Mitochondrial Dosimeter

Abstract

Mitochondrial mutations occur as a product of contact of a person with an environmental pollutant. Mitochondrial mutations are readily detectable in body fluids. Measurement of mitochondrial mutations in body fluids can be used as a dosimeter to monitor exposure to the environmental pollutant. Mitochondrial mutations can also be detected in cancer patients. Probes and primers containing mutant mitochondrial sequences can be used to monitor patient condition.

Description

[0002] This application is a continuation of Ser. No. 10/601,692 filed Jun. 23, 2003, is a division of Ser. No. 09/525,906 filed Mar. 15, 2000, is a continuation-in-part of application Ser. No. 09/377,856 filed Aug. 20, 1999, which claims priority to provisional application Ser. No. 60/097,307 filed Aug. 20, 1998. The disclosure of these prior applications is expressly incorporated herein.

TECHNICAL FIELD OF THE INVENTION

[0003] This invention is related to the field of environmental toxicology, in particular to methods for measuring the effects of environmental toxins.

BACKGROUND OF THE INVENTION

[0004] The human mitochondrial (mt) genome is small (16.5 kb) and encodes 13 respiratory chain subunits, 22 tRNAs and two rRNAs. Mitochondrial DNA is present at extremely high levels (10.sup.3-10.sup.4 copies per cell) and the vast majority of these copies are identical (homoplasmic) at birth (1). Expression of the entire complement of mt genes is required to maintain proper function of the organelle, suggesting that even slight alterations in DNA sequences could have profound effects (2). It is generally accepted that mtDNA mutations are generated endogenously during oxidative phosphorylation via pathways involving reactive oxygen species (ROS), but they can also be generated by external carcinogens or environmental toxins. These mutations may accumulate partially because mitochondria lack protective histones and highly efficient DNA repair mechanisms as seen in the nucleus (3). Recently several mtDNA mutations were found specifically in human colorectal cancer (4).

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide methods of monitoring exposure of a person to an environmental pollutant.

[0006] It is another object of the present invention to provide a kit for monitoring exposure of a person to environmental pollutants.

[0007] It is an object of the invention to provide methods to aid in the detection of cancer or metastasis.

[0008] It is an object of the invention to provide probes and primers for detecting mitochondrial mutations.

[0009] It is an object of the invention to provide a method to aid in detecting the presence of tumor cells in a patient.

[0010] These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided for monitoring exposure of a person to an environmental pollutant. The presence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is determined at two or more time points. The amounts of mutations in mtDNA at different time points are compared. The amount of mutations correlates with amount of exposure to the environmental pollutant.

[0011] According to another embodiment another method is provided for monitoring exposure of a person to an environmental pollutant. The prevalence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is measured. A measured prevalence of one or more mutations in mtDNA of greater than 1% indicates clonal expansion of cells which harbor the one or more mutations in the person.

[0012] According to still another embodiment of the invention a method is provided for monitoring exposure of a person to an environmental pollutant. One or more mutations in a D-loop of mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant are measured. The number of mutations in mtDNA correlates with exposure to the environmental pollutant.

[0013] According to yet another embodiment of the invention a kit is provided. The kit comprises one or more primers which hybridize to a mitochondrial D-loop for making a primer extension product. In addition, the kit contains written material identifying mutations which are found in the D-loop as a result of exposure to one or more environmental pollutants.

[0014] According to another embodiment of the invention an oligonucleotide probe is provided. The probe comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The probe can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA. 7 amino acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521; G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A substitution at position 6264; A insertion at position 12418; T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution at position 2299; and G.fwdarw.A at nucleotide 3054.

[0015] According to another aspect of the invention an oligonucleotide primer is provided. It comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The primer can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA. 7 amino acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521; G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A substitution at position 6264; A insertion at position 12418; T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution at position 2299; and G.fwdarw.A at nucleotide 3054.

[0016] Another aspect of the invention is a method to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is detected in a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The tumor is not a colorectal tumor. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.

[0017] Yet another embodiment of the invention is provided by another method to aid in detecting the presence of tumor cells in a patient. The presence of a mutation is determined in a D-loop of a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.

[0018] According to still another aspect of the invention a method is provided to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is determined in a mitochondrial genome of a cell sample of a patient. The mutation is found in a cancer of the patient but not in normal tissue of the patient. The cancer is selected from the group of cancers consisting of: lung, head and neck, bladder, brain, breast, lymphoma, leukaemia, skin, prostate, stomach, pancreas, liver, ovarian, uterine, testicular, and bone. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.

[0019] According to still another aspect of the invention a method is provided to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is determined in a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The cancer is selected from the group of cancers consisting of: lung, head and neck, and bladder. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.

[0020] Another embodiment of the invention provides a method to aid in detecting the presence of tumor cells in a patient. The presence of a mutation in a mitochondrial genome of a cell sample of a patient is determined. The mutation is selected from the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA. 7 amino acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521; G.fwdarw.A at nucleotide 12345; T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A substitution at position 6264; A insertion at position 12418; T.fwdarw.C substitution at position 1967; T.fwdarw.A substitution at position 2299; and G.fwdarw.A at nucleotide 3054. The patient is identified as having a tumor if one or more mutations are determined in the mitochondrial genome of the cell sample of the patient.

[0021] These and other embodiments provide the art with non-invasive tools for monitoring exposure to and the effects of environmental pollutants on the human body as well as early detection methods for cancer and metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1. Schematic representation of a linearized mt genome. Hatched bars indicate the regions sequenced in this study and solid bars indicate the positions of tRNAs (transfer RNAs). rRNA=ribosomal RNA, ND=NADH dehydrogenase, COX=cytochrome c oxidase, Cyt b=cytochrome b, ATPase=ATP synthase.

[0023] FIG. 2. Sequence detection of mutated mtDNAs in samples from tumors and bodily fluids. (FIG. 2A) The mt mutation was analyzed by direct sequencing of the tumor (T), normal (N), and corresponding urine (U) DNAs of bladder cancer patient #799. The arrow indicates a single nucleotide change (G(A) at 2056 np in the 16S rRNA gene. (FIG. 2B and FIG. 2C) Examples of somatic mutations in head and neck cancers. Both mutations at 16172 np (B) and 10822 np (C) were detected from saliva (S) samples from patients #1680 and #1708, respectively. (FIG. 2D) Mutated mtDNA at 2664 np was not detected by sequence analysis in the paired BAL fluid (B), obtained from lung cancer patient #898.

[0024] FIG. 3. Oligonucleotide-mismatch ligation assay (22) to detect mtDNA mutations in BAL. The arrows identify mutated mt sequences at 12345 np within tRNA (FIG. 3A) and at 2664 np (FIG. 3B) within 16S rRNA in the tumor DNA. More dilute signals are seen in the corresponding BAL (B) samples with no detectable signal from the paired normal (N) tissue.

[0025] FIG. 4. Highly enriched mutated mtDNA in BAL samples from lung cancer patients. Oligonucleotide (oligo) specific hybridization detected .about.2000 plaques containing WT p53 clones in the BAL from patient #1113, and only two plaques (2/2000=0.1%) with the p53 gene mutation (FIG. 4A) were found in the primary tumor. The same BAL sample demonstrated a much greater enrichment of mutated mtDNA; 445 plaques contained mtDNA mutations (FIG. 4A) at 16159 np (445/2000=22.3%; 220-fold) compared to approximately 1500 WT clones. A similar enrichment was seen in patient #1140 where oligo specific hybridization detected 12 p53 mutant plaques among 437 WT clones (2.7%, FIG. 4B), while mutant mtDNA at 16380 np (FIG. 4B) represented over 50% of the plaques (52.3%, 460/880; 19-fold) amplified from mtDNA.

[0026] FIG. 5. Pseudoclonal selection of mtDNA. A mitochondrial genome gains some replicative advantage due to a somatic mutation (such as in the D-loop region), leading to a dominant mitochondrial genotype (step 1). This mitochondrion can gain additional replicative advantage through nuclear influences: for example, a mutated sequence gains a higher binding affinity to nuclear-encoded mitochondrial trans-acting factors (step 2). Due to its stochastic segregation together with the clonal expansion of a neoplastic cell driven by nuclear mutations, mutated mitochondria overtake the entire population of tumor cells (step 3).

BRIEF DESCRIPTION OF THE TABLES

[0027] Table 1 provides a summary of mutations in mitochondria of colorectal tumors.

[0028] Table 2 provides a summary of mutations in mitochondria of bladder, lung, head and neck tumors.

[0029] Table 3 provides a summary of new polymorphisms in mitochondria of bladder, lung, head and neck tumors.

DETAILED DESCRIPTION

[0030] It is a discovery of the present inventors that mitochondrial DNA mutations can be monitored non-invasively and sensitively and used as an indicator of environmental pollutants. It is shown below that these mutations are more prevalent in body samples than nuclear mutations, and thus are detected more sensitively. Mitochondrial mutations can be monitored over time to detect changes in the amount of exposure to pollutants. In addition, the prevalence of the mitochondrial mutation in the sample indicates whether clonal proliferation has occurred. Finally, the D-loop has been identified as a hotspot of mutations within the mitochondrial genome.

[0031] Mitochondrial mutations are determined with reference to wild-type human mitochondrial sequence. Sequence information can be found at the website http://www.gen.emory.edu/mitomap.html and at SEQ ID NO: 1. However, some differences between a sample sequence and a documented wild-type sequence can be polymorphisms, not mutations. Table 3 provides a number of new polymorphisms. Other polymorphisms can be found in references 2 and 8. Polymorphisms can be distinguished from somatic mutations by comparing the sequence in the sample to the corresponding sequence in a normal body tissue of the same person. If the same variant sequence is found in the sample as in the normal body tissue it is a polymorphism. Normal tissues can be paraffin-embedded. It has been found by the present inventors that mitochondrial DNA which is paraffin-embedded remains more highly intact and amplifiable than genomic DNA. Amplifiable regions of mitochondrial DNA may be from 10 bp to about 4 kb, desirably 2 kb to 4 kb or 10 bp to about 2 kb. Other suitable sources of reference mtDNA are blood, serum, or plasma of the human being tested.

[0032] Suitable bodily fluids for testing according to the present invention include saliva, sputum, urine, and bronchoalveolar lavage (BAL). These can be collected as is known in the art. People who are prime candidates for testing and supplying such bodily fluids are those who have been episodically, periodically or chronically exposed to environmental pollutants. These include without limitation cigarette smoke, biological toxins, such as aflatoxin, cholera toxin, and botulinum toxin, radiation including UV irradiation, industrial wastes, chemicals, water-borne or air-borne pollutants, and drugs. The environmental pollutant can be known, suspected, or unidentified, as the assay depends on the effect and not on the identity of the pollutant.

[0033] The inventors have found that there are certain characteristics of the mutations which are found in mitochondrial DNA. Many mutations are found in sequences which do not encode proteins. These include the D-loop region (i.e., nucleotides 16024-526), the 16S RNA gene, and the tRNA genes. Furthermore, even where the mutations do occur in protein coding regions, they often result in silent mutations which do not affect the encoded amino acids. Other regions frequently affected include the genes for NADH dehydrogenase 4, NADH dehydrogenase 3, NADH dehydrogenase 5, and cytochrome B.

[0034] Mutation detection can be done according to any methodology which is known in the art for determining mutations. These include without limitation, nucleotide sequencing, hybridization, amplification, PCR, oligonucleotide mismatch ligation assays, primer extension assays, heteroduplex analysis, allele-specific amplification, allele-specific primer extension, SCCP, DGGE, mass spectroscopy, high pressure liquid chromatography, and combinations of these techniques.

[0035] Prevalence of a particular mutation according to the present invention can be used to monitor clonal expansion. Mutations which are present in greater than 1% of the mitochondrial DNA present in a sample have may have conferred a growth advantage on the cells harboring them. Even if no growth advantage is conferred by the mutation itself, the mutation serves as a marker for a clone which is expanding relative to the population of cells in the sample. Clonal expansion can be measured over time to monitor the growth of the clone or to monitor the efficacy of anti-proliferative agents which can be considered environmental pollutants, according to the present invention.

[0036] The inventors have also found that the presence of subtle mutations in the mitochondrial genome can be used as a means to trace the presence, spread, metastasis, growth, or recurrence of a tumor in a patient. Such subtle mutations include single basepair substitutions, single basepair insertions, and single basepair deletions. Single basepair substitutions can be either transitions or transversions, although the former are more frequent. Detection of such mutations can be useful to screen for the initial appearance of a tumor as well as the recurrence of a previously identified tumor. The methods are particularly suited to monitor anti-cancer therapy, recurrence, metastasis, and completeness of surgical removals.

[0037] A single basepair substitution is the substitution of a single nucleotide base with a different nucleotide base at the same position, with the corresponding substitution of the complementary base on the other strand of the DNA. While any single basepair substitution is conceivable within the scope of the invention, the most frequently encountered substitutions are those which are consistent with endogenous oxidative damage, such as T to C or G to A transitions, or which are consistent with a variety of external carcinogens which cause a variety of types of mutations. The mutations can appear in protein coding or non-coding regions or in regions which encode ribosomal or transfer RNAs.

[0038] The homoplasmic or near homoplasmic property of most mutant mitochondrial genomes from tumors permits the ready detection of such mutations within a sample of mitochondrial DNA from a patient. Homoplasmic mutations are those which appear in essentially all of the copies of the mitochondrial genome within a given cell or tissue. However, heteroplasmic mutations, which are those appearing in only a fraction of the mitochondrial genomes of a cell or tissue, are also suitable for use with the invention.

[0039] Any cell sample can be tested from a patient who has cancer or is suspected of having cancer. Suitable cell samples include, but are not limited to, tissue from a growth suspected or known to be cancerous, tissue adjacent to a resection of a tumor, and tissue distant from the site of a tumor, such as lymph nodes which are suspected of bearing metastatic cells. Cells can also be obtained from bodily fluids or secretions, e.g., blood, urine, sputum, saliva, or feces, which may contain cancerous cells or metastatic cells. Cell samples can also be collected from other bodily secretions and tissues as is known in the art. A cell sample can be collected from suspected or known cancerous tissue or from bodily fluids or secretions harboring cancer cells as well as from suspected or known normal tissue or bodily fluids or secretions harboring normal cells.

[0040] In order to detect mutations of the mitochondrial genome from a cell sample of a patient, mitochondrial DNA can be isolated from the cell sample using any method known in the art. One way of identifying subtle mutations involves sequencing the mitochondrial DNA. This can be done according to any method known in the art. For example, isolated mitochondrial DNA can be cleaved using endonucleases into overlapping fragments of appropriate size for sequencing, e.g., about 1-3 kilobases in length, followed by polymerase chain reaction (PCR) amplification and sequencing of the fragments. 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., Drossman, H., Kostihka, T.; and Smith, L. M., 1993, High-speed DNA sequencing by capillary gel electrophoresis, Methods Enzymol. 218:154-172. Amplification methods such as PCR can be applied to samples as small as a single cell and still yield sufficient DNA for complete sequence analysis. The combined use of PCR and sequencing of mitochondrial DNA is described in Hopgood, R., Sullivan, K. M., and Gill, P., 1992, Strategies for automated sequencing of human mitochondrial DNA directly from PCR products, Biotechniques 13:82-92 and Tanaka, M., Hayakawa, M., and Ozawa, T., 1996, Automated sequencing of mitochondrial DNA, Methods Enzymol. 264:407-21.

[0041] Mutations can first be identified by comparison to sequences present in public databases for human mitochondrial DNA, e.g., at http://www.gen.emory.edu/mitomap.html and at SEQ ID NO: 1. Any single basepair substitution identified in the sample DNA compared to a normal sequence from a database can be confirmed as being a somatic mutation as opposed to a polymorphic variant by comparing the sample mitochondrial DNA or sequences obtained from it to control cell mitochondrial DNA from the same individual or sequences obtained from it. Control cells are isolated from other apparently normal tissues, i.e., tissues which are phenotypically normal and devoid of any visible, histological, or immunological characteristics of cancer tissue. A difference between the sample and the control identifies a somatic mutation which is associated with the tumor.

[0042] An alternative to serially sequencing the entire mitochondrial genome in order to identify a single basepair substitution is to use hybridization of the mitochondrial DNA to an array of oligonucleotides. Hybridization techniques are available in the art which can rapidly identify mutations by comparing the hybridization of the sample to matched and mismatched sequences which are based on the human mitochondrial genome. Such an array can be as simple as two oligonucleotide probes, one of whose sequence matches the wild-type or mutant region containing the single base substitution (matched probe) and another whose sequence includes a single mismatched base (mismatch control probe). If the sample DNA hybridizes to the matched probe but not the mismatched probe, it is identified as having the same sequence as the matched probe. Larger arrays containing thousands of such matched/mismatched pairs of probes on a glass slide or microchip ("microarrays" or "gene chips") are available which are capable of sequencing the entire mitochondrial genome very quickly. Such arrays are commercially available. Review articles describing the use of microarrays in genome and DNA sequence analysis and links to their commercial suppliers are available at www.gene-chips.com.

[0043] The invention can be used to screen patients suspected of having cancer for the presence of tumor cells. A cell sample is first obtained from a suspected tumor of the patient, or is obtained from another source such as blood or lymph tissue, for example, if metastasis is suspected. The cell sample is tested to determine the presence of a single basepair mutation in mitochondrial DNA from the cell sample using the techniques outlined above. Optionally, a cell sample from normal, non-cancerous cells or tissue of the patient is also obtained and is tested for the presence or absence of a single basepair mutation in mitochondrial DNA. If a single basepair mutation is determined which is not present in a cell sample from normal tissue of the patient, then the mutation is a somatic mutation and the presence of tumor cells in the patient is indicated. If one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient, then the patient is identified as having a tumor. As in any diagnostic technique for cancer, to confirm or extend the diagnosis, further diagnostic techniques may be warranted. For example, conventional histological examination of a biopsy specimen can be performed to detect the presence of tumor cells, or analysis of a tumor-specific antigen in a blood or tissue sample can be performed.

[0044] The method outlined above can be practiced either in the situation where the somatic mutation is previously known or previously unknown. The method can be practiced even in the absence of prior knowledge about any particular somatic mutation. The method can also be carried out subsequent to the discovery of a somatic mutation in a mitochondrial genome of a cell of the patient or of another patient. In this case, a previous association of the somatic mutation with the presence of a tumor in the patient or in another patient strongly indicates the presence of tumor cells in the patient. It may also indicate the recurrence of a tumor or the incomplete prior removal of cancerous tissue from the patient.

[0045] The effectiveness of therapy can be evaluated when a tumor has already been identified and found to contain a single basepair substitution in the mitochondrial genome. Once a single basepair mutation has been identified in the mitochondrial DNA of a tumor of the patient, further tumor cells can be detected in tissue surrounding a resection or at other sites, if metastasis has occurred. Using the methods outlined above, the recurrence of the tumor or its incomplete removal can be assessed. Similarly, if a tumor has been treated using a non-surgical method such as chemotherapy or radiation, then the success of the therapy can be evaluated at later times by repeating the analysis. The step for determining the presence of a single basepair mutation in a mitochondrial genome of a cell sample of a patient can be performed 1, 2, 3, 4, 5, 6, 8, 10, or more times in order to monitor the development or regression of a tumor or to monitor the progress or lack of progress of therapy undertaken to eliminate the tumor.

[0046] Upon repeated analyses, the step for determining the presence of a single basepair mutation is simplified, because only a well defined and limited region of the genome need be sequenced. Using the hybridization method, for example, it is possible to evaluate the presence of the mutation with only a single matched/mismatched pair of oligonucleotide probes in the array. In the event that a mixture of genotypes is observed, it is possible to obtain quantitative information about the relative amount of each mitochondrial genotype using techniques known to the art, e.g., hybridization. Quantitative analysis can reveal changes in the relative proportion of tumor to normal cells in a tissue over time or in response to therapy.

[0047] The following examples are provided to demonstrate certain aspects of the invention but they do not define the scope of the invention.

EXAMPLE 1

[0048] This example demonstrates detection of mt mutations in tissue samples.

[0049] To determine whether mt mutations could be identified in cancer other than colorectal cancer, we studied primary bladder (n=14), head and neck (n=13), and lung (n=14) tumors (5). Eighty percent of the mt genome of all the primary tumor samples was PCR-amplified (6) and sequenced manually (FIG. 1). Tumor mtDNA was compared to mtDNA from paired blood samples in all cases, and mtDNA from corresponding normal tissue when available (7). Of the 292 sequence variants detected, 196 were previously recorded polymorphisms (2, 8), while 57 were novel polymorphisms (Table 3). The remaining 39 variants were acquired (somatic) mutations identified in 64% (9/14) of the bladder cancer, 46% (6/13) of the head and neck cancer, and 43% (6/14) of the lung cancer patients (Table 2). Most of these mutations were T-to-C and G-to-A base transitions, indicating possible exposure to ROS-derived mutagens (9). Similar to the previous observation by Polyak et al. (Table 1; 4), the majority of the somatic mutations identified here were also homoplasmic in nature. In addition, several of the bladder and head and neck cancers studied here (Table 2) had multiple mutations implying possible accumulation of mtDNA damage.

[0050] In the bladder tumors, mutation hot spots were primarily in the NADH dehydrogenase subunit 4 (ND4) gene (35%), and in the displacement-loop (D-loop) region (30%). The D-loop region is a critical site for both replication and expression of the mt genome since it contains the leading-strand origin of replication and the major promoters for transcription (10). Many (73%) of the mutations identified within protein-coding regions were silent, except for a (Val.fwdarw.Ala) substitution in the NADH dehydrogenase subunit 3 (ND3) and a 7-amino-acid deletion in cytochrome b (Cyt b). The D-loop region was also commonly mutated in head and neck cancer (67%). Two of the head and neck tumors (22%) contained mutations in the ND4 gene at nucleotide pairs (nps) 10822 and 11150, resulting in amino acid substitutions of Thr.fwdarw.Met and Ala.fwdarw.Thr, respectively. A similar tendency was observed in lung cancers, demonstrating a high concentration of mutations in the D-loop region (70%).

EXAMPLE 2

[0051] This example demonstrates detection of mt mutations in bodily fluids.

[0052] We hypothesized that the homoplasmic nature of these mutations would make them readily detectable in paired bodily fluids. To test this, we extracted and directly amplified mtDNA from urine samples from patients diagnosed with bladder cancer. All three corresponding urine samples available in this study contained the mutant mtDNA derived from tumor tissues. For example, the mtDNA from a urine sample of bladder cancer patient #799 showed the same nucleotide transition (G.fwdarw.A) as seen in the tumor (FIG. 2A). In all cases, the urine sample contained a relatively pure population of tumor-derived mtDNA, comparable to that of the micro-dissected tumor sample. Consistent with this observation, saliva samples obtained from head and neck cancer patients contained no detectable wild-type (WT) signals (FIGS. 2B, and 2C). By sequence analysis alone, we were able to detect mtDNA mutations in 67% (6/9) of saliva samples from head and neck cancer patients. In lung cancer cases, we were initially unable to identify mutant bands from paired bronchoalveolar lavage (BAL) fluids because of the significant dilution of neoplastic cells in BAL fluid (11), (FIG. 2D). We, thus, applied a more sensitive oligonucleotide-mismatch ligation assay to detect mutated mtDNA. As shown in FIGS. 3A and 3B, both lung cancer mutations (arrows) were confirmed in tumor mtDNA with more dilute signals in the corresponding BAL samples, and no signal in the corresponding normal tissues. Again, we detected the majority of mtDNA mutations (8/10) in BAL fluids with the exception of two cases where the ligation assays were not feasible due to the sequence compositions (16183 and 302 nps) adjacent to the mutations.

EXAMPLE 3

[0053] This example demonstrates the enrichment of mitochondrial mutant DNA in samples relative to nuclear mutant DNA.

[0054] To quantitate this neoplastic DNA enrichment, we compared the abundance of mt gene mutations to that of nuclear-encoded p53 mutations in bodily fluids using a quantitative plaque assay. Nuclear and mt fragments that contained a mutated sequence were PCR-amplified and cloned for plaque hybridization (12). Two BAL samples from lung cancer patients were chosen for analysis because they had mutations in both the mt and nuclear genomes. For p53 mutations, the percentages of neoplastic cells among normal cells for patients #1113 and #1140 were 0.1 and 3.0%, respectively. Remarkably, the abundance of the corresponding mutated mtDNA (MT) was 22% and 52% when compared to the wild-type (WT) mt sequence (FIG. 4). This enrichment of mtDNA is presumably due to the homoplasmic nature of these mutations and the high copy number of mt genomes in cancer cells. Enrichment was further suggested by our observations in head and neck paraffin samples where we were able to PCR-amplify 2-3 kb fragments of mtDNA, whereas we were unable to amplify nuclear p53 gene fragments of over 300 bp.

[0055] A role for mitochondria in tumorigenesis was implicated when tumor cells were found to have an impaired respiratory system and high glycolytic activity (13,14). Recent findings elucidating the role of mitochondria in apoptosis (15) and the high incidence of mtDNA mutations in colon cancer (4) further support the original hypothesis of mitochondrial participation in the initiation and progression of cancer. Although further investigation is needed to define the functional significance of mt mutations, our data clearly show that those mutations are frequent and present at high levels in all of the tumor types examined.

[0056] The homoplasmic nature of the mutated mitochondria remains puzzling. It is estimated that each cell contains several hundred-to-thousands of mitochondria and that each mitochondrion contains 1-10 genomes (16). Conceivably, certain mutated mtDNAs may gain a significant replicative advantage. For example, mutations in the D-loop regulatory region might alter the rate of DNA replication by modifying the binding affinity of important trans-acting factors. Mitochondria that undergo the most rapid replication are likely to acquire more DNA damage, leading to an accumulation of mutational events. Although the mechanism may vary for other mutations (such as silent mutations in the ND4 gene), the accumulation of a particular mtDNA mutation may become more apparent during neoplastic transformation. Even subtle mtDNA mutations may also gain significant replicative advantage, perhaps through interactions with important nuclear factors. Homoplasmic transformation of mtDNA was observed in small populations of cells in other non-neoplastic, but diseased tissues (17), sometimes associated with aging (18). We hypothesize that, in contrast to classic clonal expansion, the process may occur as "pseudoclonal" selection where stochastic segregation of mitochondria (16) together with neoplastic clonal expansion driven by nuclear mutations lead to a homogeneous population of a previously "altered" mitochondrion (FIG. 5).

[0057] The large number of mt polymorphisms identified here and elsewhere (2) likely reflects the high mutation rate of mtDNA, which is thought to be caused mainly by high levels of ROS (19). In agreement with this, our data imply that constitutive hypervariable areas such as the D-loop region represent somatic mutational hot spots. As further mutations are tabulated in primary tumors, DNA-chip technology can be harnessed to develop high-throughput analyses with sufficient sensitivity (20, 21). Due to its high copy number, mtDNA may provide a distinct advantage over other nuclear genome based methods for cancer and environmental pollutant detection.

LITERATURE CITED

[0058] 1. R. N. Lightowlers, P. F. Chinnery, D. M. Tumbull, N. Howell, Trends Genet 13, 450 (1997). [0059] 2. MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, Ga., USA. http://www.gen.emory.edu/mitomap.html [0060] 3. D. L. Croteau and V. A. Bohr, J. Biol. Chem. 272, 25409 (1997). [0061] 4. K. Polyak et al., Nature Genet. 20, 291 (1998). [0062] 5. Paired normal and tumor specimens along with blood and bodily fluids were collected following surgical resections with prior consent from patients in The Johns Hopkins University Hospital. Tumor specimens were frozen and micro-dissected on a cryostat so that the tumor samples contained greater than 70% neoplastic cells. DNA from tumor sections was digested with 1% SDS/Proteinase K, extracted by phenol-chloroform, and ethanol precipitated. Control DNA from peripheral lymphocytes, matched normal tissues, from urine, saliva, and BAL fluid were processed in the same manner as described in (11). [0063] 6. Mitochondrial DNAs were amplified using overlapping primers (4) in PCR buffer containing 6% DMSO. Approximately 5-20 ng of genomic DNA was subjected to the step-down PCR protocol: 94.degree. C. 30 sec, 64.degree. C. 1 min, 70.degree. C. 3 min, 3 cycles, 94.degree. C. 30 sec, 61.degree. C. 1 min, 70.degree. C. 3 min, 3 cycles, 94.degree. C. 30 sec, 58.degree. C. 1 min, 70.degree. C. 3.5 min 15 cycles, 94.degree. C. 30 sec, 57.degree. C. 1 min, 70.degree. C. 3.5 min, 15 cycles, and a final extension at 70.degree. C. for 5 min. PCR products were gel-purified using a Qiagen gel extraction kit (Qiagen) and sequence reactions were performed with Thermosequenase (Perkin-Elmer) using the cycle conditions (95.degree. C. 30 sec, 52.degree. C. 1 min, and 70.degree. C. 1 min for 25 cycles). [0064] 7. Corresponding normal tissues from 4 patients (#874, #915, #1684, and #1678) were available and DNA was extracted from paraffin samples as described previously (9). [0065] 8. R. M. Andrew et al., Nature Genet. 23 147, (1999) [0066] 9. J. Cadet, M. Berger, T. Douki, J. L. Ravanat, Rev. Physiol. Biochem. Pharmacol. 131, 1 (1997). [0067] 8. J. W. Taanman, Biochimica. et. Biophysica. Acta. 1410, 103 (1999). [0068] 9. S. A. Ahrendt et al., J. Natl. Cancer Inst. 91, 332 (1999). [0069] 10. Subcloning of PCR fragments into phage vector was performed according to the manufacturer's instructions (Stratagene). Titered plaques were plated and subjected to hybridization using tetramethylammonium chloride (TMAC) as a solvent. Positive signals were confirmed by secondary screenings. Oligonucleotides (Oligos) used for this assay were as follows; for patient #1113, p53 and mtDNA sequence alterations were detected using oligos containing either WT-(p53: 5'-GTATTTGGATGTCAGAAACACTT-3' (SEQ ID NO: 2)/mtDNA: 5'-ACTTCAGGGTCATAAAGCC-3'(SEQ ID NO: 3)) or MT (p53: 5'-GTATTTGGATGTCAGAAACACTT-3' (SEQ ID NO: 4)/mtDNA:5'-ACTTCAGGGCCATAAAGCC-3' (SEQ ID NO: 5)) sequences, respectively. For patient #1140, oligos 5'-ACCCGCGTCCGCGCCATGGCC-3' (SEQ ID NO: 6) and 5'-ACCCGCGTCCTCGCCATGGCC-3' (SEQ ID NO: 7) were used to detect WT and MT sequences, respectively. [0070] 11. O. Warburg, Science 123, 309 (1956). [0071] 12. J. W. Shay and H. Werbin, Mut. Res. 186, 149 (1987). [0072] 13. D. R. Green and J. C. Reed, Science 281, 1309 (1999). [0073] 14. D. C. Wallace, Annu. Rev. Biochem. 61, 1175 (1992). [0074] 15. D. C. Wallace, Proc. Natl. Acad. Sci. USA 91, 8746 (1994). [0075] 16. K. Khrapko et al, N. A. R. 27, 2434 (1999). [0076] 17. C. Richter, J. W. Park, B. N. Ames, Proc. Natl. Acad. Sci. USA 17, 6465 (1988). [0077] 18. M. Chee et al., Science 274, 610 (1996). [0078] 19. S. A. Ahrendt et al., Proc. Natl. Acad. Sci. USA 96, 7382 (1999). [0079] 20. Fragments containing mutations were PCR-amplified and then ethanol precipitated. For each mutation, discriminating oligonucleotides that contained the mutated base at the 3' end were designed (TAACCATA-3' (SEQ ID NO: 8) for patient #915 and TCTCTTACC-3' (SEQ ID NO: 9) for patient #898). Immediately adjacent [.sup.32P] end-labeled 3' sequences (5'-CACACTACTA-3' (SEQ ID NO: 10) for patient #915 and 5'-TTTAACCAG-3' (SEQ ID NO: 11) for patient #898) were used as substrate together with discriminating oligonucleotides for the ligation reaction. After a denaturing step of 95.degree. C. for 5', the reactions were incubated for 1 hr at 37.degree. in the presence of T4 DNA ligase (Life Technologies), in a buffer containing 50 mM Tris-Cl, 10 mM MgCl.sub.2, 150 mM NaCl, 1 mM Spermidine, 1 mM ATP, 5 mM DTT, and analyzed on denatured 12% polyacrylamide gels. [Jen et al., Cancer Res. 54, 5523 (1994)].

Sequence CWU 1

1

11116568DNAHomo sapiens 1gatcacaggt 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 atctacttca aattcctccc 3120tgtacgaaag gacaagagaa ataaggccta cttcacaaag cgccttcccc cgtaaatgat 3180atcatctcaa cttagtatta tacccacacc cacccaagaa cagggtttgt taagatggca 3240gagcccggta atcgcataaa acttaaaact ttacagtcag aggttcaatt cctcttctta 3300acaacatacc catggccaac ctcctactcc tcattgtacc cattctaatc gcaatggcat 3360tcctaatgct taccgaacga aaaattctag gctatataca actacgcaaa ggccccaacg 3420ttgtaggccc ctacgggcta ctacaaccct tcgctgacgc cataaaactc ttcaccaaag 3480agcccctaaa acccgccaca tctaccatca ccctctacat caccgccccg accttagctc 3540tcaccatcgc tcttctacta tgaacccccc tccccatacc caaccccctg gtcaacctca 3600acctaggcct cctatttatt ctagccacct ctagcctagc cgtttactca atcctctgat 3660cagggtgagc atcaaactca aactacgccc tgatcggcgc actgcgagca gtagcccaaa 3720caatctcata tgaagtcacc ctagccatca ttctactatc aacattacta ataagtggct 3780cctttaacct ctccaccctt atcacaacac aagaacacct ctgattactc ctgccatcat 3840gacccttggc cataatatga tttatctcca cactagcaga gaccaaccga acccccttcg 3900accttgccga aggggagtcc gaactagtct caggcttcaa catcgaatac gccgcaggcc 3960ccttcgccct attcttcata gccgaataca caaacattat tataataaac accctcacca 4020ctacaatctt cctaggaaca acatatgacg cactctcccc tgaactctac acaacatatt 4080ttgtcaccaa gaccctactt ctaacctccc tgttcttatg aattcgaaca gcataccccc 4140gattccgcta cgaccaactc atacacctcc tatgaaaaaa cttcctacca ctcaccctag 4200cattacttat atgatatgtc tccataccca ttacaatctc cagcattccc cctcaaacct 4260aagaaatatg tctgataaaa gagttacttt gatagagtaa ataataggag cttaaacccc 4320cttatttcta ggactatgag aatcgaaccc atccctgaga atccaaaatt ctccgtgcca 4380cctatcacac cccatcctaa agtaaggtca gctaaataag ctatcgggcc cataccccga 4440aaatgttggt tatacccttc ccgtactaat taatcccctg gcccaacccg tcatctactc 4500taccatcttt gcaggcacac tcatcacagc gctaagctcg cactgatttt ttacctgagt 4560aggcctagaa ataaacatgc tagcttttat tccagttcta accaaaaaaa taaaccctcg 4620ttccacagaa gctgccatca agtatttcct cacgcaagca accgcatcca taatccttct 4680aatagctatc ctcttcaaca atatactctc cggacaatga accataacca atactaccaa 4740tcaatactca tcattaataa tcataatagc tatagcaata aaactaggaa tagccccctt 4800tcacttctga gtcccagagg ttacccaagg cacccctctg acatccggcc tgcttcttct 4860cacatgacaa aaactagccc ccatctcaat catataccaa atctctccct cactaaacgt 4920aagccttctc ctcactctct caatcttatc catcatagca ggcagttgag gtggattaaa 4980ccaaacccag ctacgcaaaa tcttagcata ctcctcaatt acccacatag gatgaataat 5040agcagttcta ccgtacaacc ctaacataac cattcttaat ttaactattt atattatcct 5100aactactacc gcattcctac tactcaactt aaactccagc accacgaccc tactactatc 5160tcgcacctga aacaagctaa catgactaac acccttaatt ccatccaccc tcctctccct 5220aggaggcctg cccccgctaa ccggcttttt gcccaaatgg gccattatcg aagaattcac 5280aaaaaacaat agcctcatca tccccaccat catagccacc atcaccctcc ttaacctcta 5340cttctaccta cgcctaatct actccacctc aatcacacta ctccccatat ctaacaacgt 5400aaaaataaaa tgacagtttg aacatacaaa acccacccca ttcctcccca cactcatcgc 5460ccttaccacg ctactcctac ctatctcccc ttttatacta ataatcttat agaaatttag 5520gttaaataca gaccaagagc cttcaaagcc ctcagtaagt tgcaatactt aatttctgta 5580acagctaagg actgcaaaac cccactctgc atcaactgaa cgcaaatcag ccactttaat 5640taagctaagc ccttactaga ccaatgggac ttaaacccac aaacacttag ttaacagcta 5700agcaccctaa tcaactggct tcaatctact tctcccgccg ccgggaaaaa aggcgggaga 5760agccccggca ggtttgaagc tgcttcttcg aatttgcaat tcaatatgaa aatcacctcg 5820gagctggtaa aaagaggcct aacccctgtc tttagattta cagtccaatg cttcactcag 5880ccattttacc tcacccccac tgatgttcgc cgaccgttga ctattctcta caaaccacaa 5940agacattgga acactatacc tattattcgg cgcatgagct ggagtcctag gcacagctct 6000aagcctcctt attcgagccg agctgggcca gccaggcaac cttctaggta acgaccacat 6060ctacaacgtt atcgtcacag cccatgcatt tgtaataatc ttcttcatag taatacccat 6120cataatcgga ggctttggca actgactagt tcccctaata atcggtgccc ccgatatggc 6180gtttccccgc ataaacaaca taagcttctg actcttacct ccctctctcc tactcctgct 6240cgcatctgct atagtggagg ccggagcagg aacaggttga acagtctacc ctcccttagc 6300agggaactac tcccaccctg gagcctccgt agacctaacc atcttctcct tacacctagc 6360aggtgtctcc tctatcttag gggccatcaa tttcatcaca acaattatca atataaaacc 6420ccctgccata acccaatacc aaacgcccct cttcgtctga tccgtcctaa tcacagcagt 6480cctacttctc ctatctctcc cagtcctagc tgctggcatc actatactac taacagaccg 6540caacctcaac accaccttct tcgaccccgc cggaggagga gaccccattc tataccaaca 6600cctattctga tttttcggtc accctgaagt ttatattctt atcctaccag gcttcggaat 6660aatctcccat attgtaactt actactccgg aaaaaaagaa ccatttggat acataggtat 6720ggtctgagct atgatatcaa ttggcttcct agggtttatc gtgtgagcac accatatatt 6780tacagtagga atagacgtag acacacgagc atatttcacc tccgctacca taatcatcgc 6840tatccccacc ggcgtcaaag tatttagctg actcgccaca ctccacggaa gcaatatgaa 6900atgatctgct gcagtgctct gagccctagg attcatcttt cttttcaccg taggtggcct 6960gactggcatt gtattagcaa actcatcact agacatcgta ctacacgaca cgtactacgt 7020tgtagcccac ttccactatg tcctatcaat aggagctgta tttgccatca taggaggctt 7080cattcactga tttcccctat tctcaggcta caccctagac caaacctacg ccaaaatcca 7140tttcactatc atattcatcg gcgtaaatct aactttcttc ccacaacact ttctcggcct 7200atccggaatg ccccgacgtt actcggacta ccccgatgca tacaccacat gaaacatcct 7260atcatctgta ggctcattca tttctctaac agcagtaata ttaataattt tcatgatttg 7320agaagccttc gcttcgaagc gaaaagtcct aatagtagaa gaaccctcca taaacctgga 7380gtgactatat ggatgccccc caccctacca cacattcgaa gaacccgtat acataaaatc 7440tagacaaaaa aggaaggaat cgaacccccc aaagctggtt tcaagccaac cccatggcct 7500ccatgacttt ttcaaaaagg tattagaaaa accatttcat aactttgtca aagttaaatt 7560ataggctaaa tcctatatat cttaatggca catgcagcgc aagtaggtct acaagacgct 7620acttccccta tcatagaaga gcttatcacc tttcatgatc acgccctcat aatcattttc 7680cttatctgct tcctagtcct gtatgccctt ttcctaacac tcacaacaaa actaactaat 7740actaacatct cagacgctca ggaaatagaa accgtctgaa ctatcctgcc cgccatcatc 7800ctagtcctca tcgccctccc atccctacgc atcctttaca taacagacga ggtcaacgat 7860ccctccctta ccatcaaatc aattggccac caatggtact gaacctacga gtacaccgac 7920tacggcggac taatcttcaa ctcctacata cttcccccat tattcctaga accaggcgac 7980ctgcgactcc ttgacgttga caatcgagta gtactcccga ttgaagcccc cattcgtata 8040ataattacat cacaagacgt cttgcactca tgagctgtcc ccacattagg cttaaaaaca 8100gatgcaattc ccggacgtct aaaccaaacc actttcaccg ctacacgacc gggggtatac 8160tacggtcaat gctctgaaat ctgtggagca aaccacagtt tcatgcccat cgtcctagaa 8220ttaattcccc taaaaatctt tgaaataggg cccgtattta ccctatagca ccccctctac 8280cccctctaga gcccactgta aagctaactt agcattaacc ttttaagtta aagattaaga 8340gaaccaacac ctctttacag tgaaatgccc caactaaata ctaccgtatg gcccaccata 8400attaccccca tactccttac actattcctc atcacccaac taaaaatatt aaacacaaac 8460taccacctac ctccctcacc aaagcccata aaaataaaaa attataacaa accctgagaa 8520ccaaaatgaa cgaaaatctg ttcgcttcat tcattgcccc cacaatccta ggcctacccg 8580ccgcagtact gatcattcta tttccccctc tattgatccc cacctccaaa tatctcatca 8640acaaccgact aatcaccacc caacaatgac taatcaaact aacctcaaaa caaatgataa 8700ccatacacaa cactaaagga cgaacctgat ctcttatact agtatcctta atcattttta 8760ttgccacaac taacctcctc ggactcctgc ctcactcatt tacaccaacc acccaactat 8820ctataaacct agccatggcc atccccttat gagcgggcac agtgattata ggctttcgct 8880ctaagattaa aaatgcccta gcccacttct taccacaagg cacacctaca ccccttatcc 8940ccatactagt tattatcgaa accatcagcc tactcattca accaatagcc ctggccgtac 9000gcctaaccgc taacattact gcaggccacc tactcatgca cctaattgga agcgccaccc 9060tagcaatatc aaccattaac cttccctcta cacttatcat cttcacaatt ctaattctac 9120tgactatcct agaaatcgct gtcgccttaa tccaagccta cgttttcaca cttctagtaa 9180gcctctacct gcacgacaac acataatgac ccaccaatca catgcctatc atatagtaaa 9240acccagccca tgacccctaa caggggccct ctcagccctc ctaatgacct ccggcctagc 9300catgtgattt cacttccact ccataacgct cctcatacta ggcctactaa ccaacacact 9360aaccatatac caatgatggc gcgatgtaac acgagaaagc acataccaag gccaccacac 9420accacctgtc caaaaaggcc ttcgatacgg gataatccta tttattacct cagaagtttt 9480tttcttcgca ggatttttct gagcctttta ccactccagc ctagccccta ccccccaatt 9540aggagggcac tggcccccaa caggcatcac cccgctaaat cccctagaag tcccactcct 9600aaacacatcc gtattactcg catcaggagt atcaatcacc tgagctcacc atagtctaat 9660agaaaacaac cgaaaccaaa taattcaagc actgcttatt acaattttac tgggtctcta 9720ttttaccctc ctacaagcct cagagtactt cgagtctccc ttcaccattt ccgacggcat 9780ctacggctca acattttttg tagccacagg cttccacgga cttcacgtca ttattggctc 9840aactttcctc actatctgct tcatccgcca actaatattt cactttacat ccaaacatca 9900ctttggcttc gaagccgccg cctgatactg gcattttgta gatgtggttt gactatttct 9960gtatgtctcc atctattgat gagggtctta ctcttttagt ataaatagta ccgttaactt 10020ccaattaact agttttgaca acattcaaaa aagagtaata aacttcgcct taattttaat 10080aatcaacacc ctcctagcct tactactaat aattattaca ttttgactac cacaactcaa 10140cggctacata gaaaaatcca ccccttacga gtgcggcttc gaccctatat cccccgcccg 10200cgtccctttc tccataaaat tcttcttagt agctattacc ttcttattat ttgatctaga 10260aattgccctc cttttacccc taccatgagc cctacaaaca actaacctgc cactaatagt 10320tatgtcatcc ctcttattaa tcatcatcct agccctaagt ctggcctatg agtgactaca 10380aaaaggatta gactgaaccg aattggtata tagtttaaac aaaacgaatg atttcgactc 10440attaaattat gataatcata tttaccaaat gcccctcatt tacataaata ttatactagc 10500atttaccatc tcacttctag gaatactagt atatcgctca cacctcatat cctccctact 10560atgcctagaa ggaataatac tatcgctgtt cattatagct actctcataa ccctcaacac 10620ccactccctc ttagccaata ttgtgcctat tgccatacta gtctttgccg cctgcgaagc 10680agcggtgggc ctagccctac tagtctcaat ctccaacaca tatggcctag actacgtaca 10740taacctaaac ctactccaat gctaaaacta atcgtcccaa caattatatt actaccactg 10800acatgacttt ccaaaaaaca cataatttga atcaacacaa ccacccacag cctaattatt 10860agcatcatcc ctctactatt ttttaaccaa atcaacaaca acctatttag ctgttcccca 10920accttttcct ccgaccccct aacaaccccc ctcctaatac taactacctg actcctaccc 10980ctcacaatca tggcaagcca acgccactta tccagtgaac cactatcacg aaaaaaactc 11040tacctctcta tactaatctc cctacaaatc tccttaatta taacattcac agccacagaa 11100ctaatcatat tttatatctt cttcgaaacc acacttatcc ccaccttggc tatcatcacc 11160cgatgaggca accagccaga acgcctgaac gcaggcacat acttcctatt ctacacccta 11220gtaggctccc ttcccctact catcgcacta atttacactc acaacaccct aggctcacta 11280aacattctac tactcactct cactgcccaa gaactatcaa actcctgagc caacaactta 11340atatgactag cttacacaat agcttttata gtaaagatac ctctttacgg actccactta 11400tgactcccta aagcccatgt cgaagccccc atcgctgggt caatagtact tgccgcagta 11460ctcttaaaac taggcggcta tggtataata cgcctcacac tcattctcaa ccccctgaca 11520aaacacatag cctacccctt ccttgtacta tccctatgag gcataattat aacaagctcc 11580atctgcctac gacaaacaga cctaaaatcg ctcattgcat actcttcaat cagccacata 11640gccctcgtag taacagccat tctcatccaa accccctgaa gcttcaccgg cgcagtcatt 11700ctcataatcg cccacgggct tacatcctca ttactattct gcctagcaaa ctcaaactac 11760gaacgcactc acagtcgcat cataatcctc tctcaaggac ttcaaactct actcccacta 11820atagcttttt gatgacttct agcaagcctc gctaacctcg ccttaccccc cactattaac 11880ctactgggag aactctctgt gctagtaacc acgttctcct gatcaaatat cactctccta 11940cttacaggac tcaacatact agtcacagcc ctatactccc tctacatatt taccacaaca 12000caatggggct cactcaccca ccacattaac aacataaaac cctcattcac acgagaaaac 12060accctcatgt tcatacacct atcccccatt ctcctcctat ccctcaaccc cgacatcatt 12120accgggtttt cctcttgtaa atatagttta accaaaacat cagattgtga atctgacaac 12180agaggcttac gaccccttat ttaccgagaa agctcacaag aactgctaac tcatgccccc 12240atgtctaaca acatggcttt ctcaactttt aaaggataac agctatccat tggtcttagg 12300ccccaaaaat tttggtgcaa ctccaaataa aagtaataac catgcacact actataacca 12360ccctaaccct gacttcccta attcccccca tccttaccac cctcgttaac cctaacaaaa 12420aaaactcata cccccattat gtaaaatcca ttgtcgcatc cacctttatt atcagtctct 12480tccccacaac aatattcatg tgcctagacc aagaagttat tatctcgaac tgacactgag 12540ccacaaccca aacaacccag ctctccctaa gcttcaaact agactacttc tccataatat 12600tcatccctgt agcattgttc gttacatggt ccatcataga attctcactg tgatatataa 12660actcagaccc aaacattaat cagttcttca aatatctact catcttccta attaccatac 12720taatcttagt taccgctaac aacctattcc aactgttcat cggctgagag ggcgtaggaa 12780ttatatcctt cttgctcatc agttgatgat acgcccgagc agatgccaac acagcagcca 12840ttcaagcaat cctatacaac cgtatcggcg atatcggttt catcctcgcc ttagcatgat 12900ttatcctaca ctccaactca tgagacccac aacaaatagc ccttctaaac gctaatccaa 12960gcctcacccc actactaggc ctcctcctag cagcagcagg caaatcagcc caattaggtc 13020tccacccctg actcccctca gccatagaag gccccacccc agtctcagcc ctactccact 13080caagcactat agttgtagca ggaatcttct tactcatccg cttccacccc ctagcagaaa 13140atagcccact aatccaaact ctaacactat gcttaggcgc tatcaccact ctgttcgcag 13200cagtctgcgc ccttacacaa aatgacatca aaaaaatcgt agccttctcc acttcaagtc 13260aactaggact cataatagtt acaatcggca tcaaccaacc acacctagca ttcctgcaca 13320tctgtaccca cgccttcttc aaagccatac tatttatgtg ctccgggtcc atcatccaca 13380accttaacaa tgaacaagat attcgaaaaa taggaggact actcaaaacc atacctctca 13440cttcaacctc cctcaccatt ggcagcctag cattagcagg aatacctttc ctcacaggtt 13500tctactccaa agaccacatc atcgaaaccg caaacatatc atacacaaac gcctgagccc 13560tatctattac tctcatcgct acctccctga caagcgccta tagcactcga ataattcttc 13620tcaccctaac aggtcaacct cgcttcccca cccttactaa cattaacgaa aataacccca 13680ccctactaaa ccccattaaa cgcctggcag ccggaagcct attcgcagga tttctcatta 13740ctaacaacat ttcccccgca tcccccttcc aaacaacaat ccccctctac ctaaaactca 13800cagccctcgc tgtcactttc ctaggacttc taacagccct agacctcaac tacctaacca 13860acaaacttaa aataaaatcc ccactatgca cattttattt ctccaacata ctcggattct 13920accctagcat cacacaccgc acaatcccct atctaggcct tcttacgagc caaaacctgc 13980ccctactcct cctagaccta acctgactag aaaagctatt acctaaaaca atttcacagc 14040accaaatctc cacctccatc atcacctcaa cccaaaaagg cataattaaa ctttacttcc 14100tctctttctt cttcccactc atcctaaccc tactcctaat cacataacct attcccccga 14160gcaatctcaa ttacaatata tacaccaaca aacaatgttc aaccagtaac tactactaat 14220caacgcccat aatcatacaa agcccccgca ccaataggat cctcccgaat caaccctgac 14280ccctctcctt cataaattat tcagcttcct acactattaa agtttaccac aaccaccacc 14340ccatcatact ctttcaccca cagcaccaat cctacctcca tcgctaaccc cactaaaaca 14400ctcaccaaga cctcaacccc tgacccccat gcctcaggat actcctcaat agccatcgct 14460gtagtatatc caaagacaac catcattccc cctaaataaa ttaaaaaaac tattaaaccc 14520atataacctc ccccaaaatt cagaataata acacacccga ccacaccgct aacaatcaat 14580actaaacccc cataaatagg agaaggctta gaagaaaacc ccacaaaccc cattactaaa 14640cccacactca acagaaacaa agcatacatc attattctcg cacggactac aaccacgacc 14700aatgatatga aaaaccatcg ttgtatttca actacaagaa caccaatgac cccaatacgc 14760aaaactaacc ccctaataaa attaattaac cactcattca tcgacctccc caccccatcc 14820aacatctccg catgatgaaa cttcggctca ctccttggcg cctgcctgat cctccaaatc 14880accacaggac tattcctagc catgcactac tcaccagacg cctcaaccgc cttttcatca 14940atcgcccaca tcactcgaga cgtaaattat ggctgaatca tccgctacct tcacgccaat 15000ggcgcctcaa tattctttat ctgcctcttc ctacacatcg

ggcgaggcct atattacgga 15060tcatttctct actcagaaac ctgaaacatc ggcattatcc tcctgcttgc aactatagca 15120acagccttca taggctatgt cctcccgtga ggccaaatat cattctgagg ggccacagta 15180attacaaact tactatccgc catcccatac attgggacag acctagttca atgaatctga 15240ggaggctact cagtagacag tcccaccctc acacgattct ttacctttca cttcatcttg 15300cccttcatta ttgcagccct agcaacactc cacctcctat tcttgcacga aacgggatca 15360aacaaccccc taggaatcac ctcccattcc gataaaatca ccttccaccc ttactacaca 15420atcaaagacg ccctcggctt acttctcttc cttctctcct taatgacatt aacactattc 15480tcaccagacc tcctaggcga cccagacaat tataccctag ccaacccctt aaacacccct 15540ccccacatca agcccgaatg atatttccta ttcgcctaca caattctccg atccgtccct 15600aacaaactag gaggcgtcct tgccctatta ctatccatcc tcatcctagc aataatcccc 15660atcctccata tatccaaaca acaaagcata atatttcgcc cactaagcca atcactttat 15720tgactcctag ccgcagacct cctcattcta acctgaatcg gaggacaacc agtaagctac 15780ccttttacca tcattggaca agtagcatcc gtactatact tcacaacaat cctaatccta 15840ataccaacta tctccctaat tgaaaacaaa atactcaaat gggcctgtcc ttgtagtata 15900aactaataca ccagtcttgt aaaccggaga tgaaaacctt tttccaagga caaatcagag 15960aaaaagtctt taactccacc attagcaccc aaagctaaga ttctaattta aactattctc 16020tgttctttca tggggaagca gatttgggta ccacccaagt attgactcac ccatcaacaa 16080ccgctatgta tttcgtacat tactgccagc caccatgaat attgtacggt accataaata 16140cttgaccacc tgtagtacat aaaaacccaa tccacatcaa aaccccctcc ccatgcttac 16200aagcaagtac agcaatcaac cctcaactat cacacatcaa ctgcaactcc aaagccaccc 16260ctcacccact aggataccaa caaacctacc cacccttaac agtacatagt acataaagcc 16320atttaccgta catagcacat tacagtcaaa tcccttctcg tccccatgga tgacccccct 16380cagatagggg tcccttgacc accatcctcc gtgaaatcaa tatcccgcac aagagtgcta 16440ctctcctcgc tccgggccca taacacttgg gggtagctaa agtgaactgt atccgacatc 16500tggttcctac ttcagggtca taaagcctaa atagcccaca cgttcccctt aaataagaca 16560tcacgatg 16568223DNAHomo sapiens 2gtatttggat gtcagaaaca ctt 23319DNAHomo sapiens 3acttcagggt cataaagcc 19423DNAHomo sapiens 4gtatttggat gtcagaaaca ctt 23519DNAHomo sapiens 5acttcagggc cataaagcc 19621DNAHomo sapiens 6acccgcgtcc gcgccatggc c 21721DNAHomo sapiens 7acccgcgtcc tcgccatggc c 2188DNAHomo sapiens 8taaccata 899DNAHomo sapiens 9tctcttacc 91010DNAHomo sapiens 10cacactacta 10119DNAHomo sapiens 11tttaaccag 9

Claims

1. An oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome, wherein the oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at nucleotide 16380; G.fwdarw.A at nucleotide 75; insert C at nucleotide 302; insert C.fwdarw.G at nucleotide 514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA. 7 amino acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521; G.fwdarw.A at nucleotide 12345; G.fwdarw.A at nucleotide 3054; T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A substitution at position 6264; A insertion at position 12418; T.fwdarw.C substitution at position 1967; and T.fwdarw.A substitution at position 2299.

2. An oligonucleotide primer comprising a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome, wherein the oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T.fwdarw.C at nucleotide 114; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 386; insert T at nucleotide 16189; A.fwdarw.C at nucleotide 16265; A.fwdarw.T at nucleotide 16532; C.fwdarw.T at nucleotide 150; T.fwdarw.C at nucleotide 195; .DELTA.C at nucleotide 302; C.fwdarw.A at nucleotide 16183; C.fwdarw.T at nucleotide 16187; T.fwdarw.C at nucleotide 16519; G.fwdarw.A at nucleotide 16380; GA at nucleotide 75; insert C at nucleotide 302; insert C.fwdarw.G at nucleotide 514; T.fwdarw.C at nucleotide 16172; C.fwdarw.T at nucleotide 16292; A.fwdarw.G at nucleotide 16300; A.fwdarw.G at nucleotide 10792; C.fwdarw.T at nucleotide 10793; C.fwdarw.T at nucleotide 10822; A.fwdarw.G at nucleotide 10978; A.fwdarw.G at nucleotide 11065; G.fwdarw.A at nucleotide 11518; C.fwdarw.T at nucleotide 12049; T.fwdarw.C at nucleotide 10966; G.fwdarw.A at nucleotide 11150; G.fwdarw.A at nucleotide 2056; T.fwdarw.C at nucleotide 2445; T.fwdarw.C at nucleotide 2664; T.fwdarw.C at nucleotide 10071; T.fwdarw.C at nucleotide 10321; T.fwdarw.C at nucleotide 12519; .DELTA. 7 amino acids at nucleotide 15642; G.fwdarw.A at nucleotide 5521; G.fwdarw.A at nucleotide 12345; G.fwdarw.A at nucleotide 3054; T.fwdarw.C substitution at position 710; T.fwdarw.C substitution at position 1738; T.fwdarw.C substitution at position 3308; G.fwdarw.A substitution at position 8009; G.fwdarw.A substitution at position 14985; T.fwdarw.C substitution at position 15572; G.fwdarw.A substitution at position 9949; T.fwdarw.C substitution at position 10563; G.fwdarw.A substitution at position 6264; A insertion at position 12418; T.fwdarw.C substitution at position 1967; and T.fwdarw.A substitution at position 2299.

3.-57. (canceled)