agaacccgta tacataaaat 7440ctagacaaaa aaggaaggaa tcgaaccccc caaagctggt ttcaagccaa ccccatggcc 7500tccatgactt tttcaaaaag gtattagaaa aaccatttca taactttgtc aaagttaaat 7560tataggctaa atcctatata tcttaatggc acatgcagcg caagtaggtc tacaagacgc 7620tacttcccct atcatagaag agcttatcac ctttcatgat cacgccctca taatcatttt 7680ccttatctgc ttcctagtcc tgtatgccct tttcctaaca ctcacaacaa aactaactaa 7740tactaacatc tcagacgctc aggaaataga aaccgtctga actatcctgc ccgccatcat 7800cctagtcctc atcgccctcc catccctacg catcctttac ataacagacg aggtcaacga 7860tccctccctt accatcaaat caattggcca ccaatggtac tgaacctacg agtacaccga 7920ctacggcgga ctaatcttca actcctacat acttccccca ttattcctag aaccaggcga 7980cctgcgactc cttgacgttg acaatcgagt agtactcccg attgaagccc ccattcgtat 8040aataattaca tcacaagacg tcttgcactc atgagctgtc cccacattag gcttaaaaac 8100agatgcaatt cccggacgtc taaaccaaac cactttcacc gctacacgac cgggggtata 8160ctacggtcaa tgctctgaaa tctgtggagc aaaccacagt ttcatgccca tcgtcctaga 8220attaattccc ctaaaaatct ttgaaatagg gcccgtattt accctatagc accccctcta 8280ccccctctag agcccactgt aaagctaact tagcattaac cttttaagtt aaagattaag 8340agaaccaaca cctctttaca gtgaaatgcc ccaactaaat actaccgtat ggcccaccat 8400aattaccccc atactcctta cactattcct catcacccaa ctaaaaatat taaacacaaa 8460ctaccaccta cctccctcac caaagcccat aaaaataaaa aattataaca aaccctgaga 8520accaaaatga acgaaaatct gttcgcttca ttcattgccc ccacaatcct aggcctaccc 8580gccgcagtac tgatcattct atttccccct ctattgatcc ccacctccaa atatctcatc 8640aacaaccgac taatcaccac ccaacaatga ctaatcaaac taacctcaaa acaaatgata 8700accatacaca acactaaagg acgaacctga tctcttatac tagtatcctt aatcattttt 8760attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac cacccaacta 8820tctataaacc tagccatggc catcccctta tgagcgggca cagtgattat aggctttcgc 8880tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac accccttatc 8940cccatactag ttattatcga aaccatcagc ctactcattc aaccaatagc cctggccgta 9000cgcctaaccg ctaacattac tgcaggccac ctactcatgc acctaattgg aagcgccacc 9060ctagcaatat caaccattaa ccttccctct acacttatca tcttcacaat tctaattcta 9120ctgactatcc tagaaatcgc tgtcgcctta atccaagcct acgttttcac acttctagta 9180agcctctacc tgcacgacaa cacataatga cccaccaatc acatgcctat catatagtaa 9240aacccagccc atgaccccta acaggggccc tctcagccct cctaatgacc tccggcctag 9300ccatgtgatt tcacttccac tccataacgc tcctcatact aggcctacta accaacacac 9360taaccatata ccaatgatgg cgcgatgtaa cacgagaaag cacataccaa ggccaccaca 9420caccacctgt ccaaaaaggc cttcgatacg ggataatcct atttattacc tcagaagttt 9480ttttcttcgc aggatttttc tgagcctttt accactccag cctagcccct accccccaat 9540taggagggca ctggccccca acaggcatca ccccgctaaa tcccctagaa gtcccactcc 9600taaacacatc cgtattactc gcatcaggag tatcaatcac ctgagctcac catagtctaa 9660tagaaaacaa ccgaaaccaa ataattcaag cactgcttat tacaatttta ctgggtctct 9720attttaccct cctacaagcc tcagagtact tcgagtctcc cttcaccatt tccgacggca 9780tctacggctc aacatttttt gtagccacag gcttccacgg acttcacgtc attattggct 9840caactttcct cactatctgc ttcatccgcc aactaatatt tcactttaca tccaaacatc 9900actttggctt cgaagccgcc gcctgatact ggcattttgt agatgtggtt tgactatttc 9960tgtatgtctc catctattga tgagggtctt actcttttag tataaatagt accgttaact 10020tccaattaac tagttttgac aacattcaaa aaagagtaat aaacttcgcc ttaattttaa 10080taatcaacac cctcctagcc ttactactaa taattattac attttgacta ccacaactca 10140acggctacat agaaaaatcc accccttacg agtgcggctt cgaccctata tcccccgccc 10200gcgtcccttt ctccataaaa ttcttcttag tagctattac cttcttatta tttgatctag 10260aaattgccct ccttttaccc ctaccatgag ccctacaaac aactaacctg ccactaatag 10320ttatgtcatc cctcttatta atcatcatcc tagccctaag tctggcctat gagtgactac 10380aaaaaggatt agactgaacc gaattggtat atagtttaaa caaaacgaat gatttcgact 10440cattaaatta tgataatcat atttaccaaa tgcccctcat ttacataaat attatactag 10500catttaccat ctcacttcta ggaatactag tatatcgctc acacctcata tcctccctac 10560tatgcctaga aggaataata ctatcgctgt tcattatagc tactctcata accctcaaca 10620cccactccct cttagccaat attgtgccta ttgccatact agtctttgcc gcctgcgaag 10680cagcggtggg cctagcccta ctagtctcaa tctccaacac atatggccta gactacgtac 10740ataacctaaa cctactccaa tgctaaaact aatcgtccca acaattatat tactaccact 10800gacatgactt tccaaaaaac acataatttg aatcaacaca accacccaca gcctaattat 10860tagcatcatc cctctactat tttttaacca aatcaacaac aacctattta gctgttcccc 10920aaccttttcc tccgaccccc taacaacccc cctcctaata ctaactacct gactcctacc 10980cctcacaatc atggcaagcc aacgccactt atccagtgaa ccactatcac gaaaaaaact 11040ctacctctct atactaatct ccctacaaat ctccttaatt ataacattca cagccacaga 11100actaatcata ttttatatct tcttcgaaac cacacttatc cccaccttgg ctatcatcac 11160ccgatgaggc aaccagccag aacgcctgaa cgcaggcaca tacttcctat tctacaccct 11220agtaggctcc cttcccctac tcatcgcact aatttacact cacaacaccc taggctcact 11280aaacattcta ctactcactc tcactgccca agaactatca aactcctgag ccaacaactt 11340aatatgacta gcttacacaa tagcttttat agtaaagata cctctttacg gactccactt 11400atgactccct aaagcccatg tcgaagcccc catcgctggg tcaatagtac ttgccgcagt 11460actcttaaaa ctaggcggct atggtataat acgcctcaca ctcattctca accccctgac 11520aaaacacata gcctacccct tccttgtact atccctatga ggcataatta taacaagctc 11580catctgccta cgacaaacag acctaaaatc gctcattgca tactcttcaa tcagccacat 11640agccctcgta gtaacagcca ttctcatcca aaccccctga agcttcaccg gcgcagtcat 11700tctcataatc gcccacgggc ttacatcctc attactattc tgcctagcaa actcaaacta 11760cgaacgcact cacagtcgca tcataatcct ctctcaagga cttcaaactc tactcccact 11820aatagctttt tgatgacttc tagcaagcct cgctaacctc gccttacccc ccactattaa 11880cctactggga gaactctctg tgctagtaac cacgttctcc tgatcaaata tcactctcct 11940acttacagga ctcaacatac tagtcacagc cctatactcc ctctacatat ttaccacaac 12000acaatggggc tcactcaccc accacattaa caacataaaa ccctcattca cacgagaaaa 12060caccctcatg ttcatacacc tatcccccat tctcctccta tccctcaacc ccgacatcat 12120taccgggttt tcctcttgta aatatagttt aaccaaaaca tcagattgtg aatctgacaa 12180cagaggctta cgacccctta tttaccgaga aagctcacaa gaactgctaa ctcatgcccc 12240catgtctaac aacatggctt tctcaacttt taaaggataa cagctatcca ttggtcttag 12300gccccaaaaa ttttggtgca actccaaata aaagtaataa ccatgcacac tactataacc 12360accctaaccc tgacttccct aattcccccc atccttacca ccctcgttaa ccctaacaaa 12420aaaaactcat acccccatta tgtaaaatcc attgtcgcat ccacctttat tatcagtctc 12480ttccccacaa caatattcat gtgcctagac caagaagtta ttatctcgaa ctgacactga 12540gccacaaccc aaacaaccca gctctcccta agcttcaaac tagactactt ctccataata 12600ttcatccctg tagcattgtt cgttacatgg tccatcatag aattctcact gtgatatata 12660aactcagacc caaacattaa tcagttcttc aaatatctac tcatcttcct aattaccata 12720ctaatcttag ttaccgctaa caacctattc caactgttca tcggctgaga gggcgtagga 12780attatatcct tcttgctcat cagttgatga tacgcccgag cagatgccaa cacagcagcc 12840attcaagcaa tcctatacaa ccgtatcggc gatatcggtt tcatcctcgc cttagcatga 12900tttatcctac actccaactc atgagaccca caacaaatag cccttctaaa cgctaatcca 12960agcctcaccc cactactagg cctcctccta gcagcagcag gcaaatcagc ccaattaggt 13020ctccacccct gactcccctc agccatagaa ggccccaccc cagtctcagc cctactccac 13080tcaagcacta tagttgtagc aggaatcttc ttactcatcc gcttccaccc cctagcagaa 13140aatagcccac taatccaaac tctaacacta tgcttaggcg ctatcaccac tctgttcgca 13200gcagtctgcg cccttacaca aaatgacatc aaaaaaatcg tagccttctc cacttcaagt 13260caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 13320atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 13380aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 13440acttcaacct ccctcaccat tggcagccta gcattagcag gaataccttt cctcacaggt 13500ttctactcca aagaccacat catcgaaacc gcaaacatat catacacaaa cgcctgagcc 13560ctatctatta ctctcatcgc tacctccctg acaagcgcct atagcactcg aataattctt 13620ctcaccctaa caggtcaacc tcgcttcccc acccttacta acattaacga aaataacccc 13680accctactaa accccattaa acgcctggca gccggaagcc tattcgcagg atttctcatt 13740actaacaaca tttcccccgc atcccccttc caaacaacaa tccccctcta cctaaaactc 13800acagccctcg ctgtcacttt cctaggactt ctaacagccc tagacctcaa ctacctaacc 13860aacaaactta aaataaaatc cccactatgc acattttatt tctccaacat actcggattc 13920taccctagca tcacacaccg cacaatcccc tatctaggcc ttcttacgag ccaaaacctg 13980cccctactcc tcctagacct aacctgacta gaaaagctat tacctaaaac aatttcacag 14040caccaaatct ccacctccat catcacctca acccaaaaag gcataattaa actttacttc 14100ctctctttct tcttcccact catcctaacc ctactcctaa tcacataacc tattcccccg 14160agcaatctca attacaatat atacaccaac aaacaatgtt caaccagtaa ctactactaa 14220tcaacgccca taatcataca aagcccccgc accaatagga tcctcccgaa tcaaccctga 14280cccctctcct tcataaatta ttcagcttcc tacactatta aagtttacca caaccaccac 14340cccatcatac tctttcaccc acagcaccaa tcctacctcc atcgctaacc ccactaaaac 14400actcaccaag acctcaaccc ctgaccccca tgcctcagga tactcctcaa tagccatcgc 14460tgtagtatat ccaaagacaa ccatcattcc ccctaaataa attaaaaaaa ctattaaacc 14520catataacct cccccaaaat tcagaataat aacacacccg accacaccgc taacaatcaa 14580tactaaaccc ccataaatag gagaaggctt agaagaaaac cccacaaacc ccattactaa 14640acccacactc aacagaaaca aagcatacat cattattctc gcacggacta caaccacgac 14700caatgatatg aaaaaccatc gttgtatttc aactacaaga acaccaatga ccccaatacg 14760caaaactaac cccctaataa aattaattaa ccactcattc atcgacctcc ccaccccatc 14820caacatctcc gcatgatgaa acttcggctc actccttggc gcctgcctga tcctccaaat 14880caccacagga ctattcctag ccatgcacta ctcaccagac gcctcaaccg ccttttcatc 14940aatcgcccac atcactcgag acgtaaatta tggctgaatc atccgctacc ttcacgccaa 15000tggcgcctca atattcttta tctgcctctt cctacacatc gggcgaggcc tatattacgg 15060atcatttctc tactcagaaa cctgaaacat cggcattatc ctcctgcttg caactatagc 15120aacagccttc ataggctatg tcctcccgtg aggccaaata tcattctgag gggccacagt 15180aattacaaac ttactatccg ccatcccata cattgggaca gacctagttc aatgaatctg 15240aggaggctac tcagtagaca gtcccaccct cacacgattc tttacctttc acttcatctt 15300gcccttcatt attgcagccc tagcaacact ccacctccta ttcttgcacg aaacgggatc 15360aaacaacccc ctaggaatca cctcccattc cgataaaatc accttccacc cttactacac 15420aatcaaagac gccctcggct tacttctctt ccttctctcc ttaatgacat taacactatt 15480ctcaccagac ctcctaggcg acccagacaa ttatacccta gccaacccct taaacacccc 15540tccccacatc aagcccgaat gatatttcct attcgcctac acaattctcc gatccgtccc 15600taacaaacta ggaggcgtcc ttgccctatt actatccatc ctcatcctag caataatccc 15660catcctccat atatccaaac aacaaagcat aatatttcgc ccactaagcc aatcacttta 15720ttgactccta gccgcagacc tcctcattct aacctgaatc ggaggacaac cagtaagcta 15780cccttttacc atcattggac aagtagcatc cgtactatac ttcacaacaa tcctaatcct 15840aataccaact atctccctaa ttgaaaacaa aatactcaaa tgggcctgtc cttgtagtat 15900aaactaatac accagtcttg taaaccggag atgaaaacct ttttccaagg acaaatcaga 15960gaaaaagtct ttaactccac cattagcacc caaagctaag attctaattt aaactattct 16020ctgttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca 16080accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat 16140acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16200caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc 16260cctcacccac taggatacca acaaacctac ccacccttaa cagtacatag tacataaagc 16320catttaccgt acatagcaca ttacagtcaa atcccttctc gtccccatgg atgacccccc 16380tcagataggg gtcccttgac caccatcctc cgtgaaatca atatcccgca caagagtgct 16440actctcctcg ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16500ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct taaataagac 16560atcacgatg 16569429DNAArtificialSynthetic primer 4ttggtgcaac tccaaagcca cccctcacc 29519DNAartificialSynthetic primer. 5aggatggtgg tcaagggac 19631DNAArtificialSynthetic primer 6cccaaaaatt ttggtgcaac tccaaagcca c 31

Claims

1. A method of detecting a cancer in a subject, the method comprising: a) quantifying, in a biological sample obtained from the subject, the amount of mtDNA having a deletion in the mtDNA sequence spanning approximately nucleotides 12317 and 16254 of the human mtDNA genome; b) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value; and, c) detecting said cancer based on the results of step (b).

2. The method of claim 1 wherein the deletion has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

3. The method of claim 1 wherein the at least one known reference value is the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid, and wherein an elevated amount of the deletion in the biological sample compared to the reference sample is indicative of cancer.

4. (canceled)

5. The method of claim 3 further comprising the step of comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid.

6. The method of claim 1 wherein the at least one known reference value is the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid, wherein a similar level of the deletion in the biological sample compared to the reference sample is indicative of cancer.

7. (canceled)

8. The method of claim 7 further comprising the step of comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid.

9. (canceled)

10. The method of claim 1 wherein the step of quantifying includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region.

11. The method of claim 10 wherein a primer having ID NO: 4 is used as part of a pair of amplification primers for amplifying the target region.

12. The method of claim 1 wherein the cancer is prostate cancer or breast cancer.

13-27. (canceled)

28. A method of detecting a cancer in a subject, the method comprising: a) quantifying, in a biological sample obtained from the subject, the amount of mtDNA in the sample having a deletion set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and b) comparing the amount of mtDNA from step a) to at least one known reference value; and c) detecting said cancer based on the results of step (b).

29. The method of claim 28 wherein the at least one known reference value is the amount of SEQ ID NO: 1 or SEQ ID NO: 2 in a reference sample of mtDNA from known non-cancerous tissue or body fluid.

30. The method of claim 28 wherein the at least one known reference value is the amount of SEQ ID NO: 1 or SEQ ID NO: 2 in a reference sample of mtDNA from known cancerous tissue or body fluid.

31. (canceled)

32. The method of claim 31 wherein the step of quantifying includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region.

33. The method of claim 32 wherein one of a pair of primers used in the amplifying of the target region overlaps a rejoining site of SEQ ID NO: 1 or SEQ ID NO: 2, after the sequence has re-circularized.

34. The method of claim 28 wherein the cancer is prostate cancer or breast cancer.

35-38. (canceled)

39. A diagnostic kit for carrying out the method of claim 1 comprising: (a) at least one of material for collecting one or more biological samples, material for extracting mtDNA from one or more biological sample or reagent for conducting the method; and (b) at least one suitable primer for detecting the mtDNA deletion.

40. The kit of claim 39, wherein the at least one suitable primer overlaps a spliced region of mtDNA having the deletion.

41. The kit of claim 40, therein the at least one suitable primer is SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

42. A diagnostic kit for carrying out the method of claim 28 comprising: (a) at least one of material for collecting one or more biological samples, material for extracting mtDNA from one or more biological sample or reagent for conducting the method; and (b) at least one suitable primer for detecting the deletion set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

43. The kit of claim 43, wherein the at least one suitable primer overlaps a rejoining site of SEQ ID NO: 1 or SEQ ID NO: 2.