Patent Publication Number: US-2005123913-A1

Title: Human mitochondrial dna polymorphisms, haplogroups, associations with physiological conditions, and genotyping arrays

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
      This application claims priority to U.S. Patent Application Ser. No. 60/316,333 filed Aug. 30, 2001 and Ser. No. 60/380,546 filed May 13, 2002, and to Canadian Patent Application No. 2,356,536 filed on Aug. 31, 2001, which are hereby incorporated in their entirety by reference to the extent not inconsistent with the disclosure herein. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
      This invention was made in part with funding from the United States Government (NIH grants AG13154, HL4017, NS21328, and NS37167). The United States Government may have certain rights therein. 
    
    
     BACKGROUND OF THE INVENTION  
      Human mitochondrial DNA (mtDNA) is maternally inherited. Mutations accumulate sequentially in radiating lineages creating branches on the human evolutionary tree. Using sequences of mtDNA, human populations are divisible evolutionarily into haplogroups (Wallace, D. C. et al. (1999)  Gene  238:211-230; Ingman M. et al., (2000)  Nature  408:708-713; Maca-Meyer, N. (August 2001)  BioMed Central  2:13; T. G. Schurr et al., (1999)  American Journal of Physical Anthropology  108:1-39; and V. Macaulay et al., (1999)  American Journal of Human Genetics  64:232-249). Related haplogroups can be combined into macro-haplogroups. Haplogroups can be subdivided into subhaplogroups. The complete Cambridge mitochondrial DNA sequence may be found at MITOMAP, http://www.gen.emory.edu/cgi-gin/MITOMAP, Genbank accession no. J01415, and is provided in SEQ ID NO:2. Also see Andrews et al. (1999), “Reanalysis and Revision of the Cambridge Reference Sequence for Human Mitochondrial DNA,”  Nature Genetics  23:147.  
      Publications on the subject of mitochondrial biology include: Scheffler, I. E. (1999)  Mitochondria , Wiley-Liss, NY; Lestienne P Ed.;  Mitochondrial Diseases: Models and Methods , Springer-Verlag, Berlin;  Methods in Enzymology  (2000) 322: Section V Mitochondria and Apoptosis, Academic Press, CA;  Mitochondria and Cell Death  (1999) Princeton University Press, NJ; Papa S, Ferruciio G, and Tager J Eds.;  Frontiers of Cellular Bioenergetics: Molecular Biology, Biochemistry, and Physiopathology , Kluwer Academic/Plenum Publishers, NY; Lemasters, J. and Nieminen, A. (2001)  Mitochondria in Pathogenesis , Kluwer Academic/Plenum Publishers, NY; MITOMAP, http://www.gen.emory.edu/cgi-gin/MITOMAP; Wallace, D. C. (2001) “A mitochondrial paradigm for degenerative diseases and ageing” Novartis Foundation Symposium 235:247-266; Wallace, D. C. (1997) “Mitochondrial DNA in Aging and Disease”  Scientific American  August 277:40-47; Wallace, D. C. et al., (1998) “Mitochondrial biology, degenerative diseases and aging,” BioFactors 7:187-190; Heddi, A. et al., (1999) “Coordinate Induction of Energy Gene Expression in Tissues of Mitochondrial Disease Patients”  JBC  274:22968-22976; Wallace, D. C. (1999) “Mitochondrial Diseases in Man and Mouse”  Science  283:1482-1488; Saraste, M. (1999) “Oxidative Phosphorylation at the fin de siecle”  Science  283:1488-1493; Kokoszka et. al. (2001) “Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis”  PNAS  98:2278-2283; Wallace, D. C. (2001)  Mental Retardation and Developmental Disabilities  7:158-166; Wallace, D. C. (2001)  Am. J. Med. Gen.  106:71-93; Wei, Y-H et al. (2001)  Chinese Medical Journal  (Taipei) 64:259-270; and Wallace, D. C. (2001)  EuroMit  5 Abstract.  
      Certain mitochondrial mutations have been associated with physiological conditions (U.S. Pat. No. 6,280,966 issued on Aug. 28, 2001; U.S. Pat. No. 6,140,067 issued on Oct. 31, 2000; U.S. Pat. No. 5,670,320; U.S. Pat. No. 5,296,349; U.S. Pat. No. 5,185,244; U.S. Pat. No. 5,494,794; Wallace, D. C. (1999)  Science  283:1482-1488; Brown, M. D. et al. (2001)  American Society for Human Genetics Poster # 2332; Brown, M. D. et al., (2001)  Human Genet.  109:33-39; and Brown, M. D. et al. (January 2002)  Human Genet.  110:130-138), Wallace, D. C. et al. (1999)  Gene  238:211-230 describes analysis of LHON mutants. Grossman, L. I. et al. (2001)  Molecular Phylogenetics and Evolution  18(1):26-36, describes changes in the biochemical machinery for aerobic energy metabolism. Kalman, B. et al. (1999)  Acta Neurol. Scand.  99(1): 16-25 describes mitochondrial mutations and multiple sclerosis (MS). Wei, Y. H. et al. (2001)  Chinese Medical Journal  64:259-270 describes recent results in support of the mitochondrial theory of aging.  
      Ivanova, R. et al. (1998)  Geronotology  44:349 describes mitochondrial haplotypes and longevity in a French population. Tanaka, M. et al. (1998)  Lancet  351:185-186 describes longevity and haplogroups in a Japanese population. De Benedictis, G. et al. (1999)  FASEB  13:1532-1536 describes haplogroups and longevity in an Italian population. Rose, G. et al. (2001)  European Journal of Human Genetics  9:701-707 describes haplogroup J in centenarians. Ross, O. A. et al. (2001)  Experimental Gerontology  36(7):1161-1178 describes haplotypes and longevity in an Irish population.  
      Haplogroup T has been associated with reduced sperm motility in European males (E. Ruiz-Pesini et al., [2000 ] American Journal of Human Genetics  67:682-696), the tRNA Gln np 4336 variant in haplogroup H is associated with late-onset Alzheimer Disease (J. M. Shoffner et al., [1993 ] Genomics  17:171-184).  
      Taylor, R. W. (1997)  J. of Bioenergetics and Biomembranes  29(2):195-205 describes methods for treating mitochondrial disease. Collombet, J. and Coutelle, C. (1998)  Molecular Medicine Today  4(1):1-8 describes gene therapy for mitochondrial disorders, including using cell fusion to introduce healthy mitochondria. Owen, R. and Flotte, T. R. (2001)  Antioxidants and Redox Signaling  3(3):451-460 discuss approaches and limitations to gene therapy for mitochondrial diseases.  
      Human mitochondrial DNA sequence variation, except that which has been associated with particular diseases, has not been associated with specific phenotypic conditions, has been considered neutral, and has been used to reconstruct human phylogenies (Henry Gee, “Statistical Cloud over African Eden,” (13 Feb. 1992)  Nature  355:583; Marcia Barinaga, “African Eve Backers Beat a Retreat,” (7 Feb. 1992)  Science,  255:687; S. Blair Hedges et al., “Human Origins and Analysis of Mitochondrial DNA Sequences,” (7 Feb. 1992)  Science,  255:737-739; Allan C. Wilson and Rebecca L. Cann, “The Recent African Genesis of Humans,” (April 1992)  Scientific American,  68). The average number of base pair differences between two human mitochondrial genomes is estimated to be from 9.5 to 66 (Zeviani M. et al. (1998) “Reviews in molecular medicine: Mitochondrial disorders,”  Medicine  77:59-72).  
      The D-loop is the most variable region in the mitochondrial genome, and the most polymorphic nucleotide sites within this loop are concentrated in two ‘hypervariable segments’, HVS-I and HVS-II (Wilkinson-Herbots, H. M. et al., (1996) “Site 73 in hypervariable region II of the human mitochondrial genome and the origin of European populations,”  Ann Hum Genet  60:499-508). Population-specific, neutral mtDNA variants have been identified by surveying mtDNA restriction site variants or by sequencing hypervariable segments in the displacement loop. Restriction analysis using fourteen restriction endonucleases allowed screening of 15-20% of the mtDNA sequence for variations (Chen Y. S. et al., (1995) “Analysis of mtDNA variation in African populations reveals the most ancient of all human continent-specific haplogroups,”  Am J Hum Genet  57:133-149). The large majority of mtDNA sequence data published to date are limited to HVS-I. Bandelt, H. J. et al., (1995) “Mitochondrial portraits of human populations using median networks”  Genetics  141:743-753).  
      The coding and classification system that has been used for mtDNA haplogroups refers primarily to the information provided by RFLPs and the hypervariable segments of the control region. (Torroni, A. et al. (1996) “Classification of European mtDNAs from an analysis of three European populations,”  Genetics  144:1835-1850 and Richards M B et al., (1998) “Phylogeography of mitochondrial DNA in western Europe,”  Ann Hum Genet  62:241-260.)  
      Methods are known for testing the likelihood of neutrality of mutations (Tajima, F. (1989)  Genetics  123:585-595; Fu, Y. and Li, W. (1993)  Genetics  133:693-709; Li, W. et al. (1985)  Mol. Biol. Evol.  2(2):150-174; and Nei, M. and Gojobori, T. (198.6)  Mol. Biol. Evol.  3(5):418-426). All of the methods in these publications are used to compare datasets taken from separate groups. None of these methods are used to analyze a dataset not containing data representing an outgroup.  
      Wise, C. A. et al. (1998)  Genetics  148:409-421, describes neutrality analysis of the human mitochondrial NADH Dehydrogenase Subunit 2 gene, when compared to the NADH Dehydrogenase Subunit 2 gene from chimpanzees. Templeton, A. R. (1996)  Genetics  144:1263-1270, describes neutrality analysis of the human mitochondrial Cytochrome Oxidase II (COXII) gene when compared to the COXII gene in hominoid primates. Messier, W. and Stewart, C. (1997)  Nature  385:151-154 describes neutrality analysis of primate lysozymes. Endo, T. et al. (1996)  Mol. Biol. Evol.  13(5):685-690 describes large-scale neutrality analysis of sequences from DDBJ, EMBL, and GenBank databases. Hughes, A. L. and Nei, M. (1988)  Nature  335:167-170 describes neutrality analysis of MC Class I loci. Nachman, M. W. (1996)  Genetics  142:953-963 describes neutrality analysis of the human mitochondrial NADH Dehydrogenase subunit 3 (NADH3) gene, when compared to the NADH Dehydrogenase subunit 3 gene from chimpanzees. Nachman, M. W. et al. (1994)  Proc. Nat. Acad. Sci. USA  76:5269-5273 describes neutrality analysis of the mitochondrial NADH dehydrogenase subunit 3 gene in 3 strains of mouse. Rand, D. M. et al. (1994)  Genetics  138:741-756; Ballard, J. W. O. and Kreitman, M. (1994)  Genetics  138:757-772; and Kaneko, M. Y. et al. (1993)  Genet. Res.  61:195-204, describe neutrality analysis for mitochondrial NADH dehydrogenase subunit 5, Cytochrome b, and ATPase6 in strains of  Drosophila.    
      In the above-mentioned publications, neutrality testing, including K a /K s  analysis, has not been applied for the purpose of identifying disease-associated mutations. Populations for neutrality testing analysis were identified by observation of normal phenotypic variation. Neutrality testing has been performed to determine whether a gene is under selection. None of these publications describe neutrality analysis with the purpose of identifying phenotype-associated mutations, and no suspected phenotype-associated mutations were identified.  
      U.S. Pat. No. 6,228,586 (issued May 8, 2001) and U.S. Pat. No. 6,280,953 (issued Aug. 28, 2001) describe methods for identifying polynucleotide and polypeptide sequences in human and/or non-human primates, which may be associated with a physiological condition. The methods employ comparison of human and non-human primate sequences using statistical methods. U.S. Pat. No. 6,274,319 (issued Aug. 14, 2001) describes K a /K s  methods for identifying polynucleotide and polypeptide sequences that may be associated with commercially or aesthetically relevant traits in domesticated plants or animals. The methods employ comparison of homologous genes from the domesticated organism and its wild ancestor to identify evolutionarily significant changes. In the above-mentioned publications, neutrality testing, including K a /K s  analysis, is only applied to interspecific, not intraspecific, comparisons, and only genes from the nuclear genome, not from organelle genomes, are analyzed.  
      Methods for constructing peptide and nucleotide libraries are well known to the art, e.g. as described in U.S. Pat. Nos. 6,156,511 and 6,130,092. Sequencing methods are also known to the art, e.g., as described in U.S. Pat. No. 6,087,095. Arrays of nucleic acid have been used for sequencing and for identifying exceptional alleles including disease-associated alleles. Nucleic acid arrays have been described, e.g., in patent nos.: U.S. Pat. Nos. 5,837,832, 5,807,522, 6,007,987, 6,110,426, WO 99/05324, 99/05591, WO 00/58516, WO 95/11995, WO 95/35505A1, WO 99/42813, JP10503841T2, GR3030430T3, ES2134481T3, EP804731B1, DE69509925C0, CA2192095AA, AU2862995A1, AU709276B2, AT180570, EP 1066506, and AU 2780499. Computational methods are useful for analyzing hybridization results, e.g., as described in PCT Publication WO 99/05574, and U.S. Pat. Nos. 5,754,524; 6228,575; 5,593,839; and 5,856,101. Methods for screening for disease markers are also known to the art, e.g. as described in U.S. Pat. Nos. 6,228,586; 6,160,104; 6,083,698; 6,268,398; 6,228,578; and 6,265,174.  
      The development of microarray technologies has stemmed from the desire to examine very large numbers of nucleic acid probe sequences simultaneously, in an effort to obtain information about genetic mutations, gene expression or nucleic acid sequences. Microarray technologies are intimately connected with the Human Genome Project, which has development of rapid methods of nucleic acid sequencing and genome analysis as key objectives (E. Marshall, (1995)  Science  268:1270), as well as elucidation of sequence-function relationships (M. Schena et al., (1996)  Proc. Nat&#39;l. Acad. Sci. USA,  93:10614). Microarray hybridization of PCR-amplified fragments to allele-specific oligonucleotide (ASO) probes is widely used in large-scale single nucleotide polymorphism (SNP) genotyping (Huber M. et al. (2002)  Analytical Biochemistry  303:25-33 and Southern, E. M. (1996)  Trends Genet.  12:110-115).  
      The Affymetrix GeneChip® HuSNP™ Array enables whole-genome surveys by simultaneously tracking nearly 1,500 genetic variations, known as single nucleotide polymorphisms (SNPs), dispersed throughout the genome. The HuSNP Affymetrix Array is being used for familial linkage studies that aim to map inherited disease or drug susceptibilities as well as for tracking de novo genetic alterations. For genotyping, arrays rely on multiple probes to interrogate individual nucleotides in a sequence. The identity of a target base can be deduced using four identical probes that vary only in the target position, each containing one of the four possible bases. Alternatively, the presence of a consensus sequence can be tested using one or two probes representing specific alleles. To genotype heterozygous or genetically mixed samples, arrays with many probes can be created to provide redundant information.  
      Arrays, also called DNA microarrays or DNA chips, are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes (Phimister, B. (1999)  Nature Genetics  21 s: 1-60) with known identity are used to determine complementary binding. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously. There are several steps in the design and implementation of a DNA array experiment. Many strategies have been investigated at each of these steps: 1) DNA types; 2) Chip fabrication; 3) Sample preparation; 4) Assay; 5) Readout; and 6) Software (informatics).  
      There are two major application forms for the array technology: 1) Determination of expression level (abundance) of genes; and 2) Identification of sequence (gene/gene mutation). There appear to be two variants of the array technology, in terms of intellectual property, of arrayed DNA sequence with known identity: Format I consists of probe cDNA (500˜5,000 bases long) immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, “traditionally” called DNA microarray, is widely considered as having been developed at Stanford University. (R. Ekins and F. W. Chu “Microarrays: their origins and applications,” [1999 ] Trends in Biotechnology,  17:217-218). Format II consists of an array of oligonucleotide (20˜80-mer oligos) or peptide nucleic acid (PNA) probes synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences is determined. This method, “historically” called DNA chips, was developed at Affymetrix, Inc., which sells its photolithographically fabricated products under the GeneChip® trademark. Many companies are manufacturing oligonucleotide-based chips using alternative in-situ synthesis or depositioning technologies.  
      Probes on arrays can be hybridized with fluorescently-labeled target polynucleotides and the hybridized array can be scanned by means of scanning fluorescence microscopy. The fluorescence patterns are then analyzed by an algorithm that determines the extent of mismatch content identifies polymorphisms, and provides some general sequencing information (M. Chee et al., [1996 ] Science  274:610). Selectivity is afforded in this system by low stringency washes to rinse away non-selectively adsorbed materials. Subsequent analysis of relative binding signals from array elements determines where base-pair mismatches may exist. This method then relies on conventional chemical methods to maximize stringency, and automated pattern recognition processing is used to discriminate between fully complementary and partially complementary binding.  
      Devices such as standard nucleic acid microarrays or gene chips, require data processing algorithms and the use of sample redundancy (i.e., many of the same types of array elements for statistically significant data interpretation and avoidance of anomalies) to provide semi-quantitative analysis of polymorphisms or levels of mismatch between the target sequence and sequences immobilized on the device surface.  
      Labels appropriate for array analysis are known in the art. Examples are the two-color fluorescent systems, such as Cy3/Cy5 and Cy3.5/Cy5.5 phosphoramidites (Glen Research, Sterling Va.). Patents covering cyanine dyes include: U.S. Pat. No. 6,114,350 (Sep. 5, 2000); U.S. Pat. No. 6,197,956 (Mar. 6, 2001); U.S. Pat. No. 6,204,389 (Mar. 20, 2001) and U.S. Pat. No. 6,224,644 (May 1, 2001). Array printers and readers are available in the art.  
      A process of using arrays is described in Grigorenko, E. V. ed., (2002)  DNA Arrays: Technologies and Experimental Strategies , CRC Press, NY; Vrana, K. E. et al., (May 2001) Microarrays and Related Technologies: Miniaturization and Acceleration of Genomics Research, CHI, Upper Falls, Mass.; and Branca, M. A. et al., (February 2002) DNA Microarray Informatics: Key Technological Trends and Commercial Opportunities, CHI, Upper Falls, Mass.  
      All publications referred to herein are incorporated by reference to the extent not inconsistent herewith. The mention of a publication in this Background Section does not constitute an admission that it is prior art.  
     SUMMARY OF INVENTION  
      The high mitochondrial DNA mutation rate of human mitochondrial DNA has been thought to result in the accumulation of a wide range of neutral, population-specific base substitutions in mtDNA. These have accumulated sequentially along radiating maternal lineages that have diverged approximately on the same time scale as human populations have colonized different geographical regions of the world.  
      About 76% of all African mtDNAs fall into haplogroup L, defined by an HpaI restriction site gain at bp 3592.77% of Asian mtDNAs are encompassed within a super-haplogroup defined by a DdeI site gain at bp 10394 and an AluI site gain at bp 10397. Essentially all native American mtDNAs fall into four haplogroups, A-D. Haplogroup A is defined by a HaeIII site gain at bp 663, B by a 9 bp deletion between bp 8271 to bp 8281, C by a HincII site loss at bp 13259, and D defined by an AluI site loss at bp 5176. Ten haplogroups encompass almost all mtDNAs in European populations. The ten-mtDNA haplogroups of Europeans can be surveyed by using a combination of data from RFLP analysis of the coding region and sequencing of the hypervariable segment I. About 99% of European mtDNAs fall into one of ten haplogroups: H, I, J, K, M, T, U, V, W or X.  
      This invention provides human mtDNA polymorphisms that are diagnostic of all the major human haplogroups and methods of diagnosing those haplogroups and selected sub-haplogroups.  
      This invention also provides methods for identifying evolutionarily significant mitochondrial DNA genes, nucleotide alleles, and amino acid alleles. Evolutionarily significant genes and alleles are identified using one or two populations of a single species. The process of identifying evolutionarily significant nucleotide alleles involves identifying evolutionarily significant genes and then evolutionarily significant nucleotide alleles in those genes, and identifying evolutionarily significant amino acid alleles involves identifying amino acids encoded by all nonsynonymous alleles. Synonymous codings of the nucleotide alleles encoding evolutionarily significant amino acid alleles of this invention are equivalent to the evolutionarily significant amino acid alleles disclosed herein and are included within the scope of this invention. Synonymous codings include alleles at neighboring nucleotide loci that are within the same codon.  
      This invention also provides methods for associating haplogroups and evolutionarily significant nucleotide and amino acid alleles with predispositions to physiological conditions. Methods for diagnosing predisposition to LHON, and methods for diagnosing increased likelihood of developing blindness, centenaria, and increased longevity that are not dependent on the geographical location of the individual being diagnosed are provided herein. Diagnosis of an individual with a predisposition to an energy metabolism-related physiological condition is dependent on the geographic region of the individual. Physiological conditions diagnosable by the methods of this invention include healthy conditions and pathological conditions. Physiological conditions that are associated with haplogroups and with alleles provided by this invention include energetic imbalance, metabolic disease, abnormal energy metabolism, abnormal temperature regulation, abnormal oxidative phosphorylation, abnormal electron transport, obesity, amount of body fat, diabetes, hypertension, and cardiovascular disease.  
      Molecules having sequences provided by this invention are provided in libraries and on genotyping arrays. This invention provides methods of making and using the genotyping arrays of this invention. The arrays of this invention are useful for determining the presence and absence of nucleotide alleles of this invention, for determining a haplogroup, and for diagnosis.  
      This invention also provides machine-readable storage devices and program devices for storing data and programmed methods for diagnosing haplogroups and physiological conditions.  
      The arrays of this invention are useful for determining the presence and absence of nucleotide alleles of this invention, for determining a haplogroup, and for diagnosis. This invention also provides machine-readable storage devices and program devices for storing data and programmed methods for diagnosing haplogroups and physiological conditions. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       FIG. 1  shows a consensus neighbor-joining tree of 104 human mtDNA complete sequences and two primate sequences. Numbers correspond to bootstrap values (% of 500 total bootstrap replicates) (Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) 3.53c. Distributed by author, Department of Genetics, University of Washington, Seattle, Wash.). Maximum Likelihood (ML) and UPGMA yielded consistent branching orders with respect to continent-specific mtDNA haplogroups. Sequences: 11-53: Genbank AF346963-AF347015 (4); E21U: Genbank X93334, A1L1a: Genbank D38112, cam revise: Genbank NC — 001807 corrected according to (R. M. Andrews et al., Nature Genetics 23, 147 (1999)); the rest are 48 sequences generated in this invention using an ABI 377. Specific mutations in patient samples that have been implicated in disease were excluded from this analysis, as well as gaps and deletions, with the exception of the 9 bp deletion (nucleotide position (np) 8272 to 8281). Haplogroups A, B, C, D, and X were drawn from both Eurasia and the Americas. Haplogroup names are designated with capital letters.  P. paniscus  and  P. troglodytes  mtDNA sequences were used as outgroups. Haplogroups L0 and L1 encompass previously assigned L1a and L1b mtDNAs, respectively (Y. S. Chen et al., American Journal of Human Genetics 66, 1362-1383 (2000)).  
       FIG. 2  shows the migrations of human haplogroups around the world. +/−, +/+, or −/− equals Dde I 10394 and Alu I 10397. * equals Rsa I 16329. The mutation rate is 2.2-2.9% per million years. Time estimates are YBP (years before present).  
       FIG. 3  shows a cladogram listing nucleotide alleles describing 21 major human haplogroups, 21 sub-haplogroups, and several macro-haplogroups. The groups on the left are described by the alleles to their right. A vertical bar designates that each group to the left of the bar has all of the alleles to the right of the bar.  
       FIG. 4  shows the selective constraint (k C  values) of mtDNA protein genes with comparisons among mammalian species. Statistical significance (P&lt;0.05) was determined using ANOVA, t-tests or the Tukey-Kramer Multiple Comparisons tests. Most programs used are from DNAsp (J. Rozas and R. Rozas, (1999)  Bioinformatics  15:174-5). DNA sequence divergence was analyzed using the DIVERGE program (Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.). For all thirteen mtDNA genes, data is shown for human, human compared to P. troglodytes, human compared to  P. paniscus , and nine species of primates. For only ATP6 and ATP8, data is also shown for fourteen species of mammals. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Table 1 shows human mitochondrial nucleotide alleles, which have been associated with physiological conditions. In Table 1, columns three (nucleotide locus), five (physiological condition nucleotide allele), and column two (physiological condition) make up the set of Human Mitochondrial Nucleotide Alleles Known to be Associated with Physiological Conditions.  
               TABLE 1 1                            Human Mitochondrial Alleles Known to be Associated with Physiological Conditions                                                         Physiological       Physiological                   Cambridge   Condition   Cambridge   Condition               Nucleotide   Nucleotide   Nucleotide   Amino Acid   Amino Acid       Gene   Physiological Condition   Locus   Allele   Allele   Allele   Allele                                                 MTND1   *MELAS   3308   T   C   M   T       MTND1   *NIDDM; LHON; PEO   3316   G   A   A   T       MTND1   *LHON   3394   T   C   Y   H       MTND1   *NIDDM   3394   T   C   Y   H       MTND1   *ADPD   3397   A   G   M   V       MTND1   *LHON   3460   G   A   A   T       MTND1   *LHON   3496   G   T   A   S       MTND1   *LHON   3497   C   T   A   V       MTND1   *LHON   4136   A   G   Y   C       MTND1   *LHON   4160   T   C   L   P       MTND1   *LHON   4216   T   C   Y   H       MTND2   *LHON   4917   A   G   D   N       MTND2   *LHON   5244   G   A   G   S       MTND2   *AD   5460   G   A   A   T       MTND2   *AD   5460   G   T   A   S       MTCO1   *Myoglobinuria, Exercise Intolerance   5920   G   A   W   Ter       MTCO1   *Multisystem Disorder   6930   G   A   G   Ter       MTCO1   *LHON   7444   G   A   Ter   K       MTCO2   *Mitochondrial Encephalomyopathy   7587   T   C   M   T       MTCO2   *MM   7671   T   A   M   K       MTCO2   *Multisystem Disorder   7896   G   A   W   Ter       MTCO2   *Lactic Acidosis   8042   AT   2 nt del (AT)   M   Ter       MTATP6   *NARP   8993   T   G   L   R       MTATP6   *NARP/Leigh Disease   8993   T   C   L   P       MTATP6   *LHON   9101   T   C   I   T       MTATP6   *FBSN/Leigh Disease   9176   T   C   L   P       MTATP6   *Leigh Disease   9176   T   G   L   R       MTCO3   *LHON   9438   G   A   G   S       MTCO3   *Leigh-like   9537   C   C ins   Q   frameshift       MTCO3   *LHON   9738   G   T   A   S       MTCO3   *LHON   9804   G   A   A   T       MTCO3   *Mitochondrial Encephalopathy   9952   G   A   W   Ter       MTCO3   *PEM; MELAS   9957   T   C   F   L       MTND3   *ESOC   10191   T   C   S   P       MTND4   *MELAS   11084   A   G   T   A       MTND4   *LHON   11778   G   A   R   H       MTND4   *Exercise Intolerance   11832   G   A   W   Ter       MTND4   *DM   12026   A   G   I   V       MTND5   *MELAS   13513   G   A   D   N       MTND5   *MELAS   13514   A   G   D   G       MTND5   *LHON-like   13528   A   G   T   A       MTND5   *LHON   13708   G   A   A   T       MTND5   *LHON   13730   G   A   G   E       MTND6   *MELAS   14453   G   A   A   V       MTND6   *LDYT   14459   G   A   A   V       MTND6   *LHON   14484   T   C   M   V       MTND6   *LHON   14495   A   G   L   S       MTND6   *LHON   14568   C   T   G   S       MTCYB   *PD/MELAS   14787   TTAA   4 nt del   I   frameshift                       (TTAA)       MTCYB   *MM   15059   G   A   G   Ter       MTCYB   *Exercise Intolerance   15150   G   A   W   Ter       MTCYB   *Exercise Intolerance   15197   T   C   S   P       MTCYB   *Mitochondrial Encephalomyopathy   15242   G   A   G   Ter       MTCYB   *LHON   15257   G   A   D   N       MTCYB   *Exercise Intolerance   15615   G   A   G   D       MTCYB   *MM   15762   G   A   G   E       MTCYB   *LHON   15812   G   A   V   M                   1 (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA, USA. http://www.gen.emory.edu/mitomap.html, 2001).            *Definitions:            LHON Leber Hereditary Optic Neuropathy            MM Mitochondrial Myopathy            AD Alzheimer&#39;s Disease            LIMM Lethal Infantile Mitochondrial Myopathy            ADPD Alzheimer&#39;s Disease and Parkinson&#39;s Disease            MMC Maternal Myopathy and Cardiomyopathy            NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; alternate phenotype at this locus is reported as Leigh Disease            FICP Fatal Infantile Cardiomyopathy Plus a MELAS-associated Cardiomyopathy            MELAS Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes LDYT Leber&#39;s hereditary optic neuropathy and DYsTonia            MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers            MHCM Maternally inherited Hypertrophic CardioMyopathy            CPEO Chronic Progressive External Ophthalmoplegia            KSS Kearns Sayre Syndrome            DM Diabetes Mellitus            DMDF Diabetes Mellitus + DeaFness            CIPO Chronic Intestinal Pseudoobstructton with myopathy and Ophthalmoplegia            DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness            PEM Progressive encephalopathy            SNHL SensoriNeural Hearing Loss             
 
      Thirteen protein-coding mitochondrial genes are known (MitoMap, http://www.gen.emory.edu/cgi-bin/MITOMAP).  
               TABLE 2                          Protein-coding Human MtDNA Genes                             Gene   Map Locus a     Abbreviation   Location b                 NADH dehydrogenase 1   MTND1   ND1   3307-4262       NADH dehydrogenase 2   MTND2   ND2   4470-5511       NADH dehydrogenase 3   MTND3   ND3   10059-10404       NADH dehydrogenase 4L   MTND4L   ND4L   10470-10766       NADH dehydrogenase 4   MTND4   ND4   10760-12137       NADH dehydrogenase 5   MTND5   ND5   12337-14148       NADH dehydrogenase 6   MTND6   ND6   14149-14673       Cytochrome b   MTCYB   Cytb   14747-15887       Cytochrome c oxidase I   MTCO1   COI   5904-7445       Cytochrome c oxidase II   MTCO2   COII   7586-8269       Cytochrome c oxidase III   MTCO3   COIII   9207-9990       ATP synthase 6   MTATP6   ATP6   8527-9207       ATP synthase 8   MTATP8   ATP8   8366-8572                   a,b As defined on MitoMap, http://www.gen.emory.edu/cgi-bin/MITOMAP, which is numbered relative to the Cambridge Sequence (Genbank accession no. J01415 and Andrews et al. (1999), A Reanalysis and Revision of the Cambridge Reference Sequence for Human Mitochondrial DNA, Nature Genetics 23: 147.             
 
      Codon usage for mtDNA differs slightly from the universal code. For example, UGA codes for typtophan instead of termination, AUA codes for methionine instead of isoleucine, and AGA and AGG are terminators instead of coding for arginine.  
      As used herein “printing” refers to the process of creating an array of nucleic acids on known positions of a solid substrate. The arrays of this invention can be printed by spotting, e.g., applying arrays of probes to a solid substrate, or to the synthesis of probes in place on a solid substrate. As used herein “glass slide” refers to a small piece of glass of the same dimensions as a standard microscope slide. As used herein, “prepared substrate” refers to a substrate that is prepared with a substance capable of serving as an attachment medium for attaching the probes to the substrate, such as poly Lysine. As used herein, “sample” refers to a composition containing human mitochondrial DNA that can be genotyped. As used herein, “quantitative hybridization” refers to hybridization performed under appropriate conditions and using appropriate materials such that the sequence of one nucleotide allele (a single nucleotide polymorphism) can be determined, such as by hybridization of a molecule containing that allele to two or more probes, each containing different alleles at that nucleotide locus, all as is known in the art.  
      As used herein, “physiological condition” includes diseased conditions, healthy conditions, and cosmetic conditions. Diseased conditions include, but are not limited to, metabolic diseases such as diabetes, hypertension, and cardiovascular disease. Healthy conditions include, but are not limited to, traits such as increased longevity. Physiological conditions include cosmetic conditions. Cosmetic conditions include, but are not limited to, traits such as amount of body fat. Physiological conditions can change health status in different contexts, such as for the same organism in a different environment. Such different environments for humans are different cultural environments or different climatic contexts such as are found on different continents.  
      As used herein, “neutrality analysis” refers to analysis to determine the neutrality of one or more nucleotide alleles and/or the gene containing the allele(s) using at least two alleles of a sequence. Commonly, the alleles in a sequence to be analyzed are divided into two groups, synonymous and nonsynonymous. Codon usage tables showing which codons encode which amino acids are used in this analysis. Codon usage tables for many organisms and genomes are available in the art. If a gene is determined to not be neutral, the gene is determined to have had selection pressure applied to it during evolution, and to be evolutionarily significant. The alleles that change amino acids in the gene (nonsynonymous) are then determined to be non-neutral and evolutionarily significant.  
      As used herein, “K a /K s ” refers to a ratio of the proportion of nonsynonymous differences to the proportion of synonymous differences in a DNA sequence analysis, as is known to the art. The proportion of nonsynonymous differences is the number of nonsynonymous nucleotide substitutions in a sequence per site at which a nonsynonymous substitution could occur. The proportion of synonymous differences is the number of synonymous nucleotide substitutions in a sequence per site at which synonymous substitutions could occur. Alternatively, instead of only including the number of sites in the denominator of each proportion, the number of alternative substitutions that could occur at each site are also included. Either definition may be used as long as similar definitions are used for both K a  and K s  in an analysis. K C  is K a /K s .  
      As used herein “nonsynonymous” refers to mutations that result in changes to the encoded amino acid. As used herein, “synonymous” refers to mutations that do not result in changes to the encoded amino acids.  
      As used herein, “haplogroup” refers to radiating lineages on the human evolutionary tree, as is known in the art. As used herein, “macro-haplogroup” refers to a group of evolutionarily related haplogroups. As used herein, “sub-haplogroup” refers to an evolutionarily related subset of a haplogroup. An individual&#39;s haplotype is the haplogroup to which he belongs.  
      As used herein, “extended longevity” or “extended lifespan” refers to living longer than the average expected lifespan for the population to which one belongs. As used herein, “centenaria” refers to an extended lifespan that is at least 100 years.  
      As used herein, “abnormal energy metabolism” in an individual who is non-native to the geographical region in which he lives refers to energy metabolism that differs from that of the population that is native to where the individual lives. As used herein, “abnormal temperature regulation” in such an individual refers to temperature regulation that differs from that of the population that is native to where he lives. As used herein, “abnormal oxidative phosphorylation” in such an individual refers to oxidative phosphorylation that differs from that of the population that is native to where he lives. As used herein, “abnormal electron transport” in such an individual refers to electron transport that differs from that of the population that is native to where he lives. As used herein “metabolic disease” of such an individual refers to metabolism that differs from that of the population that is native to where he lives. As used herein, “energetic imbalance” of such an individual refers to a balance of energy generation or use that differs from that of the population that is native to where he lives. As used herein, “obesity” of such an individual refers to a body weight that, for the height of the individual, is 20% higher than the average body weight that is recommended for the population native to where the individual lives. As used herein, “amount of body fat” of such an individual refers to a low or high percentage of body fat relative to what is recommended for the population that is native to where he lives.  
      As used herein, an isolated nucleic acid is a nucleic acid outside of the context in which it is found in nature. The term covers, for example: (a) a DNA which has the sequence of part of a naturally-occurring genomic DNA molecule but is not flanked by both of the coding or noncoding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally-occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, or a modified gene having a sequence not found in nature.  
      As used herein, “nucleotide locus” refers to a nucleotide position of the human mitochondrial genome. The Cambridge sequence SEQ ID NO:2 is used as a reference sequence, and the positions of the mitochondrial genome referred to herein are assigned relative to that sequence. As used herein, “loci” refers to more than one locus. As used herein, “nucleotide allele” refers to a single nucleotide at a selected nucleotide locus from a selected sequence when different bases occur naturally at that locus in different individuals. The nucleotide allele information is provided herein as the nucleotide locus number and the base that is at that locus, such as 3796C, which means that at human mitochondrial position 3796 in the Cambridge sequence, there is a cytosine (C). As used herein, “amino acid allele” refers to the amino acid that is at a selected amino acid location in the human mitochondrial genome when different amino acids occur naturally at that location in different individuals. There are thirteen protein-coding genes in the human mitochondria. For each gene, the encoded protein consists of amino acids that are numbered starting at one. ND1 304H, means that there is a histidine at amino acid 304 in the ND1 protein. Amino acids are encoded by codons. As used herein, “codon” refers to the group of three nucleotides that encode an amino acid in a protein, as is known in the art. An amino acid allele can be referred to by one or more of the nucleotide loci that code for it. For example, ntl 15884 P means that there is a proline (P) encoded by the codon containing nucleotide locus 15884.  
      As used herein, “evolutionarily significant gene” refers to a gene that has statistically significantly more nonsynonymous nucleotide changes, when compared to the corresponding gene in another individual, than would be expected by chance. As used herein, “evolutionarily significant nucleotide allele” refers to a nucleotide allele that is located in a gene that has been determined to be evolutionarily significant using that nucleotide allele, or an equivalent nucleotide allele in a corresponding gene in another individual. As used herein, “intraspecific” means within one species. As used herein, “subpopulation” refers to a population within a larger population. A subpopulation can be as small as one individual. As used herein, “geographic region” refers to a geographic area in which a statistically significant number of individuals have the same haplotype. As used herein, being “native” to a geographic region refers to having the haplotype associated with that geographic region. The haplotype associated with a geographic region is that which originated in the region or of many individuals who settled historically in the region with respect to human evolution.  
      As used herein, “target” or “target sample” refers to the collection of nucleic acids used as a sample for array analysis. The target is interrogated by the probes of the array. A “target” or “target sample” may be a mixture of several samples that are combined. For example, an experimental target sample may be combined with a differently labeled control target sample and hybridized to an array, the combined samples being referred to as the “target” interrogated by the probes of the array during that experiment. As used herein, “interrogated” means tested. Probes, targets, and hybridization conditions are chosen such that the probes are capable of interrogating the target, i.e., of hybridizing to complementary sequences in the target sample.  
      As used herein, “increased likelihood of developing blindness” refers to a higher than normal probability of losing the ability to see normally and/or of losing the ability to see normally at a younger age.  
      All sequences defined herein are meant to encompass the complementary strand as well as double-stranded polynucleotides comprising the given sequence.  
      This invention provides a list of human mtDNA polymorphisms found in all the major human haplogroups. Example 1 summarizes data from sequencing over 100 human mtDNA genomes that are representative of the major human haplogroups around the world. The summary includes over 900 point mutations and one nine-base pair deletion. Table 3, Human MtDNA Nucleotide Alleles, lists the alleles identified in 103 such sequences in the third column, the corresponding alleles of the Cambridge mtDNA sequence in the second column and the nucleotide loci (position in the Cambridge sequence), in the first column. Table 3 lists the set of human mtDNA nucleotide alleles that occur naturally in different haplogroups. Table 3 does not include alleles previously known to be associated with disease (i.e., does not include the alleles of Table 1). The nucleotide alleles listed in column three of Table 3, together with the corresponding nucleotide loci in column one, make up the set of non-Cambridge human mtDNA nucleotide alleles. Table 4 lists the nucleotide alleles identified by the inventors hereof in 48 human mtDNA genomes in column three, and the corresponding Cambridge alleles in column two. Columns one and three of Table 4 make up the set of non-Cambridge human mtDNA nucleotide alleles in 48 genomes.  
      The nucleotide alleles listed in Table 3, including the Cambridge nucleotide alleles, being naturally occurring, are useful for identifying alleles that are associated with abnormal physiological conditions. These nucleotide alleles can be ignored during analysis steps when performing methods for identifying novel alleles associated with selected physiological conditions.  
      As described below, certain alleles of Table 3 are useful for identifying physiological conditions related to energy metabolism such as energetic imbalance, metabolic disease, abnormal energy metabolism, abnormal temperature regulation, abnormal oxidative phosphorylation, abnormal electron transport, obesity, amount of body fat, diabetes, hypertension, and cardiovascular disease when the affected individuals have the abnormal physiological condition because they are in a geographical region that is not native for their haplogroup.  
      The nucleotide alleles listed in Table 3, including the Cambridge nucleotide alleles, are also useful for identifying mtDNA sequences associated with and diagnostic of human haplogroups. Example 2 summarizes phylogenetic analyses of the sequence data of the 103 individuals and the Cambridge sequence along with two chimpanzee mtDNA sequences. The results are shown in  FIG. 1  in a cladogram. Calculations of the time since the most recent common ancestor (MRCA) are shown in Table 5. The 104 individuals were chosen from known haplogroups, and the corresponding haplogroups are labeled on the figure. Combining the sequence data of the 104 individuals with  FIG. 1  and the geographic regions native to human haplogroups, as is known in the art, results in  FIG. 2  (Example 3), which tracks human mtDNA migrations. Analysis of several mtDNA genomic sequences representing each haplogroup demonstrated which alleles are segregating within a haplogroup as well as which alleles are present in every individual within one or more haplogroups. The alleles that are present in every individual within each haplogroup are shown in  FIG. 3  (Example 4). On the left, sub-haplogroups and haplogroups are listed. Macrohaplogroups are shown in parentheses. Nucleotide loci and alleles that are present in all the members of each group (sub-haplo or haplo) are listed. A vertical bar designates that all of the alleles to the right are present in all of the haplogroups and/or sub-haplogroups to the left.  FIG. 3  is drawn as a cladogram. For example,  FIG. 3  demonstrates that the macrohaplogroup (R) individuals all contain 12705C and 16223C, and no other individuals are known to have these alleles, therefore macro-haplogroup (R) can be diagnosed by identifying in a sample containing mtDNA, the presence of either 12705C or 16223C. Similarly, macro-haplogroup (N) can be diagnosed by identifying the presence of 8701A, 9540T, or 10873T.  
      Analysis of the data in  FIG. 3  demonstrated sets of alleles useful for diagnosing the haplogroups (Example 5). These alleles are listed by haplogroup in Tables 6 and 7, and by sub-haplogroup in Tables 8 and 9. A set of alleles useful for diagnosing all of the haplogroups and sub-haplogroups in  FIG. 3  is listed in Table 10. Table 10 lists the nucleotide loci in column one and the nucleotide alleles useful for diagnosing haplogroups in column two. Table 10 contains some alleles from the Cambridge sequence. There are many equivalent methods for diagnosing the haplogroups. Methods for diagnosing haplogroups that require testing only one or a few loci are listed in Example 5. The presence of only one particular allele is usually sufficient for diagnosing a haplogroup, however, often it is not known which locus needs to be tested. By determining the allele at each nucleotide locus listed in Table 10, the haplogroup of an unknown sample can be diagnosed. Alternatively, macro-haplogroups can be diagnosed or excluded first, thereby decreasing the number of loci that need to be tested to distinguish between the remaining, possible haplogroups. Alleles useful for diagnosing macro-haplogroups by methods that require testing only one or a few loci are included in Table 11. Further analysis of the data provided by this invention will demonstrate which sets of alleles identify additional sub-haplogroups and additional macro-haplogroups.  
      Diagnosing the haplogroup of a sample is useful in criminal investigations and forensic analyses. Identifying a sample as belonging to a particular haplogroup, and knowing which alleles have not been associated with a selected physiological condition and context, are useful when identifying novel alleles associated with a selected physiological condition, as described above and in Example 6. Diagnosing the haplogroup of a sample is also useful for identifying a novel allele associated with a selected physiological condition when the novel allele causes the physiological condition only in the genetic context of a particular haplogroup, as shown in Example 6. In example 6, the list of alleles associated with haplogroups found in Russia was used in the sequence analysis of two Russian LHON families. By eliminating alleles listed in Table 3, two novel mutations were identified that are associated with LHON. These new complex I mutations, 3635A and 4640C, are useful for diagnosing a predisposition to Leber Hereditary Optic Neuropathy (LHON).  
      Example 7 demonstrates the identification of a new primary LHON mutation, 10663C, in complex I, that appears to cause a predisposition to LHON only when associated with haplogroup J. Haplogroup J is defined by a nonsynonymous difference that is useful for diagnosing haplogroup J, 458T in ND5. This invention provides a method of diagnosing a person with a predisposition to LHON and/or to developing early onset blindness by identifying, in a sample containing mtDNA from the person, the nucleotide allele, or a synonymous nucleotide allele of 10663C and also identifying alleles diagnostic of haplogroup J, such as 458T in ND5. Because ND5458T is a missense mutation in all haplogroup J individuals, this particular mutation may be directly involved in causing LHON. ND1 304H is another missense mutation that is present in all haplogroup J individuals, and may also be directly involved in causing LHON. 458T is also present in haplogroup T individuals. Haplogroup J is also associated with a predisposition to centenaria and an extended lifespan. ND5 458T and ND1 304H may also be directly involved in causing the predisposition to centenaria and extended lifespan.  
      Example 8 demonstrates the importance of demographic factors in intercontinental mtDNA sequence radiation. Haplogroups are combined and separated into various populations for statistical analyses.  
      Previously in the art, it has been thought that polymorphisms in human mtDNA, such as the nucleotide alleles listed in Table 3, were neutral in all contexts and could not be associated with physiological conditions. It has been thought that differences in human mtDNA diversity associated with inter-continental migrations were due to random genetic drift (e.g. founder effects followed by rapid population expansion). In this invention, the biological and clinical significance of these human mtDNA polymorphisms are disclosed. The neutrality of the nucleotide alleles listed in Table 3 was tested using neutrality analysis (Examples 9-12).  
      Some of the nucleotide loci in Table 3 are located in the mitochondrial protein-coding genes (Table 2). Of those loci, some of the identified nucleotide alleles alter the protein encoded by the codon in which the nucleotide locus resides. This is determined using the mitochondrial codon usage table, as is known in the art. Nucleotide alleles that change an amino acid are called missense mutations, missense polymorphisms, or nonsynomymous differences. Missense polymorphisms alter the protein sequence relative to a compared sequence, but they still may be neutral because they do not affect the function of the encoded protein. Without performing biochemical studies on the affected proteins, statistical analyses can be performed to determine whether a polymorphism is neutral, whether evolution imposed selection on the encoding allele, and whether that selection is positive. This invention provides results of the statistical analyses of the polymorphisms in Table 3 and provides a list of which alleles are not neutral, and therefore evolutionarily significant.  
      Neutrality testing of nucleotide alleles first requires neutrality testing of the genes containing those nucleotide alleles. Neutrality testing of one or more genes by comparing two sets of allelic genes from two intraspecific populations was performed, as described in Example 9. Haplogroups were combined to make populations for the comparison. In example 9, nucleotide alleles from the entire coding region of the mtDNA genome, representing haplogroups native to a geographic region, were combined to make a first population and first set of sequences. Nucleotide alleles of the entire coding region of the mtDNA genome, from haplogroups native to a different geographic region, were combined to make the second population and the second set of sequences. Nucleotide alleles were divided into those encoding synonymous and non-synonymous differences. The ratio of K a /K s  for each gene, separated by the population containing the allele, is shown in Table 12. Neutrality testing of genes by comparing one set of at least two nucleotide alleles of at least one gene from one population of one species was performed in Example 10. In Example 10, sequences of the entire coding region of the mtDNA genome, of haplogroups in all geographic regions on earth, were combined to make one population and set of sequences for analysis.  FIG. 4  shows the results of the comparison of one set of sequences from one population of only one species, 104 human sequences. Example 11 includes comparisons of sets of sequences between two populations, human vs.  P. paniscus , human vs.  P. troglodytes , human vs. eight other primate species, and human vs. thirteen mammalian species.  
      To identify an evolutionarily significant gene, two sets of nucleotide sequences, each set from a different population, are compared to each other. Nucleotide sequences representing parts of genes or one or more whole genes are useful. The sets of sequences are compared to each other by neutrality analysis. Differences in the sequences from each set are determined to be synonymous or nonsynonymous differences. The proportion of nonsynonymous differences is compared to the proportion of synonymous differences (K a /K s ). The results of the analysis are compiled in a data set and the data set is analyzed, as is known in the art, to identify one or more evolutionarily significant genes. When the nonsynonymous differences occur significantly more often than is expected by chance than the synonymous differences, the gene or part of the gene is determined to be evolutionarily significant. When the synonymous differences occur significantly more often than is expected by chance than the nonsynonymous differences, the gene or part of the gene is determined to be conserved. When the ratio is as expected by chance, then there is no evidence of selection or evolutionary significance.  
      To identify an evolutionarily significant gene, only one set of nucleotide sequences (from only one population) may also be analyzed, e.g., the nucleotide sequences representative of humans living on one continent. When only one set of sequences is analyzed, the set must contain at least two corresponding nucleotide alleles (i.e., there must be sequence polymorphism). Corresponding sequences are sequences of the same gene or gene part from at least two individuals. The sequences from different individuals within the population must contain polymorphisms with respect to each other. Differences in the sequences relative to each other are determined to be synonymous or nonsynonymous. Neutrality analysis is performed to generate a data set. The data set is analyzed to identify an evolutionarily significant gene. If an analysis determines that none of the analyzed genes are evolutionarily significant, the set of nucleotide sequences can be increased, such as by increasing the size of the population from which the sequences are derived, to determine if one or more genes are evolutionarily significant in the enlarged population.  
      Example 12 is similar to example 9 except that the data is further analyzed by manipulating K a /K s  to K C . Examples 9-12 demonstrate that all but one mtDNA gene are not neutral and therefore are evolutionarily significant. Genes are determined to not be neutral by statistical significance tests known in the art. Some genes are only evolutionarily significant when comparing selected populations. For example, ND4 was demonstrated to be significant when comparing Native American sequences to African sequences and when comparing all human sequences to each other, but not when comparing European to African sequences. ND4L is the only mtDNA gene not shown to be evolutionarily significant by the current analyses. ND4L might be demonstrated to be evolutionarily significant by the methods of this invention using one or more different populations or using only part of the gene sequence. In examples 9-12, the entire sequence of each gene was used for analysis, however portions of genes are also useful in the methods of this invention. The statistical significance tests prevent too small a gene portion from being used to determine non-neutrality.  
      After identifying evolutionarily significant genes, evolutionarily significant nucleotide alleles can be identified. To identify an evolutionarily significant nucleotide allele, the steps for identifying an evolutionarily significant gene, using one or two populations, are performed with the addition of a step of analyzing the sequence data set to determine an evolutionarily significant nucleotide allele. An evolutionarily significant nucleotide allele is part of a sequence incoding an allelic amino acid in an evolutionarily significant gene or part of a gene. Examples 13 and 14 demonstrate identification of evolutionary significant nucleotide alleles and evolutionarily significant amino acid alleles in the evolutionarily significant genes identified in Examples 9-12. Evolutionarily significant amino acid alleles are the amino acids encoded by the codons containing evolutionarily significant nucleotide alleles. In these examples, nucleotides at loci not listed in Table 3 are identical to the Cambridge sequence so that the entire codon containing an evolutionarily significant nucleotide allele and the amino acid encoded by that codon can be determined. All nucleotide alleles that are part of a codon encoding the same amino acid as an evolutionarily significant amino acid allele identified herein, or identified by methods of this invention, are also evolutionarily significant and are intended to be within the scope of this invention. An evolutionarily significant amino acid allele may include more than one nucleotide allele, such as at two neighboring nucleotide loci. Evolutionarily significant nucleotide alleles and evolutionarily significant amino acid alleles in human mitochondrial sequences, identified by the methods of this invention, are listed in Table 14. In column one, Table 14 lists the gene containing the alleles, column two indicates the locus of the nucleotide allele, column three lists the Cambridge nucleotide allele at that nucleotide locus, column four lists a non-Cambridge allele of this invention, column five lists the amino acid encoded by the codon containing the Cambridge nucleotide allele (when other Cambridge nucleotides are present at the other nucleotide loci of the codon), and column six lists the amino acid encoded by the codon containing the non-Cambridge allele (when Cambridge nucleotides are present at the other nucleotide loci of the codon). Columns two, three, and four make the set of evolutionarily significant human mitochondrial nucleotide alleles. Columns two, five, and six make the set of evolutionarily significant human mitochondrial amino acid alleles. Table 14 designates the nucleotide locus of the listed alleles. For the amino acid alleles listed in columns five and six, the relevant loci are all three nucleotide loci in the encoding codon containing the nucleotide locus listed in column two.  
      To identify an evolutionarily significant amino acid allele, the steps for identifying an evolutionarily significant gene, using one or two populations, are performed with the addition of two steps: 1) analyzing the data set to determine an evolutionarily significant nucleotide allele; and 2) determining the encoded amino acid allele. An evolutionarily significant amino acid allele is a different amino acid, representing a nonsynonymous difference, relative to the corresponding amino acid allele against which it was compared, wherein the gene has been determined to be evolutionarily significant in the corresponding one or more populations.  
      In this invention it is demonstrated that amino acid substitution mutations (nonsynonymous differences) are much more common in human mtDNAs than would be expected by chance, and that most of them are evolutionarily significant. This invention demonstrates that these alleles have become fixed by selection. The mitochondrial genes encode proteins that are responsible for generating energy and for generating heat to maintain body temperature. As humans migrated to different parts of the world, they encountered changes in diet and climate. The high mutation rate of mtDNA and the central role of mitochondrial proteins in cellular energetics make the mtDNA an ideal system for permitting rapid mammalian adaptation to varying climatic and dietary conditions. The increased amino acid sequence variability that has been found among human mtDNA genes is due to the fact that natural selection favored mtDNA alleles that altered the coupling efficiency between the electron transport chain (ETC) and ATP synthesis, determined by the mitochondrial inner membrane proton gradient (AT). The coupling efficiency between the ETC and ATP synthesis is mediated to a considerable extent by the proton channel of the ATP synthase, which is composed of the mtDNA-encoded ATP6 protein and the nuclear DNA-encoded ATP9 protein. Mutations in the ATP6 gene, which create a more leaky ATP synthase proton channel, reduced ATP production but increased heat production for each calorie consumed. Such a change in energy balance was beneficial in a temperate or arctic climate, but deleterious in a tropical climate. Humans acquiring mtDNA alleles enabling better adaptation to the encountered changes in diet and climate experienced a higher genetic fitness and those alleles were selected for. In particular, these alleles were established genetically because they had an adaptive advantage as humans moved from the African tropics into the EurAsian temperate zone and on into the arctic ( FIG. 2 ). The lack of recombination of the maternally inherited mtDNAs favored the rapid segregation, expression and adaptive selection of advantageous mtDNA alleles. The apparent non-randomness of the differences in non-synonymous versus synonymous mtDNA variation between continents demonstrates that selection also influenced inter-continental colonization. Random genetic hitchhiking, such as in the synonymous alleles, then resulted in identifiable continent-specific haplogroups.  
      Modern mtDNA variation has been shaped by adaptation as our ancestors moved into different environmental conditions. Variants that are advantageous in one climatic and dietary environment are maladaptive when individuals locate to a different environment. The methods of this invention associate mtDNA nucleotide alleles with haplogroups and combine this data with native haplogroup geographic regions as is known in the art, to diagnose individuals as having predispositions to late-onset clinical disorders such as obesity, diabetes, hypertension, and cardiovascular disease when those individuals live in climatic and dietary environments that are disadvantageous with respect to their mtDNA alleles. When humans having regional mtDNA alleles move into a different thermal and/or dietary environment from the one in which the alleles were selected, they are energetically imbalanced with their environment, and as a result are predisposed to having metabolic diseases such as diabetes, hypertension, cardiovascular disease, and other diseases known to the art to be associated with metabolism and mitochondrial functions. The above-mentioned late-onset clinical disorders are rapidly becoming epidemic around the world in members of our globally mobile society. This invention provides a method of diagnosing a human with a predisposition to a physiological condition such as, but not limited to, energetic imbalance, metabolic disease, abnormal energy metabolism, abnormal temperature regulation, abnormal oxidative phosphorylation, abnormal electron transport, obesity, amount of body fat, diabetes, hypertension, and cardiovascular disease. The method involves testing a sample containing mitochondrial nucleic acid from an individual in a geographic region to determine the haplogroup of the sample and therefore of the individual, comparing the haplogroup of the individual to the set of haplogroups known to be native to that geographic region, and diagnosing the individual human with a predisposition to the above-mentioned conditions if the haplogroup of the individual is not in the set of haplogroups native to that geographic region. This invention enables treatment of one of the above-mentioned conditions that is diagnosed by the above-mentioned method, comprising relocating the diagnosed human to a geographic region that is of similar climate as the region(s) native to the human&#39;s haplogroup and/or changing the diagnosed human&#39;s diet to more closely match the diet historically available in the region(s) native to the human&#39;s haplogroup.  
      The above-described method for diagnosing a predisposition to a physiological condition is also useful for associating an amino acid allele with the physiological condition The evolutionarily significant amino acid alleles present in the haplogroup of the diagnosed individual and not in the haplogroups native to the individual&#39;s geographic location are associated with the physiological condition by the methods of this invention. Amino acid alleles, and the corresponding nucleotide alleles, useful for diagnosing haplogroups, and the haplogroup they are useful for diagnosing, are listed in Table 15. The amino acid alleles and corresponding nucleotide alleles listed in Table 15, and synonymously coding nucleotide alleles, are associated with the above-mentioned physiological conditions. Table 15 lists the set of amino acid alleles useful for diagnosing haplogroups. Column one of Table 15 lists the gene, column two lists the nucleotide locus, column three lists the useful nucleotide allele, column four lists the useful amino acid allele encoded by the useful nucleotide allele when Cambridge nucleotides are present at the other nucleotide loci of the encoding codon, and column five lists the haplogroups or sub-haplogroups, in parentheses, that contain the corresponding alleles. The amino acid alleles (column four) can be identified by the codon containing the nucleotide locus (column two). For example, the proline in the ND1 gene is identified as ntl 3796 P, where ntl signifies the codon containing the nucleotide locus (ntl) 3796. When an individual of one of the haplogroups listed in column five of Table 15 is diagnosed with one of the above-mentioned physiological conditions by the above-mentioned method, the physiological condition is associated with the presence of one of the alleles listed in Table 15. When the haplogroup of the individual is haplogroup G, the amino acid allele likely to have caused the physiological condition is ntl 4833 A. When the haplogroup of the individual is haplogroup T, the amino acid allele is selected from the group consisting of ntl 14917 D, ntl 8701 T, and ntl 15452 I. When the haplogroup is haplogroup W, the amino acid allele is selected from the group consisting of ntl 5046 I, ntl 5460 T, ntl 8701 T, and ntl 15884 P. When the haplogroup is haplogroup D, the amino acid allele is selected from the group consisting of ntl 5178 M and ntl 8414 F. When the haplogroup is haplogroup L0, the amino acid allele is selected from the group consisting of ntl 5442 L, ntl 7146 A, ntl 9402 P, ntl 13105 V, and ntl 13276 V. When the haplogroup is haplogroup L1, the amino acid allele is selected from the group consisting of ntl 7146 A, ntl 7389H, ntl 13105 V, ntl 13789H, and ntl 14178 V. When the haplogroup is haplogroup C the amino acid allele is selected from the group consisting of ntl 8584 T and ntl 14318 S. When the haplogroup is selected from the group consisting of haplogroups A, I, X, B, F, Y, and U the amino acid allele is ntl 8701 T. When the haplogroup is haplogroup J the amino acid allele is selected from the group consisting of ntl 8701 T, ntl 13708 T, and ntl 15452 I. When the haplogroup is haplogroup selected from the group consisting of haplogroups V and H, the amino acid allele is selected from the group consisting of ntl 8701 T and ntl 14766 T.  
      Evolutionarily significant nucleotide and amino acid alleles also exist in nuclear-encoded ATP9 that are useful for diagnosing predisposition to an energy metabolism-related physiological condition such as energetic imbalance, metabolic disease, abnormal energy metabolism, abnormal temperature regulation, abnormal oxidative phosphorylation, abnormal electron transport, obesity, centenaria, diabetes, hypertension, and cardiovascular disease. These alleles may be identified by methods of this invention.  
      The evolutionarily significant amino acid alleles and corresponding nucleotide alleles are candidates for alleles causing a physiological condition for which a predisposition is diagnosable by the methods of this invention. The evolutionarily significant amino acid and nucleotide alleles identified by the methods of this invention (Table 19) are useful for gene therapy and mitochondrial replacement therapy to treat the corresponding physiological conditions. The evolutionarily significant genes, amino acid alleles, and nucleotide alleles identified by the methods of this invention are useful for identifying targets for traditional therapy, and for designing corresponding therapeutic agents. The evolutionarily significant genes and amino acid and nucleotide changes identified by the methods of this invention are useful for generating animal models of the corresponding human physiological conditions.  
      As is known to the art, individuals may contain more than one mitochondrial DNA allele at any given nucleotide locus. One cell contains many mitochondria, and one cell or different cells within one organism may contain genetically different mitochondria. Heteroplasmy is the occurrence of more than one type of mitochondria in an individual or sample. Varying degrees of heteroplasmy are associated with varying degrees of the physiological conditions described herein. Heteroplasmy may be identified by means known to the art, and the severity of the physiological condition associated with specific nucleotide alleles is expected to vary with the percentage of such associated alleles within the individual.  
      The methods of this invention are used to analyze the human mitochondrial genome in the listed examples, but the methods are also useful for analyzing other genomes and other species. The methods of this invention are useful for identifying evolutionarily significant protein-coding genes and the correspondingly encoded mutations in other genomes in addition to mitochondrial genomes, such as in nuclear and chloroplast genomes. Using human haplogroups as populations ( FIG. 1 ), the methods of this invention are useful for identifying evolutionarily significant protein-coding genes and the corresponding evolutionarily significant alleles in human nuclear genes. The methods of this invention are also useful for identifying evolutionarily significant protein-coding genes and the corresponding alleles in many species. For example, the methods of this invention are applicable to varieties of beef or dairy cattle, or pig lines. Corn lines are divisible by phenotypic and/or molecular markers into heterotic groups that are useful populations in the methods of this invention. Using corn heterotic groups as populations, the methods of this invention are useful for identifying evolutionarily significant protein-coding genes and the corresponding mutations in the nuclear, chloroplast, and mitochondrial genomes of corn.  
      This invention provides isolated nucleic acid molecules containing novel nucleotide alleles of this invention in libraries. The libraries contain at least two such molecules. Preferably the molecules have unique sequences. The molecules typically have a length from about 7 to about 30 nucleotides. “About” as used herein means within about 10% (e.g., “about 30 nucleotides” means 27-33 nucleotides). However, the molecules may be longer, such as about 50 nucleotides long. A library of this invention contains at least two isolated nucleic acid molecules each containing at least one non-Cambridge nucleotide allele of this invention. A library of this invention may contain at least ten, twenty-five, fifty, 100, 500 or more isolated nucleic acid molecules, at least one of which contains a nucleotide allele of this invention. A library of this invention may contain molecules having at least two to all of the nucleotide alleles of this invention, including synonymous codings of evolutionarily significant amino acid alleles. The nucleotide alleles of this invention are defined by a nucleotide locus, the nucleotide location in the human mitochondrial genome, and by the A G C T (or U) nucleotide. An isolated nucleic acid molecule, in a library of this invention, can be identified as containing a nucleotide allele of this invention, because the nucleotide allele of this invention is bounded on at least one side by its context in the mitochondrial genome. Statistically, to be unique in the human mitochondrial genome, such a molecule would need to be at least about seven nucleotides long. Statistically, to be unique in the total human genome, including the mitochondrial genome, such a molecule would need to be at least about fifteen nucleotides long. Examples of isolated nucleic acid molecules of this invention are molecules containing the following nucleotide alleles: 1) Cambridge alleles at human mtDNA nucleotide loci 168-170, non-Cambridge alleles at locus 171A, and Cambridge alleles at human mtDNA nucleotide loci 172-174; and 2) Cambridge alleles at 11940-11946, non-Cambridge alleles at 11947G, and Cambridge alleles at 11948-11954. An isolated nucleic acid molecule of this invention may contain more than one nucleotide allele of this invention. The nucleotide allele of this invention may be at any position in the isolated nucleic acid molecule. Often it is useful to have the relevant nucleotide allele in the center of the isolated nucleic acid molecule or on the 3′ end of the molecule. Isolated nucleic acid molecules of this invention are useful for interrogating, determining the presence or absence of, a nucleotide allele at the corresponding nucleotide locus in the mitochondrial genome in a sample containing mitochondrial nucleic acid from a human, using any method known in the art. Methods for determining the presence of absence of the nucleotide allele include allele-specific PCR and nucleic acid array hybridization or sequencing.  
      The alleles and libraries of this invention are useful for designing probes for nucleic acid arrays. This invention provides nucleic acid arrays having two or more nucleic acid molecules or spots (each spot comprising a plurality of substantially identical isolated nucleic acid molecules), each molecule having the sequence of an allele of this invention. The molecules on the arrays of this invention are usually about 7 to about 30 nucleotides long. The arrays are useful for detecting the presence or absence of alleles. Arrays of this invention are also useful for sequencing human mtDNA. Alleles may be selected from sets of nucleotide alleles including human mtDNA nucleotide alleles, non-Cambridge human mtDNA nucleotide alleles, human mtDNA nucleotide alleles in 48 genomes and the Cambridge sequence, non-Cambridge human mtDNA nucleotide alleles in 48 genomes, nucleotide alleles useful for diagnosing human haplogroups and macro-haplogroups, nucleotide alleles useful for diagnosing human haplogroups, and evolutionarily significant human mitochondrial nucleotide alleles as listed in the various Tables and portions of tables hereof. Arrays of this invention may contain molecules capable of interrogating all of the alleles in one of the above-mentioned sets of alleles. A genotyping array useful for detecting sequence polymorphisms, such as are provided by this invention, are similar to Affymetrix (Santa Clara, Calif., USA) genotyping arrays containing a Perfect Match probe (PM) and a corresponding Mismatch probe (MM). A PM probe could comprise a non-Cambridge allele at a selected nucleotide locus and the corresponding MM probe could comprise the corresponding Cambridge allele at the selected nucleotide locus. Arrays of this invention include sequencing arrays for human mtDNA.  
      As used herein, “array” refers to an ordered set of isolated nucleic acid molecules or spots consisting of pluralities of substantially identical isolated nucleic acid molecules. Preferably the molecules are attached to a substrate. The spots or molecules are ordered so that the location of each (on the substrate) is known and the identity of each is known. Arrays on a microscale can be called microarrays. Microarays on solid substrates, such as glass or other ceramic slides, can be called gene chips or chips.  
      Arrays are preferably printed on solid substrates. Before printing, substrates such as glass slides are prepared to provide a surface useful for binding, as is known to the art. Arrays may be printed using any printing techniques and machines known in the art. Printing involves placing the probes on the substrate, attaching the probes to the substrate, and blocking the substrate to prevent non-specific hybridization Spots are printed at known locations. Arrays may be printed on glass microscope slides. Alternatively, probes may be synthesized in known positions on prepared solid substrates (Affymetrix, Santa Clara, Calif., USA).  
      Arrays of this invention may contain as few as two spots, or more than about ten spots, more than about twenty-five spots, more than about one hundred spots, more than about 1000 spots, more than about 65,000 spots, or up to about several hundred thousand spots.  
      Using microarrays may require amplification of target sequences (generation of multiple copies of the same sequence) of sequences of interest, such as by PCR or reverse transcription. As the nucleic acid is copied, it is tagged with a fluorescent label that emits light like a light bulb. The labeled nucleic acid is introduced to the microarray and allowed to react for a period of time. This nucleic acid sticks to, or hybridizes, with the probes on the array when the probe is sufficiently complementary to the labeled, amplified, sample nucleic acid. The extra nucleic acid is washed off of the array, leaving behind only the nucleic acid that has bound to the probes. By obtaining an image of the array with a fluorescent scanner and using software to analyze the hybridized array image, it can be determined if, and to what extent, genes are switched on and off, or whether or not sequences are present, by comparing fluorescent intensities at specific locations on the array. The intensity of the signal indicates to what extent a sequence is present. In expression arrays, high fluorescent signals indicate that many copies of a gene are present in a sample, and lower fluorescent signal shows a gene is less active. By selecting appropriate hybridization conditions and probes, this technique is useful for detecting single nucleotide polymorphisms (SNPs) and for sequencing. Methods of designing and using microarrays are continuously being improved (Relogio, A. et al. (2002)  Nuc. Acids. Res.  30(11): e51; Iwasaki, H et al. (2002)  DNA Res.  9(2):59-62; and Lindroos, K. et al. (2002)  Nuc. Acids. Res.  30(14):E70).  
      Arrays of this invention may be made by any array synthesis methods known in the art such as spotting technology or solid phase synthesis. Preferably the arrays of this invention are synthesized by solid phase synthesis using a combination of photolithography and combinatorial chemistry. Some of the key elements of probe selection and array design are common to the production of all arrays. Strategies to optimize probe hybridization, for example, are invariably included in the process of probe selection. Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and by using empirical rules that correlate with desired hybridization behaviors. Computer models may be used for predicting the intensity and concentration-dependence of probe hybridization.  
      Detecting a particular polymorphism can be accomplished using two probes. One probe is designed to be perfectly complementary to a target sequence, and a partner probe is generated that is identical except for a single base mismatch in its center. In the Affymetrix system, these probe pairs are called the Perfect Match probe (PM) and the Mismatch probe (MM). They allow for the quantitation and subtraction of signals caused by non-specific cross-hybridization. The difference in hybridization signals between the partners, as well as their intensity ratios, serve as indicators of specific target abundance, and consequently of the sequence.  
      Arrays can rely on multiple probes to interrogate individual nucleotides in a sequence. The identity of a target base can be deduced using four identical probes that vary only in the target position, each containing one of the four possible bases. Alternatively, the presence of a consensus sequence can be tested using one or two probes representing specific alleles. To genotype heterozygous or genetically mixed samples, arrays with many probes can be created to provide redundant information, resulting in unequivocal genotyping.  
      Probes fixed on solid substrates and targets (nucleotide sequences in the sample) are combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the substrate is washed free of extraneous materials, leaving the nucleic acids on the target bound to the fixed probe molecules allowing for detection and quantitation by methods known in the art such as by autoradiograph, liquid scintillation counting, and/or fluorescence. As improvements are made in hybridization and detection techniques, they can be readily applied by one of ordinary skill in the art. As is well known in the art, if the probe molecules and target molecules hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and target nucleic acid are essentially identical, or almost completely complementary if the annealing and washing steps are carried out under conditions of high stringency. The detectable label provides a means for determining whether hybridization has occurred.  
      When using oligonucleotides or polynucleotides as hybridization probes, the probes may be labeled. In arrays of this invention, the target may instead be labeled by means known to the art. Target may be labeled with radioactive or non-radioactive labels. Targets preferably contain fluorescent labels.  
      Various degrees of stringency of hybridization can be employed. The more stringent the conditions are, the greater the complementarity that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Hybridization experiments are often conducted under moderate to high stringency conditions by techniques well know in the art, as described, for example in Keller, G. H., and M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170, hereby incorporated by reference. However, sequencing arrays typically use lower hybridization stringencies, as is known in the art.  
      Moderate to high stringency conditions for hybridization are known to the art. An example of high stringency conditions for a blot are hybridizing at 68° C. in 5×SSC/5× Denhardt&#39;s solution/0.1% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature. An example of conditions of moderate stringency are hybridizing at 68° C. in 5×SSC/5× Denhardt&#39;s solution/0.1% SDS and washing at 42° C. in 3×SSC. The parameters of temperature and salt concentration can be varied to achieve the desired level of sequence identity between probe and target nucleic acid. See, e.g., Sambrook et al. (1989) vide infra or Ausubel et al. (1995)  Current Protocols in Molecular Biology , John Wiley &amp; Sons, NY, N.Y., for further guidance on hybridization conditions.  
      The melting temperature is described by the following formula (Beltz, G. A. et al., [1983 ] Methods of Enzymology , R. Wu, L. Grossman and K. Moldave [Eds.] Academic Press, New York 100:266-285).  
      T m =81.5o C+16.6 Log[Na+]+0.41(+G+C)−0.61(% formamide)−600/length of duplex in base pairs.  
      Washes can typically be carried out as follows: twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash), and once at TM-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).  
      Nucleic acid useful in this invention can be created by Polymerase Chain Reaction (PCR) amplification. PCR products can be confirmed by agarose gel electrophoresis. PCR is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos.  4 , 683 , 195 ,  4 , 683 , 202 , and 4,800,159; Saiki et al. [1985 ] Science  230:1350-1354). PCR is used to enzymatically amplify a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as the Taq polymerase, which is isolated from the thermophilic bacterium  Thermus aquaticus , the amplification process can be completely automated. Other enzymes that can be used are known to those skilled in the art.  
      Polynucleotide sequences of the present invention can be truncated and/or mutated such that certain of the resulting fragments and/or mutants of the original full-length sequence can retain the desired characteristics of the full-length sequence. A wide variety of restriction enzymes that are suitable for generating fragments from larger nucleic acid molecules are well known. In addition, it is well known that Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA. See, for example, Maniatis (1982)  Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, New York, pages 135-139, incorporated herein by reference. See also Wei et al. (1983)  J. Biol. Chem.  258:13006-13512. By use of Bal31 exonuclease (commonly referred to as “erase-a-base” procedures), the ordinarily skilled artisan can remove nucleotides from either or both ends of the subject nucleic acids to generate a wide spectrum of fragments that are functionally equivalent to the subject nucleotide sequences. One of ordinary skill in the art can, in this manner, generate hundreds of fragments of controlled, varying lengths from locations all along the original molecule. The ordinarily skilled artisan can routinely test or screen the generated fragments for their characteristics and determine the utility of the fragments as taught herein. It is also well known that the mutant sequences can be easily produced with site-directed mutagenesis. See, for example, Larionov, O. A. and Nikiforov, V. G. (1982)  Genetika  18(3):349-59; and Shortle, D. et al., (1981)  Annu. Rev. Gene.  15:265-94, both incorporated herein by reference. The skilled artisan can routinely produce deletion-, insertion-, or substitution-type mutations and identify those resulting mutants that contain the desired characteristics of wild-type sequences, or fragments thereof.  
      Percent sequence identity of two nucleic acids may be determined using the algorithm of Karlin and Altschul (1990)  Proc. Natl. Acad. Sci. USA  87:2264-2268, modified as in Karlin and Altschul (1993)  Proc. Natl. Acad. Sci. USA  90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990)  J. Mol. Biol.  215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997)  Nucl. Acids. Res.  25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See http://www.ncbi.nih.gov.  
      Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques useful herein are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)  Meth. Enzymol.  218, Part I; Wu (ed.) (1979)  Meth. Enzymol.  68; Wu et al. (eds.) (1983)  Meth. Enzymol.  100 and 101; Grossman and Moldave (eds.)  Meth. Enzymol.  65; Miller (ed.) (1972)  Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981)  Principles of Gene Manipulation , University of California Press, Berkeley; Schleif and Wensink (1982)  Practical Methods in Molecular Biology ; Glover (Ed.) (1985)  DNA Cloning  Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) (1985)  Nucleic Acid Hybridization , IRL Press, Oxford, UK; Setlow and Hollaender (1979)  Genetic Engineering: Principles and Methods , Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992)  Current Protocols in Molecular Biology , Greene/Wiley, New York, N.Y. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.  
      This invention provides machine-readable storage devices and program storage devices having data and methods for diagnosing haplogroups and physiological conditions. One program storage device provided by this invention contains the program steps: a) determining the haplogroup of a sample from an individual using nucleotide sequence data from nucleic acid in the sample; b) associating the haplogroup with information identifying the geographic region of the individual; c) comparing the haplogroup and geographic region of the sample to the set of haplogroups native to the geographic region of the individual; and d) diagnosing the individual with a predisposition to an energy metabolism-related physiological condition if the haplogroup of the individual is not within the set of haplogroups native to the geographic region of the individual; all said program steps being encoded in machine readable form, and all said information encoded in machine readable form. This invention also provides a data set, encoded in machine-readable form, containing nucleotide alleles listed in Table 19, with each allele associated with encoded information identifying a physiological condition in humans. These physiological conditions are energy-metabolism-related conditions including energetic imbalance, metabolic disease, abnormal energy metabolism, abnormal temperature regulation, abnormal oxidative phosphorylation, abnormal electron transport, obesity, amount of body fat, diabetes, hypertension, and cardiovascular disease. This storage device may also contain information associating each allele with one or more native geographic regions. A program storage device provided by this invention contains input means for inputting the haplogroup of an individual and the geographic region of that individual, and contains information associating alleles with native geographic regions, and program steps for diagnosing the individual with a predisposition to a physiological condition. A storage device containing a data set in machine readable form provided by this invention may include encoded information comprising amino acid alleles listed in Table 19, with each allele associated with a physiological condition in humans.  
      It will be appreciated by those of ordinary skill in the art that populations, subpopulations, organelles, and amino acid and nucleotide sequence comparison methods, neutrality test methods, nucleotide sequencing methods, codons, samples, sample collection techniques, sample preparation techniques, probes, probe generation techniques, genes involved in mitochondrial biology, hybridization techniques, array printing techniques, physiological conditions, cell lines, mutant strains, organisms, tissues, solid substrates, machine-readable storage devices, program devices, and methods of data analyses other than those specifically disclosed herein are available in the art and can be employed in the practice of this invention. All art-known functional equivalents are intended to be encompassed within the scope of this invention.  
      The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed here. Any variations in the compositions and methods exemplified that occur to the skilled artisan are intended to fall within the scope of the present invention.  
     EXAMPLES  
     Example 1  
      This invention provides human mtDNA polymorphisms found in all the major human haplogroups. Table 3 shows naturally occurring nucleotide alleles identified in the complete mtDNA sequences of 103 individuals, as compared to the mtDNA Cambridge sequence. All nucleotide sequences not listed are identical to the Cambridge sequence. Nucleotide alleles previously known to be associated with disease conditions, such as those listed in Table 1, are not listed in Table 3. Some deletion or rearrangement polymorphisms have also been excluded. All polymorphisms listed are nucleotide substitutions except for a nine-adenine nucleotide deletion at positions 8271-8279.  
               TABLE 3                          Human MtDNA Nucleotide Alleles                                 non-       nucleotide   Cambridge   Cambridge       locus   alleles   alleles                                 64   C   T       72   T   C       73   A   G       89   T   C       93   A   G       95   A   C       114   C   T       143   G   A       146   T   C       150   C   T       151   C   T       152   T   C       153   A   G       171   G   A       180   T   C       182   C   T       183   A   G       185   G   A       185   G   T       186   C   A       189   A   C       189   A   G       194   C   T       195   T   A       195   T   C       198   C   T       199   T   C       200   A   G       204   T   C       207   G   A       208   T   C       210   A   G       212   T   C       215   A   G       217   T   C       225   G   A       227   A   G       228   G   A       235   A   G       236   T   C       247   G   A       250   T   C       252   T   C       263   A   G       291   A   G       295   C   T       297   A   G       316   G   A       317   C   A       317   C   G       320   C   T       325   C   T       340   C   T       357   A   G       373   A   G       400   T   G       408   T   A       418   C   T       456   C   T       462   C   T       465   C   T       467   C   T       471   T   C       480   T   C       482   T   C       489   T   C       493   A   G       499   G   A       508   A   G       593   T   C       597   C   T       663   A   G       678   T   C       680   T   C       709   G   A       710   T   C       721   T   C       750   A   G       769   G   A       825   T   A       827   A   G       850   T   C       921   T   C       930   G   A       961   T   C       961   T   G       1018   G   A       1041   A   G       1048   C   T       1119   T   C       1189   T   C       1243   T   C       1290   C   T       1382   A   C       1406   T   C       1415   G   A       1420   T   C       1438   A   G       1442   G   A       1503   G   A       1598   G   A       1700   T   C       1703   C   T       1706   C   T       1709   G   A       1715   C   T       1719   G   A       1736   A   G       1738   T   C       1780   T   C       1811   A   G       1888   G   A       1927   G   A       2000   C   T       2060   A   G       2092   C   T       2245   A   C       2245   A   G       2263   C   A       2308   A   G       2332   C   T       2352   T   C       2358   A   G       2380   C   T       2416   T   C       2483   T   C       2581   A   G       2639   C   T       2650   C   T       2706   A   G       2755   A   G       2758   G   A       2768   A   G       2789   C   T       2792   A   G       2834   C   T       2836   C   A       2857   T   C       2863   T   C       2885   T   C       3010   G   A       3083   T   C       3197   T   C       3200   T   A       3202   T   C       3204   C   T       3206   C   T       3221   A   G       3290   T   C       3308   T   C       3316   G   A       3372   T   C       3394   T   C       3438   G   A       3450   C   T       3480   A   G       3505   A   G       3513   C   T       3516   C   A       3516   C   G       3547   A   G       3549   C   T       3552   T   A       3552   T   C       3565   A   G       3594   C   T       3644   T   C       3666   G   A       3693   G   A       3699   C   T       3720   A   G       3756   A   G       3796   A   G       3796   A   T       3796   A   C       3808   A   G       3816   A   G       3834   G   A       3843   A   G       3847   T   C       3866   T   C       3918   G   A       3921   C   A       3927   A   G       3970   C   T       3981   A   G       4025   C   T       4040   C   T       4044   A   G       4048   G   A       4086   C   T       4104   A   G       4117   T   C       4122   A   G       4123   A   G       4158   A   G       4203   A   G       4216   T   C       4221   C   T       4225   A   G       4232   T   C       4248   T   C       4312   C   T       4336   T   C       4370   T   C       4388   A   G       4454   T   A       4491   G   A       4506   A   G       4508   C   T       4512   G   A       4529   A   C       4529   A   T       4541   G   A       4580   G   A       4586   T   C       4596   G   A       4646   T   C       4655   G   A       4688   T   C       4695   T   C       4715   A   G       4742   T   C       4767   A   G       4769   A   G       4820   G   A       4824   A   G       4833   A   G       4841   G   A       4883   C   T       4907   T   C       4917   A   G       4960   C   T       4977   T   C       4994   A   G       5004   T   C       5027   C   T       5036   A   G       5043   G   T       5046   G   A       5063   T   C       5096   T   C       5108   T   C       5147   G   A       5153   A   G       5178   C   A       5231   G   A       5237   G   A       5255   C   T       5262   G   A       5263   C   T       5285   A   G       5300   C   T       5330   C   A       5331   C   A       5390   A   G       5393   T   C       5417   G   A       5426   T   C       5442   T   C       5460   G   A       5465   T   C       5471   G   A       5492   T   C       5495   T   C       5580   T   C       5581   A   G       5601   C   T       5603   C   T       5606   C   T       5633   C   T       5655   T   C       5711   A   G       5773   G   A       5811   A   G       5814   T   C       5821   G   A       5826   T   C       5843   A   G       5951   A   G       5984   A   G       5987   C   T       6026   G   A       6029   C   T       6045   C   T       6071   T   C       6077   C   T       6104   C   T       6150   G   A       6152   T   C       6164   C   T       6167   T   C       6182   G   A       6185   T   C       6185   T   C       6221   T   C       6227   T   C       6253   T   C       6257   G   A       6324   G   A       6366   G   A       6371   C   T       6392   T   C       6473   C   T       6491   C   A       6524   T   C       6548   C   T       6587   C   T       6607   T   C       6680   T   C       6713   C   T       6719   T   C       6734   G   A       6752   A   G       6770   A   G       6776   T   C       6815   T   C       6827   T   C       6875   C   A       6938   C   T       6962   G   A       6989   A   G       7028   C   T       7052   A   G       7055   A   G       7058   T   A       7076   A   G       7146   A   G       7154   A   G       7175   T   C       7196   C   A       7202   A   G       7226   G   A       7256   C   T       7257   A   G       7271   A   G       7274   C   T       7319   T   C       7337   G   A       7347   G   A       7389   T   C       7403   A   G       7424   A   G       7444   G   A       7476   C   T       7493   C   T       7521   G   A       7561   T   C       7571   A   G       7600   G   A       7624   T   A       7645   T   C       7648   C   T       7660   T   C       7664   G   A       7673   A   G       7675   C   T       7693   C   T       7694   C   T       7697   G   A       7744   T   C       7765   A   G       7768   A   G       7771   A   G       7858   C   T       7861   T   C       7864   C   T       7867   C   T       7933   A   G       7948   C   T       7999   T   C       8014   A   G       8020   G   A       8027   G   A       8080   C   T       8087   T   C       8113   C   A       8142   C   T       8149   A   G       8152   G   A       8155   G   A       8185   T   C       8200   T   C       8206   G   A       8248   A   G       8251   G   A       8260   T   C       8269   G   A       8271-8279   A   DEL       8286   T   C       8292   G   A       8298   T   C       8344   A   G       8387   G   A       8389   A   G       8392   G   A       8404   T   C       8414   C   T       8428   C   T       8448   T   C       8460   A   G       8468   C   T       8472   C   T       8473   T   C       8485   G   A       8545   G   A       8553   C   T       8563   A   G       8566   A   G       8577   A   G       8584   G   A       8618   T   C       8655   C   T       8697   G   A       8701   A   G       8703   C   T       8705   T   C       8709   C   T       8721   A   G       8733   T   C       8764   G   A       8781   C   A       8784   A   G       8790   G   A       8793   T   C       8794   C   T       8805   A   G       8836   A   G       8838   G   A       8856   G   A       8860   A   G       8875   T   C       8877   T   C       8911   T   C       8913   A   G       8928   T   C       8943   C   T       8962   A   G       8994   G   A       9042   C   T       9053   G   A       9055   G   A       9072   A   G       9077   T   C       9090   T   C       9093   A   C       9103   T   C       9114   A   G       9120   A   G       9123   G   A       9123   G   A       9136   A   G       9151   A   G       9156   A   G       9174   T   C       9221   A   G       9237   G   A       9242   A   G       9248   C   T       9263   A   G       9272   C   T       9296   C   T       9311   T   C       9325   T   C       9335   C   T       9347   A   G       9355   A   G       9356   C   T       9377   A   G       9402   A   C       9449   C   T       9456   A   G       9477   G   A       9509   T   C       9536   C   T       9540   T   C       9545   A   G       9548   G   A       9554   G   A       9559   C   G       9575   G   A       9591   G   A       9599   C   T       9632   A   G       9647   T   C       9667   A   G       9682   T   C       9698   T   C       9755   G   A       9818   C   T       9822   C   A       9824   T   A       9911   C   T       9932   G   A       9950   T   C       9957   T   C       9966   G   A       9977   T   C       10034   T   C       10086   A   G       10086   A   C       10115   T   C       10118   T   C       10142   C   T       10151   A   G       10152   G   C       10172   G   A       10182   G   C       10197   G   A       10238   T   C       10253   T   C       10256   T   C       10310   G   A       10313   A   G       10321   T   C       10325   G   A       10358   A   G       10370   T   C       10398   A   G       10400   C   T       10410   T   C       10414   G   T       10427   G   A       10463   T   C       10499   A   G       10505   T   C       10550   A   G       10586   G   A       10589   G   A       10609   T   C       10637   C   T       10640   T   C       10646   G   A       10659   C   T       10664   C   T       10667   T   C       10688   G   A       10736   C   T       10790   T   C       10792   A   G       10793   C   T       10804   A   G       10810   T   C       10819   A   G       10828   T   C       10873   T   C       10876   A   G       10894   C   T       10915   T   C       10920   C   T       10939   C   T       10966   T   C       10984   C   G       11002   A   G       11016   G   A       11017   T   C       11023   A   G       11078   A   G       11092   A   G       11147   T   C       11150   G   A       11167   A   G       11172   A   G       11176   G   A       11177   C   T       11215   C   T       11251   A   G       11257   C   T       11296   C   T       11299   T   C       11332   C   T       11362   A   G       11365   T   C       11377   G   A       11467   A   G       11476   C   T       11536   C   T       11590   A   G       11611   G   A       11641   A   G       11653   A   G       11654   A   G       11674   C   T       11701   T   C       11719   G   A       11722   T   C       11767   C   T       11812   A   G       11854   T   C       11884   A   G       11887   G   A       11893   A   G       11899   T   C       11909   A   G       11914   G   A       11944   T   C       11947   A   G       11959   A   G       11963   G   A       11969   G   A       12007   G   A       12049   C   T       12070   G   A       12083   T   G       12121   T   C       12134   T   C       12153   C   T       12172   A   G       12175   T   C       12234   A   G       12236   G   A       12239   C   T       12248   A   G       12308   A   G       12346   C   T       12358   A   G       12361   A   G       12372   G   A       12373   A   G       12397   A   G       12406   G   A       12414   T   C       12477   T   C       12501   G   A       12507   A   G       12519   T   C       12528   G   A       12540   A   G       12612   A   G       12630   G   A       12633   C   T       12635   T   C       12669   C   T       12672   A   G       12693   A   G       12705   C   T       12720   A   G       12738   T   C       12768   A   G       12771   G   A       12810   A   G       12822   A   G       12850   A   G       12879   T   C       12882   C   T       12930   A   T       12940   G   A       12948   A   G       12967   A   C       12972   A   G       12999   A   G       13020   T   C       13059   C   T       13068   A   G       13101   A   C       13104   A   G       13105   A   G       13135   G   A       13143   T   C       13145   G   A       13149   A   G       13194   G   A       13197   C   T       13212   C   T       13221   A   G       13263   A   G       13276   A   G       13281   T   C       13368   G   A       13440   C   G       13477   G   A       13485   A   G       13494   C   T       13500   T   C       13500   T   G       13506   C   T       13512   A   G       13563   A   G       13590   G   A       13594   A   G       13602   T   C       13611   A   G       13617   T   C       13641   T   C       13650   C   T       13651   A   G       13660   A   G       13708   G   A       13722   A   G       13734   T   C       13759   G   A       13780   A   G       13789   T   C       13803   A   G       13812   T   C       13818   T   C       13819   T   C       13827   A   G       13880   C   A       13886   T   C       13914   C   A       13924   C   T       13927   A   T       13928   G   C       13958   G   C       13965   T   C       13966   A   G       13980   G   A       14000   T   A       14016   G   A       14020   T   C       14022   A   G       14025   T   C       14034   T   C       14059   A   G       14070   A   T       14070   A   G       14088   T   C       14094   T   C       14097   C   T       14118   A   G       14128   A   G       14148   A   G       14152   A   G       14167   C   T       14178   T   C       14182   T   C       14200   T   C       14203   A   G       14209   A   G       14212   T   C       14215   T   C       14221   T   C       14233   A   G       14272   C   G       14284   C   T       14308   T   C       14311   T   C       14318   T   C       14319   T   C       14371   T   C       14374   T   C       14384   G   C       14455   C   T       14459   G   A       14470   T   C       14484   T   C       14488   T   C       14502   T   C       14560   G   A       14566   A   G       14569   G   A       14571   T   A       14580   A   G       14587   A   G       14605   A   G       14668   C   T       14693   A   G       14766   C   T       14769   A   G       14783   T   C       14793   A   G       14798   T   C       14812   C   T       14836   A   G       14861   G   A       14862   C   T       14905   G   A       14911   C   T       14971   T   C       14974   C   G       14979   T   C       15016   C   T       15034   A   G       15043   G   A       15110   G   A       15113   A   G       15115   T   C       15136   C   T       15172   G   A       15204   T   C       15217   G   A       15218   A   C       15229   T   C       15238   C   G       15244   A   G       15257   G   A       15261   G   A       15301   G   A       15317   G   A       15318   C   T       15323   G   A       15326   A   G       15346   G   A       15358   A   G       15431   G   A       15442   A   G       15452   C   A       15466   G   A       15470   T   C       15487   A   T       15497   G   A       15514   T   C       15519   T   C       15535   C   T       15607   A   G       15626   C   T       15629   T   C       15646   C   T       15661   C   T       15663   T   C       15670   T   C       15724   A   G       15731   G   A       15746   A   G       15766   A   G       15784   T   C       15793   C   T       15803   G   A       15806   G   A       15812   G   A       15824   A   G       15833   C   T       15849   C   T       15884   G   C       15900   T   C       15904   C   T       15907   A   G       15924   A   G       15927   G   A       15928   G   A       15930   G   A       15932   T   C       15939   C   T       15941   T   C       15942   T   C       15968   T   C       16017   T   C       16038   A   G       16051   A   G       16069   C   T       16071   C   T       16075   T   C       16086   T   C       16093   T   C       16108   C   T       16111   C   T       16114   C   A       16124   T   C       16126   T   C       16129   G   A       16129   G   C       16140   T   C       16144   T   C       16145   G   A       16147   C   T       16148   C   T       16153   G   A       16162   A   G       16163   A   C       16166   A   C       16167   C   T       16168   C   T       16169   C   T       16171   A   G       16172   T   C       16175   A   G       16176   C   T       16182   A   C       16183   A   C       16184   C   T       16185   C   T       16186   C   T       16187   C   T       16188   C   A       16188   C   G       16189   T   C       16192   C   T       16193   C   T       16207   A   G       16209   T   C       16212   A   G       16213   G   A       16214   C   T       16217   T   C       16219   A   G       16223   C   T       16224   T   C       16227   A   G       16229   T   C       16230   A   G       16231   T   C       16232   C   T       16234   C   T       16235   A   G       16239   C   T       16241   A   G       16242   C   T       16243   T   C       16245   C   T       16247   A   G       16249   T   C       16254   A   C       16255   G   A       16256   C   T       16257   C   T       16258   A   G       16260   C   T       16261   C   T       16264   C   T       16265   A   C       16266   C   T       16268   C   T       16270   C   T       16271   T   C       16274   G   A       16278   C   T       16284   A   G       16286   C   G       16287   C   T       16288   T   C       16290   C   T       16291   C   T       16292   C   T       16293   A   G       16294   C   T       16296   C   T       16298   T   C       16304   T   C       16309   A   G       16311   T   C       16316   A   G       16317   A   T       16318   A   T       16319   G   A       16320   C   T       16324   T   C       16325   T   C       16326   A   G       16327   C   T       16343   A   G       16344   C   T       16354   C   T       16355   C   T       16356   T   C       16357   T   C       16360   C   T       16362   T   C       16366   C   T       16368   T   C       16390   G   A       16391   G   A       16399   A   G       16438   G   A       16439   C   A       16483   G   A       16519   T   C       16527   C   T                  
 
      Table 4 lists the nucleotide alleles identified in 48 mitochondrial genomes as compared to the Cambridge sequence.  
               TABLE 4                          Human MtDNA Nucleotide Alleles in 48 Genomes                                  non-       nucleotide   Cambridge   Cambridge       locus   alleles   alleles                                 64   C   T       72   T   C       73   A   G       89   T   C       93   A   G       95   A   C       114   C   T       146   T   C       150   C   T       151   C   T       152   T   C       153   A   G       171   G   A       180   T   C       182   C   T       185   G   A       185   G   T       186   C   A       189   A   C       194   C   T       195   T   C       198   C   T       199   T   C       200   A   G       204   T   C       207   G   A       210   A   G       217   T   C       225   G   A       227   A   G       228   G   A       235   A   G       236   T   C       247   G   A       250   T   C       263   A   G       295   C   T       297   A   G       316   G   A       317   C   G       320   C   T       325   C   T       340   C   T       357   A   G       400   T   G       418   C   T       456   C   T       462   C   T       467   C   T       482   T   C       489   T   C       493   A   G       499   G   A       508   A   G       597   C   T       663   A   G       680   T   C       709   G   A       710   T   C       750   A   G       769   G   A       825   T   A       827   A   G       921   T   C       930   G   A       961   T   C       961   T   G       1018   G   A       1048   C   T       1189   T   C       1243   T   C       1290   C   T       1406   T   C       1415   G   A       1438   A   G       1442   G   A       1598   G   A       1700   T   C       1703   C   T       1706   C   T       1709   G   A       1715   C   T       1719   G   A       1736   A   G       1738   T   C       1780   T   C       1811   A   G       1888   G   A       2092   C   T       2245   A   C       2245   A   G       2308   A   G       2332   C   T       2352   T   C       2358   A   G       2416   T   C       2581   A   G       2639   C   T       2706   A   G       2758   G   A       2768   A   G       2789   C   T       2834   C   T       2857   T   C       2885   T   C       3010   G   A       3083   T   C       3197   T   C       3200   T   A       3202   T   C       3221   A   G       3308   T   C       3316   G   A       3394   T   C       3450   C   T       3480   A   G       3505   A   G       3516   C   A       3516   C   G       3547   A   G       3552   T   A       3552   T   C       3565   A   G       3594   C   T       3644   T   C       3666   G   A       3693   G   A       3720   A   G       3756   A   G       3796   A   G       3796   A   T       3796   A   C       3808   A   G       3816   A   G       3834   G   A       3843   A   G       3847   T   C       3866   T   C       3921   C   A       3970   C   T       3981   A   G       4025   C   T       4040   C   T       4044   A   G       4086   C   T       4104   A   G       4122   A   G       4123   A   G       4158   A   G       4216   T   C       4221   C   T       4225   A   G       4232   T   C       4248   T   C       4312   C   T       4336   T   C       4370   T   C       4454   T   A       4529   A   C       4529   A   T       4580   G   A       4586   T   C       4596   G   A       4646   T   C       4715   A   G       4767   A   G       4769   A   G       4820   G   A       4824   A   G       4833   A   G       4841   G   A       4883   C   T       4907   T   C       4917   A   G       4960   C   T       4977   T   C       5027   C   T       5036   A   G       5043   G   T       5046   G   A       5096   T   C       5108   T   C       5147   G   A       5153   A   G       5178   C   A       5231   G   A       5300   C   T       5331   C   A       5390   A   G       5393   T   C       5417   G   A       5426   T   C       5442   T   C       5460   G   A       5465   T   C       5471   G   A       5495   T   C       5581   A   G       5601   C   T       5603   C   T       5606   C   T       5633   C   T       5711   A   G       5773   G   A       5814   T   C       5951   A   G       5984   A   G       6026   G   A       6029   C   T       6045   C   T       6071   T   C       6152   T   C       6185   T   C       6221   T   C       6227   T   C       6257   G   A       6371   C   T       6392   T   C       6473   C   T       6491   C   A       6607   T   C       6680   T   C       6713   C   T       6734   G   A       6752   A   G       6776   T   C       6815   T   C       6827   T   C       6962   G   A       6989   A   G       7028   C   T       7052   A   G       7055   A   G       7146   A   G       7154   A   G       7175   T   C       7196   C   A       7256   C   T       7271   A   G       7274   C   T       7389   T   C       7424   A   G       7476   C   T       7521   G   A       7561   T   C       7600   G   A       7624   T   A       7664   G   A       7694   C   T       7765   A   G       7771   A   G       7864   C   T       7867   C   T       7933   A   G       7999   T   C       8027   G   A       8080   C   T       8087   T   C       8113   C   A       8142   C   T       8149   A   G       8152   G   A       8155   G   A       8185   T   C       8200   T   C       8206   G   A       8248   A   G       8251   G   A       8260   T   C       8269   G   A       8271-8279   A   DEL       8286   T   C       8298   T   C       8344   A   G       8387   G   A       8389   A   G       8392   G   A       8414   C   T       8428   C   T       8448   T   C       8460   A   G       8468   C   T       8472   C   T       8545   G   A       8553   C   T       8563   A   G       8566   A   G       8584   G   A       8618   T   C       8655   C   T       8697   G   A       8701   A   G       8705   T   C       8709   C   T       8721   A   G       8790   G   A       8794   C   T       8836   A   G       8856   G   A       8860   A   G       8875   T   C       8913   A   G       8962   A   G       8994   G   A       9042   C   T       9053   G   A       9055   G   A       9072   A   G       9077   T   C       9090   T   C       9093   A   C       9114   A   G       9120   A   G       9123   G   A       9151   A   G       9221   A   G       9237   G   A       9325   T   C       9335   C   T       9347   A   G       9355   A   G       9377   A   G       9402   A   C       9449   C   T       9456   A   G       9477   G   A       9540   T   C       9545   A   G       9548   G   A       9559   C   G       9575   G   A       9632   A   G       9682   T   C       9698   T   C       9755   G   A       9818   C   T       9822   C   A       9911   C   T       9932   G   A       9950   T   C       9957   T   C       9966   G   A       10034   T   C       10086   A   G       10086   A   C       10115   T   C       10151   A   G       10152   G   C       10172   G   A       10182   G   C       10238   T   C       10256   T   C       10310   G   A       10321   T   C       10325   G   A       10398   A   G       10400   C   T       10414   G   T       10463   T   C       10550   A   G       10586   G   A       10589   G   A       10609   T   C       10637   C   T       10646   G   A       10659   C   T       10664   C   T       10688   G   A       10790   T   C       10810   T   C       10828   T   C       10873   T   C       10876   A   G       10915   T   C       10966   T   C       10984   C   G       11002   A   G       11078   A   G       11092   A   G       11147   T   C       11167   A   G       11172   A   G       11176   G   A       11177   C   T       11215   C   T       11251   A   G       11257   C   T       11299   T   C       11332   C   T       11362   A   G       11377   G   A       11467   A   G       11476   C   T       11536   C   T       11590   A   G       11641   A   G       11674   C   T       11719   G   A       11767   C   T       11812   A   G       11854   T   C       11899   T   C       11914   G   A       11944   T   C       11947   A   G       11969   G   A       12007   G   A       12083   T   G       12121   T   C       12172   A   G       12234   A   G       12236   G   A       12308   A   G       12358   A   G       12361   A   G       12372   G   A       12373   A   G       12397   A   G       12406   G   A       12414   T   C       12501   G   A       12507   A   G       12519   T   C       12528   G   A       12540   A   G       12612   A   G       12633   C   T       12669   C   T       12672   A   G       12693   A   G       12705   C   T       12720   A   G       12738   T   C       12810   A   G       12822   A   G       12882   C   T       12930   A   T       12948   A   G       12967   A   C       12972   A   G       13020   T   C       13068   A   G       13101   A   C       13104   A   G       13105   A   G       13194   G   A       13263   A   G       13276   A   G       13368   G   A       13440   C   G       13485   A   G       13494   C   T       13500   T   G       13506   C   T       13512   A   G       13563   A   G       13590   G   A       13617   T   C       13650   C   T       13708   G   A       13734   T   C       13759   G   A       13780   A   G       13789   T   C       13803   A   G       13812   T   C       13827   A   G       13880   C   A       13886   T   C       13914   C   A       13924   C   T       13928   G   C       13958   G   C       13966   A   G       14000   T   A       14016   G   A       14034   T   C       14059   A   G       14070   A   G       14088   T   C       14118   A   G       14128   A   G       14148   A   G       14167   C   T       14178   T   C       14200   T   C       14203   A   G       14215   T   C       14221   T   C       14233   A   G       14272   C   G       14284   C   T       14308   T   C       14318   T   C       14374   T   C       14459   G   A       14470   T   C       14484   T   C       14488   T   C       14502   T   C       14560   G   A       14566   A   G       14569   G   A       14668   C   T       14693   A   G       14766   C   T       14783   T   C       14793   A   G       14798   T   C       14836   A   G       14861   G   A       14905   G   A       14911   C   T       14974   C   G       15034   A   G       15043   G   A       15110   G   A       15115   T   C       15136   C   T       15172   G   A       15204   T   C       15217   G   A       15218   A   C       15238   C   G       15257   G   A       15261   G   A       15301   G   A       15317   G   A       15318   C   T       15323   G   A       15326   A   G       15431   G   A       15442   A   G       15452   C   A       15466   G   A       15487   A   T       15497   G   A       15519   T   C       15535   C   T       15607   A   G       15661   C   T       15724   A   G       15766   A   G       15784   T   C       15793   C   T       15806   G   A       15812   G   A       15824   A   G       15833   C   T       15849   C   T       15884   G   C       15900   T   C       15904   C   T       15907   A   G       15924   A   G       15928   G   A       15930   G   A       15939   C   T       15941   T   C       15968   T   C       16017   T   C       16051   A   G       16069   C   T       16086   T   C       16093   T   C       16108   C   T       16111   C   T       16114   C   A       16124   T   C       16126   T   C       16129   G   A       16129   G   C       16145   G   A       16148   C   T       16153   G   A       16162   A   G       16163   A   C       16167   C   T       16168   C   T       16172   T   C       16176   C   G       16182   A   C       16183   A   C       16184   C   T       16185   C   T       16186   C   T       16187   C   T       16188   C   A       16188   C   G       16189   T   C       16192   C   T       16193   C   T       16212   A   G       16213   G   A       16214   C   T       16217   T   C       16219   A   G       16223   C   T       16224   T   C       16227   A   G       16229   T   C       16230   A   G       16231   T   C       16232   C   T       16234   C   T       16235   A   G       16239   C   T       16243   T   C       16245   C   T       16249   T   C       16254   A   C       16255   G   A       16256   C   T       16258   A   G       16260   C   T       16261   C   T       16264   C   T       16265   A   C       16266   C   T       16270   C   T       16274   G   A       16278   C   T       16284   A   G       16290   C   T       16291   C   T       16292   C   T       16293   A   G       16294   C   T       16296   C   T       16298   T   C       16304   T   C       16309   A   G       16311   T   C       16317   A   T       16318   A   T       16319   G   A       16320   C   T       16325   T   C       16327   C   T       16355   C   T       16356   T   C       16360   C   T       16362   T   C       16366   C   T       16368   T   C       16390   G   A       16391   G   A       16399   A   G       16519   T   C                  
 
     Example 2  
      The mtDNA sequences of Example 1 were chosen because they represent all of the major haplogroup lineages in humans. Analysis of these sequences has reaffirmed that all human mtDNAs belong to a single maternal tree, rooted in Africa (R. L. Cann et al.,  Nature  325:31-36 (1987); M. J. Johnson et al., (1983)  Journal of Molecular Evolution  19:255-271; D. C. Wallace et al., “Global Mitochondrial DNA Variation and the Origin of Native Americans” in  The Origin of Humankind , M. Aloisi, B. Battaglia, E. Carafoli, G. A. Danieli, Eds., Venice (IOS Press, 2000); M. Ingman et al., (2000)  Nature  408:708-13; and D. C. Wallace et al., (1999)  Gene  238:211-230). A cladogram of these mtDNA sequences is shown in  FIG. 1 . Haplogroups are designated on branches of the tree. A calibration of the sequence evolution rate for the coding regions of the mtDNA, based on a human-chimpanzee divergence time of 6.5 million years ago (MYA) (M. Goodman et al., (1998)  Mol Phylogenet. Evol.  9:585-98), has permitted an estimate of the time to the most recent common ancestor (MRCA) of the human mtDNA phylogeny at ˜200,000 years before present (YBP), and an estimate of the time of the MRCA for each major haplogroup (Table 5).  
               TABLE 5                          Coalescence dates for haplogroups*                                     Time to MRCA ±               Sample   s.e. (×10 −4     Time to MRCA ±       Haplogroup   sizes   mutations per np)  a     s.e. (×10 3  years)  b                 chimp + human   1 + 104   818.05 ± 0.75    6,500       humans   104   24.88 ± 0.90    198 ± 19       L0   8   17.92 ± 1.87    142 ± 17       L1   9   17.81 ± 1.77    142 ± 17       L2   7   11.57 ± 1.30     91.9 ± 11.8       N   50   8.09 ± 0.53   64.3 ± 5.8       A   4   4.06 ± 0.92   32.3 ± 7.6       R   37   7.66 ± 0.51   60.9 ± 5.5       HV   15   3.61 ± 0.73   28.7 ± 6.1       H   11   2.40 ± 0.40   19.1 ± 3.4       V   3   1.71 ± 0.60   13.6 ± 4.8       JT   7   6.29 ± 0.74   50.0 ± 6.7       J   4   4.33 ± 0.87   34.4 ± 7.2       T   3   1.40 ± 0.55   11.1 ± 4.4       U   4   6.51 ± 0.66   51.7 ± 6.2       M   22   8.15 ± 0.74   64.8 ± 7.1       CZ   10   5.91 ± 0.87   47.0 ± 7.6       C   9   3.56 ± 0.65   28.3 ± 5.5       D   6   4.19 ± 0.67   33.3 ± 5.7       G   3   4.75 ± 0.93   37.7 ± 7.8                 * The high probability of reverse mutations in the control region led us to calculate the times to the MRCAs using the entire mtDNA, excluding the control region (np 577-16023).              a  Based on this value we estimated the average sequence evolution rate as (1.26 ± 0.08) × 10 −8  per nucleotide per year, using the HKY85 model (M. Hasegawa et al., (1985) J Mol. Evol. 22: 160-74 (1985)).              b  Standard errors calculated from the inverse hessian at the maximum of the likelihood do not include any uncertainty in the calibration point, and were calculated using the delta method. The coalescence times of the various haplogroups may well be underestimated because of their small sample size.             
 
     Example 3  
      Inter-Continental Founder Events  
      The most striking feature of the mtDNA tree is the remarkable reduction in the number of mtDNA lineages that are associated with the transition from one continent to another. For example, when humans moved to Eurasia from Africa, the number of mitochondrial lineages was reduced from dozens to two lineages. While northeastern Africa encompasses the entire range of African mtDNA variation from the exclusively African haplogroups L0-L2 to the progenitors of the European and Asian mtDNA lineages, only two African mtDNA lineages, macro-haplogroups M and N, which arose about 65,000 YBP, left Africa to colonize Eurasia. Moreover, the times of the MRCAs of macro-haplogroups M and N as well as sub-macro-haplogroup R are similar, suggesting rapid population expansion associated with the colonization of Eurasia.  
      Similarly, when humans later moved from Central Asia to the Americas, the number of lineages was again reduced from dozens to about five. There is great mtDNA diversity in Asia, yet this diversity is substantially reduced in Siberia, and only five mtDNA haplogroups (A, B, C, D, and X), which arose in Asia about 28,000-34,000 YBP, successfully crossed the Bering land bridge to occupy the Americas. Human mtDNA haplogroup migrations are depicted in  FIG. 2 .  
     Example 4  
      Further analysis demonstrated which alleles are descriptive of the major haplogroups, selected sub-haplogroups, and selected macro-haplogroups. The mtDNA nucleotide positions and the relevant alleles are shown in  FIG. 3 . The data is arranged as a cladogram, such that a group on the left contains all of the alleles to its right. A vertical bar designates that the alleles to the right of the bar are present in all of the groups to the left of the bar. The haplogroup data in  FIG. 3  is summarized in Tables 6 and 7. The sub-haplogroup data is summarized in Tables 8 and 9. Each group contains the alleles listed below it.  
                                               TABLE 6                       L0   L1   L2   L3   C   D   E   G   Z                  1048T   2352C   325T   2352C   3552C   4883T   16227G   4833G   11078G       3516A   3796C   680C   8618C   4715G   5178A       8200C   16185T       4312T   5951G   2416C   10086C   7196A   8414T       16017C   16224C       4586C   5984G   2758G   10398A   8584A   14668T       16129A   16260T       5442C   6071C   4158G   10819G   9545G   15487T       6185C   9072G   8206A   14212C   13263G   16362C   16362C       8113A   10586A   9221G   16124C   14318C       8251A   12810G   11944C   16278T   16298C               16298C       9347G   13485G   13803G   16362C   16327T       9402C   3666A   13958C       489C   489C   489C   489C   489C       9818T   7055G   16278T       10400T   10400T   10400T   10400T   10400T       10589A   7389C   16390G       14783C   14783C   14783C   14783C   14783C       10664T   13789C           15043A   15043A   15043A   15043A   15043A       10915C   14178C   15301A   15301A   15301A   15301A   15301A   15301A   15301A       12007A       13276G       13506T       825A   825A       2758A   2758A       2885C   2885C       7146G   7146G       8468T   8468T       8655T   8655T       10688A   10688A       10810C   10810C       13105G   13105G       769A   769A       1018A   1018A       3594T   3594T       4104G   4104G       7256T   7256T       7521A   7521A       13650T   13650T                  
 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
               
               
                 A 
                 I 
                 W 
                 X 
                 B 
                 F 
                 Y 
                 V 
                 H 
                 U 
                 J 
                 T 
               
               
                   
               
             
            
               
                 663G 
                 4529T 
                 204C 
                 1719A 
                   
                 12406A 
                 7933G 
                 72C 
                 2706A 
                 3197C 
                 295T 
                 11812G 
               
               
                 16290T 
                 10034C 
                 207A 
                 3516G 
                   
                 16304C 
                 8392A 
                 4580A 
                 7028C 
                 4646C 
                 489C 
                 12633T 
               
               
                 16319A 
                 16129A 
                 1243C 
                 6221C 
                   
                   
                 16126C 
                 15904T 
                   
                 7768G 
                 12612G 
                 14233G 
               
               
                   
                 16391A 
                 5046A 
                 14470C 
                   
                   
                 16231C 
                 16298C 
                   
                 9055A 
                 13708A 
                 16163C 
               
               
                   
                   
                 5460A 
                 16189C 
                 16189C 
                   
                 16266T 
                 73A 
                 73A 
                 11332T 
                 16069T 
                 16186T 
               
               
                   
                   
                 8251A 
                 16278T 
                   
                   
                   
                 11719G 
                 11719G 
                 11467G 
                   
                 16189C 
               
               
                   
                   
                 8994A 
                   
                   
                   
                   
                 14766C 
                 14766C 
                 12308G 
                   
                 1888A 
               
               
                   
                   
                 11947G 
                   
                   
                   
                   
                   
                   
                 12372A 
                   
                 4917G 
               
               
                   
                   
                 15884C 
                   
                   
                   
                   
                   
                   
                 13104G 
                   
                 8697A 
               
               
                   
                   
                 16292T 
                   
                   
                   
                   
                   
                   
                 14070G 
                   
                 10463C 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 15907G 
                   
                 13368A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16051G 
                   
                 14905A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16129C 
                   
                 15607G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16172C 
                   
                 15928A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16189C 
                   
                 16294T 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16219G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16224C 
                 4216C 
                 4216C 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16249C 
                 11251G 
                 11251G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16270T 
                 15452A 
                 15452A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16311T 
                 16126C 
                 16126C 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16318T 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16343G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 16356C 
               
               
                   
                   
                   
                   
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
               
               
                   
                   
                   
                   
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
               
               
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
               
               
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
               
               
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
               
               
                 L1a1 
                 L1a2 
                 L1b1 
                 L1b2 
                 L2a 
                 L2b 
                 L2c 
                 L3a 
                 L3b 
                 L3c 
                 L3d 
               
               
                   
               
             
            
               
                 4586C 
                 8113A 
                 2352C 
                 3796C 
                 13803G 
                 4158G 
                 325T 
                 23252C 
                 8618C 
                 10086C 
                 10398 
               
               
                 9818T 
                 8251A 
                   
                 5951G 
                   
                   
                 680C 
                 10819G 
                 16124C 
                 16124C 
               
               
                 1048T 
                 1048T 
                   
                 5984G 
                   
                   
                 13958C 
                 14212C 
                   
                 16278T 
               
               
                 3516A 
                 3516A 
                   
                 6071C 
                 2416C 
                 2416C 
                 2416C 
                   
                   
                 16362C 
               
               
                 4312T 
                 4312T 
                   
                 9072G 
                 2758G 
                 2758G 
                 2758G 
               
               
                 5442C 
                 5442C 
                   
                 10586A 
                 8206A 
                 8206A 
                 8206A 
               
               
                 6185C 
                 6185C 
                   
                 12810G 
                 9221G 
                 9221G 
                 9221G 
               
               
                 9402C 
                 9402C 
                   
                 13485G 
                 11944C 
                 11944C 
                 11944C 
               
               
                 9347G 
                 9347G 
                 3666A 
                 3666A 
                 16278T 
                 16278T 
                 16278T 
               
               
                 10589A 
                 10589A 
                 7055G 
                 7055G 
                 16390G 
                 16390G 
                 16390G 
               
               
                 10664T 
                 10664T 
                 7389C 
                 7389C 
                 15301A 
                 15301A 
                 15301A 
                 15301A 
                 15301A 
                 15301A 
                 15301A 
               
               
                 10915C 
                 10915C 
                 13789C 
                 13789C 
               
               
                 12007A 
                 12007A 
                 14178C 
                 14178C 
               
               
                 13276G 
                 13276G 
               
               
                 13506T 
                 13506T 
               
               
                 825A 
                 825A 
                 825A 
                 825A 
               
               
                 2758A 
                 2758A 
                 2758A 
                 2758A 
               
               
                 2885C 
                 2885C 
                 2885C 
                 2885C 
               
               
                 7146G 
                 7146G 
                 7146G 
                 7146G 
               
               
                 8468T 
                 8468T 
                 8468T 
                 8468T 
               
               
                 8655T 
                 8655T 
                 8655T 
                 8655T 
               
               
                 10688A 
                 10688A 
                 10688A 
                 10688A 
               
               
                 10810C 
                 10810C 
                 10810C 
                 10810C 
               
               
                 13105G 
                 13105G 
                 13105G 
                 13105G 
               
               
                 769A 
                 769A 
                 769A 
                 769A 
                 769A 
                 769A 
                 769A 
               
               
                 1018A 
                 1018A 
                 1018A 
                 1018A 
                 1018A 
                 1018A 
                 1018A 
               
               
                 3594T 
                 3594T 
                 3594T 
                 3594T 
                 3594T 
                 3594T 
                 3594T 
               
               
                 4104G 
                 4104G 
                 4104G 
                 4104G 
                 4104G 
                 4104G 
                 4104G 
               
               
                 7256T 
                 7256T 
                 7256T 
                 7256T 
                 7256T 
                 7256T 
                 7256T 
               
               
                 7521A 
                 7521A 
                 7521A 
                 7521A 
                 7521A 
                 7521A 
                 7521A 
               
               
                 13650T 
                 13650T 
                 13650T 
                 13650T 
                 13650T 
                 13650T 
                 13650T 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                   
               
               
                 UK 
                 U7 
                 U6 
                 U5 
                 U4 
                 U3 
                 U2 
                 U1 
                 T* 
                 T1 
               
               
                   
               
             
            
               
                 9055A 
                 16318T 
                 16172C 
                 3197C 
                 4646C 
                 16343G 
                 15907G 
                 13104G 
                 11812G 
                 12633T 
               
               
                 16224C 
                   
                 16219G 
                 7768G 
                 11332T 
                   
                 16051G 
                 14070G 
                 14233G 
                 16163C 
               
               
                 16311T 
                   
                   
                 16270T 
                 16356C 
                   
                 16129C 
                 16189C 
                   
                 16186T 
               
               
                   
                   
                   
                   
                   
                   
                   
                 16249C 
                   
                 16189C 
               
               
                 11467G 
                 11467G 
                 11467G 
                 11467G 
                 11467G 
                 11467G 
                 11467G 
                 11467G 
                 1888A 
                 1888A 
               
               
                 12308G 
                 12308G 
                 12308G 
                 12308G 
                 12308G 
                 12308G 
                 12308G 
                 12308G 
                 4917G 
                 4917G 
               
               
                 12372A 
                 12372A 
                 12372A 
                 12372A 
                 12372A 
                 12372A 
                 12372A 
                 12372A 
                 8697A 
                 8697A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 10463C 
                 10463C 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 13368A 
                 13368A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 14905A 
                 14905A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 15607G 
                 15607G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 15928A 
                 15928A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 16294T 
                 16294T 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 4216C 
                 4216C 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 11251G 
                 11251G 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 15452A 
                 15452A 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 16126C 
                 16126C 
               
               
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
                 12705C 
               
               
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
                 16223C 
               
               
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
                 8701A 
               
               
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
                 9540T 
               
               
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
                 10873T 
               
               
                   
               
            
           
         
       
     
     Example 5  
      Further analysis of the data in  FIG. 3  demonstrated sets of nucleotide alleles useful for diagnosing the haplogroups. A set of nucleotide alleles useful for diagnosing all of the haplogroups and sub-haplogroups in  FIG. 3  is listed in Table 10. There are many equivalent methods for diagnosing the haplogroups. Examples of methods requiring testing only or a few loci follow. Alleles are identified in human samples containing mtDNA. Haplogroup L0 can be diagnosed by identifying 4586C, 9818T, or 8113A. Haplogroup L1 can be diagnosed by identifying 825A, 2758A, 2885C, 7146G, 8468T, 8655T, 10688A, 10810C, or 13105G. Haplogroup L2 can be diagnosed by identifying 2416C, 2758G, 8206A, 9221G, 11944C, or 16390G. Haplogroup L3 can be diagnosed by identifying 10819G, 14212C, 8618C, 10086C, 16362C, 10398A, or 16124C. Haplogroup C can be diagnosed by identifying 3552C, 4715G, 7196A, 8584A, 9545G, 13263G, 14318C, or 16327T. Haplogroup D can be diagnosed by identifying 4883T, 5178A, 8414T, 14668T, or 15487T. Haplogroup E can be diagnosed by identifying 16227G. Haplogroup G can be diagnosed by identifying 4833G, 8200C, or 16017C. Haplogroup Z can be diagnosed by identifying 11078G, 16185T, or 16260T. Haplogroup A can be diagnosed by identifying 663G, 16290T, or 16319A. Haplogroup I can be diagnosed by identifying 4529T, 10034C, or 16391A. Haplogroup W can be diagnosed by identifying 204C, 207A, 1243C, 5046A, 5460A, 8994A, 11947G, 15884C, or 16292T. Haplogroup X can be diagnosed by identifying 1719A, 3516G, 6221C, or 14470C. Haplogroup F can be diagnosed by identifying 12406A or 16304C. Haplogroup Y can be diagnosed by identifying 7933G, 8392A, 16231C, or 16266T. Haplogroup U can be diagnosed by identifying 3197C, 4646C, 7768G, 9055A, 11332T, 13104G, 14070G, 15907G, 16051G, 16129C, 16172C, 16219G, 16249C, 16270T, 16311T, 16318T, 16343G, or 16356C. Haplogroup J can be diagnosed by identifying 295T, 12612G, 13708A, or 16069T. Haplogroup T can be diagnosed by identifying 11812G, 12633T, 14233G, 16163C, 16186T, 1888A, 4917G, 8697A, 10463C, 13368A, 14905A, 15607G, 15928A, or 16294T. Haplogroup V can be diagnosed by identifying 72C, 4580A, or 15904T. Haplogroup H can be diagnosed by identifying 2706A or 7028C. Diagnosis of haplogroup B is more complicated, requiring three steps. Haplogroup B can be diagnosed by identifying 16189C; and by identifying the absence of 1719A, 3516G, 6221C, 14470C, or 16278T; and by identifying the absence of 1888A, 4216C, 4917G, 8697A, 10463C, 11251G, 11467G, 12308G, 12372A, 12633T, 13104G, 13368A, 14070G, 14905A, 15452A, 15607G, 15928A, 16126C, 16163C, 16186T, 16249C, or 16294T.  
               TABLE 10                       Nucleotide Alleles Useful for Diagnosing Human Haplogroups                                        72   C       204   C       207   A       295   T       663   G       825   A       1243   C       1719   A       1888   A       2416   C       2706   A       2758   A       2758   G       2885   C       3197   C       3516   G       3552   C       4216   C       4529   T       4580   A       4586   C       4646   C       4715   G       4833   G       4883   T       4917   G       5046   A       5178   A       5460   A       6221   C       7028   C       7146   G       7196   A       7768   G       7933   G       8113   A       8200   C       8206   A       8392   A       8414   T       8468   T       8584   A       8618   C       8655   T       8697   A       8994   A       9055   A       9221   G       9545   G       9818   T       10034   C       10086   C       10398   A       10463   C       10688   A       10810   C       10819   G       11078   G       11251   G       11332   T       11467   G       11812   G       11944   C       11947   G       12308   G       12372   A       12406   A       12612   G       12633   T       13104   G       13105   G       13263   G       13368   A       13708   A       14070   G       14212   C       14233   G       14318   C       14470   C       14668   T       14905   A       15301   A       15452   A       15487   T       15607   G       15884   C       15904   T       15907   G       15928   A       16017   C       16051   G       16069   T       16124   C       16126   C       16129   C       16163   C       16172   C       16185   T       16186   T       16219   G       16227   G       16231   C       16249   C       16260   T       16266   T       16270   T       16278   T       16290   T       16292   T       16294   T       16304   C       16311   T       16318   T       16319   A       16327   T       16343   G       16356   C       16362   C       16390   G       16391   A                  
 
      Additional alleles are included in Table 11. These alleles are useful for designing equivalent methods, to those described above, for diagnosing the haplogroups. Alleles in Table 11 are useful for designing efficient methods for diagnosing macro-haplogroups. The data in Tables 10 and 11 and  FIG. 3  are also useful for identifying sub-haplogroups. This invention provides a method for diagnosing sub-haplogroup L1a1 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 4586C and 9818T. This invention provides a method for diagnosing sub-haplogroup L1a2 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 8113A and 8251A. This invention provides a method for diagnosing sub-haplogroup L1b1 by identifying in a human sample, the nucleotide allele 2352C and one of the nucleotide alleles selected from the group consisting of 3666A, 7055G, 7389C, 13789C, and 14178C. This invention provides a method for diagnosing sub-haplogroup L1b2 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 3796C, 5951G, 5984G, 6071C, 9072G, 10586A, 12810G, and 13485G. This invention provides a method for diagnosing sub-haplogroup L2a by identifying in a human sample the nucleotide allele 13803G. This invention provides a method for diagnosing sub-haplogroup L2b by identifying in a human sample the nucleotide allele 4158G. This invention provides a method for diagnosing sub-haplogroup L2c by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 325T, 680C, and 13958C. This invention provides a method for diagnosing sub-haplogroup L3a by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 2325C, 10819G, and 14212C. This invention provides a method for diagnosing sub-haplogroup L3b by identifying in a human sample the nucleotide allele 8618C. This invention provides a method for diagnosing sub-haplogroup L3c by identifying in a human sample the nucleotide allele 10086C. This invention provides a method for diagnosing sub-haplogroup L3d by identifying in a human sample the nucleotide allele 10398A. This invention provides a method for diagnosing sub-haplogroup Uk by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 9055A and 16311T. This invention provides a method for diagnosing sub-haplogroup U7 by identifying in a human sample the nucleotide allele 16318T. This invention provides a method for diagnosing sub-haplogroup U6 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 16172C and 16219G. This invention provides a method for diagnosing sub-haplogroup U5 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 3197C, 7768G, and 16270T. This invention provides a method for diagnosing sub-haplogroup U4 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 4646C, 11332T, 16356C. This invention provides a method for diagnosing sub-haplogroup U3 by identifying in a human sample the nucleotide allele 16343G. This invention provides a method for diagnosing sub-haplogroup U2 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 15907G, 16051G, and 16129C. This invention provides a method for diagnosing sub-haplogroup U1 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 13104G, 14070G, 16189C, and 16249C. This invention provides a method for diagnosing sub-haplogroup T* by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 11812G and 14233G. This invention provides a method for diagnosing sub-haplogroup T1 by identifying in a human sample, one of the nucleotide alleles selected from the group consisting of 12633T, 16163C, and 16186T.  
               TABLE 11                       Nucleotide Alleles Useful for Diagnosing       Human Haplogroups and Macro-Haplogroups                                        72   C       73   A       204   C       207   A       295   T       325   T       489   C       663   G       680   C       769   A       825   A       1018   A       1048   T       1243   C       1719   A       1888   A       2352   C       2416   C       2706   A       2758   A       2758   G       2885   C       3197   C       3516   A       3516   G       3552   C       3594   T       3666   A       3796   C       4104   G       4158   G       4216   C       4312   T       4529   T       4580   A       4586   C       4646   C       4715   G       4833   G       4883   T       4917   G       5046   A       5178   A       5442   C       5460   A       5951   G       5984   G       6071   C       6185   C       6221   C       7028   C       7055   G       7146   G       7196   A       7256   T       7389   C       7521   A       7768   G       7933   G       8113   A       8200   C       8206   A       8251   A       8392   A       8414   T       8468   T       8584   A       8618   C       8655   T       8697   A       8701   A       8994   A       9055   A       9072   G       9221   G       9347   G       9402   C       9540   T       9545   G       9818   T       10034   C       10086   C       10398   A       10400   T       10463   C       10586   A       10589   A       10664   T       10688   A       10810   C       10819   G       10873   T       10915   C       11078   G       11251   G       11332   T       11467   G       11719   G       11812   G       11944   C       11947   G       12007   A       12308   G       12372   A       12406   A       12612   G       12633   T       12705   C       12810   G       13104   G       13105   G       13263   G       13276   G       13368   A       13485   G       13506   T       13650   T       13708   A       13789   C       13803   G       13958   C       14070   G       14178   C       14212   C       14233   G       14318   C       14470   C       14668   T       14766   C       14783   C       14905   A       15043   A       15301   A       15452   A       15487   T       15607   G       15884   C       15904   T       15907   G       15928   A       16017   C       16051   G       16069   T       16124   C       16126   C       16129   A       16129   C       16163   C       16172   C       16185   T       16186   T       16189   C       16219   G       16223   C       16224   C       16227   G       16231   C       16249   C       16260   T       16266   T       16270   T       16278   T       16290   T       16292   T       16294   T       16298   C       16304   C       16311   T       16318   T       16319   A       16327   T       16343   G       16356   C       16362   C       16390   G       16391   A                  
 
      An equivalent method for diagnosing a haplogroup is diagnosing haplogroup L0 by identifying the presence of one of 825A, 2758A, 2885C, 7146G, 8468T, 8655T, 10688A, 10810C, or 13105G; and identifying the absence of one of 3666A, 7055G, 7389C, 13789C, or 14178C. Other equivalent methods can be derived from the data in  FIG. 3 , and are within the scope of this invention.  
     Example 6  
      Lebers Hereditary Optic Neuropathy (LHON) is a form of blindness caused by mitochondrial DNA (mtDNA) mutations. Four mutations, 3460A, 11778A, 14484C, and 14459A, account for over 90% of LHON worldwide and are designated “primary” mutations. Primary mutations strongly predispose carriers to LHON, are not found in controls, are all in Complex I genes, and do not co-occur with each other. It has been demonstrated that the 11778A and 14484C mutations occurred more frequently than expected in association with European mtDNA haplogroup J (found in 9% of European-derived mtDNAs), suggesting a synergistic interaction among mtDNA mutations increased the probability of disease expression. Sequence analysis of two Russian LHON families without primary LHON mutations, including removal of nucleotide alleles listed in Table 3, demonstrated two new complex I mutations, 3635A and 4640C. Venous blood samples were obtained from the family members. Genomic DNA was isolated from the buffy coat blood fraction using Chelex 100 (Cetus, Emberyville, Calif., USA). mtDNA was amplified by PCR in 2-3 kb fragments, purified on Centricon 100 columns, and cycle-sequenced using BigDye Terminators (ABI/Perkin Elmer Cetus) and an ABI Prism 377 automated DNA sequencer. The mutations were confirmed using mutation-specific restriction enzyme digestion following mismatched-primer PCR amplification of white blood cell mtDNA (Brown M. D. et al., (1995)  Human Mutat.  6:311-325).  
     Example 7  
      A new primary LHON mtDNA mutation, 10663C, affecting a Complex I gene was homoplasmic in 3 Caucasian LHON families, all of which belonged to haplogroup J. These 3 families were the only haplogroup J-associated LHON families (out of 17) that did not harbor a known, primary LHON mutation. Comprehensive phylogenetic analysis of haplogroup J using complete mtDNA sequences demonstrated that the 10663C variant has arisen 3 independent times on this background. This mutation was not present in over 200 non-haplogroup J European controls, 74 haplogroup J patient and control mtDNAs, or 36 putative LHON patients without primary mutations. A partial Complex I defect was found in 10663C-containing lymphoblast and cybrid mitochondria. Thus, the 10663C mutation has occurred three independent times, each time on haplogroup J and only in LHON patients without a known LHON mutation. This makes the 10663C mutation unique among all pathogenic mtDNA mutations in that it appears to require the genetic background provided by haplogroup J for expression. These results provide further evidence for the predisposing role of haplogroup J and for the paradigm of “mild” mtDNA mutations interacting in an additive way to precipitate disease expression. Europeans with the mild ND6 np 14484 and ND3 np 10663 Leber&#39;s Hereditary Optic Neuropathy (LHON) missense mutations are more prone to blindness if they also possess the mtDNA haplogroup J.  
     Example 8  
      To assess the importance of demographic factors in inter-continental mtDNA sequence radiation, deviations from the standard neutral model were tested for in the distribution of mtDNA sequence variants using the Tajima&#39;s D and Fu and L1 D* tests (Y. X. Fu, W. H. L1, (1993)  Genetics  133:693-709. and F. Tajima, (1989)  Genetics  123, 585-95). The standard neutral model of population genetics assumes a random-mating population of constant size, with all mutations uniquely arising and selectively neutral. The continental frequency distribution of pairwise mtDNA sequence differences was calculated to test for rapid population expansion using the method of A. R. Rogers, H. Harpending, (1992)  Mol. Biol. Evol.  9:552-569.  
      For the African mtDNA sequences (n=32), the results did not significantly deviate from the standard neutral model, and the frequency distribution of pairwise sequence difference counts was broad and ragged. Both of these results are consistent with the model that the African population has been relatively stable for a long time. By contrast, the non-African mtDNAs (n=72) showed a highly significant deviation from neutrality (Tajima&#39;s D=−2.43, P&lt;0.01; Fu and L1 D*=−5.09, P&lt;0.02), as well as a bell-shaped frequency distribution of pairwise sequence differences. Thus, these results are consistent with population expansions having distorted the frequency distribution (L. Excoffier,  J. Mol. Evol.  30:125-39 (1990) and D. A. Merriwether et al. (1991)  J. Mol. Evol  33:543-555).  
      To better define the regional distribution of these demographic influences, the Eurasian samples were divided into European and Asian plus Native American. Analysis of all European mtDNAs also revealed significant deviations from the standard neutral model (Tajima&#39;s D=−2.19, P&lt;0.01; Fu and Li D*=−3.31, P&lt;0.02). The distribution of pairwise sequence differences for the European mtDNAs revealed two sharp peaks, hinting at two major expansion phases. The most recent of these peaks was lost when haplogroup H and V mtDNAs were deleted from the sample. Hence, haplogroup H, which represents 40% of modern European mtDNAs (A. Torroni et al., American Journal of Human Genetics 62, 1137-1152 (1998)) and has a MRCA of 19,000 YBP, came to predominate in Europe relatively recently.  
      Analysis of the aggregated Asian and Native American mtDNAs (n=41) also revealed significant deviations from the standard neutral model (Tajima&#39;s D=−2.28, P&lt;0.01, Fu and Li D*=−4.31; P&lt;0.02) as well as revealing a broad, bell-shaped distribution of pairwise differences consistent with rapid population expansion.  
      When the Asian-Native American haplogroups A, B, C, D and X mtDNAs (n=26) were analyzed separately, they also showed significant deviation from neutrality for the Fu and Li D* test (D*=−2.65, P&lt;0.05), although not for the Tajima&#39;s D test (D=−1.60, ns). Their distribution of pairwise sequence differences was also strongly uni-modal, indicating that the population expanded as people moved through Siberia and Beringia and into the Americas.  
     Example 9  
      Variable Replacement Mutation Rates in Human mtDNA Genes  
      To determine if selection was an important factor in causing the sudden shifts in mtDNA sequence variation between continents, the number of non-synonymous to synonymous base substitutions was analyzed for all 13 mtDNA protein genes of those haplogroups which contributed to the colonization of each of the major continental spaces: African, European, and Native American. For example, for the “Native Americans” the mtDNAs from the Asian-Native American haplogroups A, B, C, D and X were combined. The Asian-Native American mtDNAs from the haplogroups were combined because random mutations accumulate in founder populations and those mtDNAs which prove advantageous in new environments are enriched. Hence, the founding mutations of the haplogroup are important in the continental success of the lineage. We then tested for possible selective effects during the colonization of each continent by comparing the ratio of non-synonymous versus synonymous nucleotide substitutions for each mtDNA gene. An increase in the non-synonymous to synonymous mutation ratio suggests that selection has favored the propagation of a functionally altered protein.  
      The comparison of the ratio of nonsynonymous to synonymous mutations, counting each change only once, revealed great variation between continents for several genes (Table 12). Marked increases in the accumulation of non-synonymous mutations were seen for ND3 in Africans, Cytb and COIII in Europeans, and ATP6 in Native Americans. The number of non-synonymous and synonymous mutations for each gene was also compared between the different continents by computing the P value using a Two-tailed Fisher Exact Test. This revealed significant differences between Africans and both Europeans and Native Americans for COIII, between Africans and Native Americans for ATP6, and between Africans and Europeans for the sum of all mtDNA genes (Table 12). Hence, this analysis supports the hypothesis that selection has played a role in shaping continental mtDNA protein variation.  
                           TABLE 12*                                          Two-Tail FET           Number of Polymorphic Sites   P-value                                             African   European   Native American   Afr   Afr   Eur                                                                     N-               N-           N-       vs   vs   vs       Gene   syn   Syn   Ratio   Syn   syn   Ratio   Syn   syn   Ratio   Eur   Am   Am                                                                         ND1   10   17   0.59   5   5         4   4         0.71   0.69   1.00       ND2   9   22   0.41   4   9   0.44   3   7   0.43   1.00   1.00   1.00       ND3   6   2         1   3   0.33   1   4   0.25   0.22   0.10   1.00       ND4L   0   7   0.00   0   1   0.00   1   4   0.25   1.00   0.42   1.00       ND4   4   35   0.11   2   13   0.15   3   12   0.25   1.00   0.38   1.00       ND5   15   31   0.48   8   20   0.40   2   14   0.14   0.80   0.19   0.28       ND6   2   14   0.14   1   6   0.17   3   5   0.60   1.00   0.29   0.57       Cytb   11   19   0.58   14   9         5   12   0.42   0.10   0.75   0.60       COI   7   30   0.23   0   9   0.00   0   13   0.00   0.32   0.17   1.00       COII   3   19   0.16   0   4   0.00   2   6   0.33   1.00   0.59   0.52       COIII   1   13   0.08   6   5         7   10   0.70               0.70       ATP6   3   15   0.20   5   6   0.83   7   5         0.20         0.68       ATP8   2   3   0.67   2   0       1   3   0.33   0.43   1.00   0.40       Total   73   227   0.32   48   90   0.53   39   99   0.39         0.41   0.30                 *Replacement versus synonymous mutation numbers of mtDNA genes. Rplmt = replacement mutations, ratio = rplmt/silent. FET = Fisher Exact Test. Afr = Africa, Eur = Europe, Am = Native American. The ratios of polymorphic sites in bold-italics highlight some of the higher values observed. Those in bold-italics under Two-Tailed FET indicate comparisons that are significant at the 0.05 level.             
 
     Example 10  
      Since the above analysis counts each mutation only once, irrespective of its frequency within the haplogroup, it under-emphasizes the importance of nodal mutations and over-emphasizes the importance of terminal private polymorphisms. As an alternative to this approach, we calculated the corrected non-synonymous (K a ) and synonymous (K s ) mutation frequencies and then determined the relative selective constraints acting on that gene by calculating the k C  value {k C =−1n(K a /K s )}. A high k C  value is indicative of high protein sequence conservation and low amino acid variation, while a low value is indicative of low protein conservation and high amino acid variation (N. Neckelmann et al., (1987)  Proc. Natl. Acad. Sci. USA  84:7580-7584).  
      The k C  values for each human mtDNA gene were compared across the total global collection of human mtDNA sequences ( FIG. 4 ). The ATP6 gene was the least conserved gene in the human mtDNA, though previously it had been shown to be relatively highly conserved in inter-specific comparisons (N. Neckelmann et al., (1987)  Proc. Natl. Acad. Sci. USA  84:7580-7584).  
     Example 11  
      The higher inter-specific conservation of ATP6 was confirmed by comparing the k C  values of human versus chimpanzee ( Pan troglodytes ) and bonobo ( Pan paniscus ); human versus eight primate species (baboon, Bomeo and Sumatran orangutan, gibbon, gorilla, lowland gorilla, bonobo, and chimpanzee); and human versus 13 diverse mammalian species (bovine, mouse, cat, dog, pig, rat, rhinoceros, horse, gibbon, gorilla, orangutan, bonobo, chimpanzee) ( FIG. 3 ). Thus, while ATP6 is highly conserved between species, it is very poorly conserved within humans. These results are consistent with the reduced intra-specific versus inter-specific conservation observed for other genes (C. A. Wise et al., (1998)  Genetics  148:409-21), and with the hypothesis that mitochondrial protein variation is accelerated in humans and other primates, as seen in cytochrome c oxidase genes (L. I. Grossman et al., (2001)  Mol. Phylogenet. EvoL.  18:26-36).  
     Example 12  
      To further investigate the possibility that individual mtDNA protein genes differ in their selective constraints in different human continental populations, k C  values for all 13 mtDNA protein genes from each set of continental haplogroups were calculated: African, European, and the Native American. The cumulative selective pressure that separated the mtDNAs of pairs of continents by pair-wise comparison of the k C  values was calculated for the genes of each mtDNA (Table 13). Comparison of mtDNA protein k C  values in Europeans versus Africans revealed that three genes (ND1, cytb and COIII) had significantly lower sequence conservation in Europeans. A comparison of the kc values of Native American versus African mtDNA genes revealed six genes (ND4, ND6, COII, COIII, ATP6 and ATP8) that had significantly lower sequence conservation in Native Americans. Finally, comparison of the k C  values of Africans versus Europeans or Native Americans revealed four mtDNA genes (ND3, ND5, cytb, and COI) had significantly lower sequence conservation in Africans. The greatest differences in k C  values were seen for the comparisons of COIII and ATP6 between Africans and Native Americans and for COIII between African and Europeans (Table 13).  
                                   TABLE 13*                                       Native American               African   European       sequences           sequences   sequences   T-test   {A, B, C, D, X}   T-test       GENES   (n = 32)   (n = 31)   P value   (n = 26)   P value                  ND1   2.08 ± 1.18   0.27 ± 1.90   P &lt; 0.0001   2.07 ± 1.92   NS       ND2   1.72 ± 1.07   1.57 ± 1.85   NS   1.81 ± 1.11   NS       ND3   0.51 ± 1.87   0.91 ± 2.32   NS   1.70 ± 1.32   P &lt; 0.01       ND4L   *   *   NS   2.41 ± 3.83   *       ND4   3.49 ± 1.34   3.39 ± 2.23   NS   2.20 ± 1.19   P &lt; 0.001       ND5   1.78 ± 0.71   2.20 ± 1.20   NS   3.63 ± 3.56   P &lt; 0.01       ND6   2.51 ± 1.19   3.13 ± 3.99   NS   1.15 ± 1.52   P &lt; 0.001       Cytb   1.89 ± 0.96   0.34 ± 1.51   P &lt; 0.0001   2.46 ± 1.15   P &lt; 0.05       COI   2.37 ± 0.95   3.85 ± 3.93   P &lt; 0.05   *   *       COII   2.73 ± 1.32   *   *   1.74 ± 2.12   P &lt; 0.05       COIII   4.65 ± 3.94   0.94 ± 2.08   P &lt; 0.0001   2.11 ± 1.26   P &lt; 0.01       ATP6   2.31 ± 1.28   1.48 ± 2.28   NS   −0.14 ± 1.34     P &lt; 0.0001       ATP8   2.62 ± 1.89   *   *   1.25 ± 1.94   P &lt; 0.01                 *Estimates of coefficients of selective constraint (k c ) stratified by gene and region. k c  values and standard deviations calculated for African, European and Asian-American haplogroups A, B, C, D and X mtDNA protein-coding genes.            * indicates that k c  values could not be calculated, since either K s  or K a  were 0, Haplogroup X is represented only by the Native-American sequence, the European X sequence being excluded.             
 
      Taken together, these data show that different selective forces have acted on individual mtDNA genes as humans colonized different continents. Moreover, the observed differences in mtDNA protein sequence correlate with the climatic transitions that humans would have experienced as they migrated out of tropical and sub-tropical Africa and into temperate Eurasia and arctic Siberia and Beringia. The mtDNA genes that showed the highest amino acid sequence variation between continents were COM and ATP6.  
     Example 13  
      The nucleotide alleles in Table 3 residing in evolutionarily significant genes identified in Examples 9-12 were analyzed for evolutionary significance. Evolutionarily significant alleles reside in evolutionarily significant genes and cause amino acid changes. A list of the evolutionarily significant nucleotide alleles in ND1, ND2, ND3, ND4, ND5, ND6, Cytb, COI, COII, COIII, ATP6, and ATP8 appear in Table 14. The Cambridge nucleotide alleles in Table 14 are evolutionarily significant. These amino acid alleles, including the Cambridge alleles, are evolutionarily significant. The locations of the amino acid alleles are identified by the location of the nucleotide allele listed in Table 3. Other evolutionarily significant nucleotide alleles not listed in Table 14, include alleles at neighboring nucleotide loci that are within the same codon and code for the same amino acids that are listed in Table 14.  
               TABLE 14                          Evolutionarily Significant Human Mitochondrial       Nucleotide and Amino Acid Allele                                                 Non-                           Cambridge   Cambridge   Non-           Genome   Cambridge   Nucl.   Amino   Cambridge       Gene   Location   Nucleotide   Allele   Acid   AA Allele                                             ND1   3308   T   C   M   T       ND1   3316   G   A   A   T       ND1   3394   T   C   Y   H       ND1   3505   A   G   T   A       ND1   3547   A   G   I   V       ND1   3565   A   G   T   A       ND1   3644   T   C   V   A       ND1   3796   A   T   T   A       ND1   3796   A   G   T   S       ND1   3796   A   C   T   P       ND1   3808   A   G   T   A       ND1   3866   T   C   I   T       ND1   4025   C   T   T   M       ND1   4040   C   T   T   M       ND1   4048   G   A   D   N       ND1   4123   A   G   I   V       ND1   4216   T   C   Y   H       ND1   4225   A   G   M   V       ND1   4232   T   C   I   T       ND2   4491   G   A   V   I       ND2   4506   A   G   I   V       ND2   4512   G   A   A   T       ND2   4596   G   A   V   I       ND2   4695   T   C   V   I       ND2   4767   A   G   M   V       ND2   4824   A   G   T   A       ND2   4833   A   G   T   A       ND2   4917   A   G   N   D       ND2   4960   C   T   A   G       ND2   5043   G   T   A   S       ND2   5046   G   A   V   I       ND2   5178   C   A   L   M       ND2   5262   G   A   A   T       ND2   5263   C   T   A   V       ND2   5331   C   A   L   I       ND2   5442   T   C   F   L       ND2   5460   G   A   A   T       COI   6150   G   A   V   I       COI   6253   T   C   M   T       COI   6324   G   A   A   T       COI   6366   G   A   V   I       COI   6607   T   C   F   S       COI   7146   A   G   T   A       COI   7257   A   G   I   V       COI   7347   G   A   V   I       COI   7389   T   C   Y   H       COI   7444   G   A   TER   K       COII   7664   G   A   A   T       COII   7673   A   G   I   V       COII   7697   G   A   V   I       COII   8027   G   A   A   T       COII   8142   C   T   A   V       ATP8   8387   G   A   V   M       ATP8   8414   C   T   L   F       ATP8   8448   T   C   M   T       ATP8   8460   A   G   N   S       ATP8   8472   C   T   P   L       ATP8   8553   C   T   S   L       ATP6   8545   G   A   A   T       ATP6   8563   A   G   T   A       ATP6   8566   A   G   I   V       ATP6   8584   G   A   A   T       ATP6   8618   T   C   I   T       ATP6   8701   A   G   T   A       ATP6   8705   T   C   M   T       ATP6   8764   G   A   A   T       ATP6   8794   C   T   H   Y       ATP6   8836   A   G   M   V       ATP6   8860   A   G   T   A       ATP6   8875   T   C   F   L       ATP6   8962   A   G   T   A       ATP6   9053   G   A   S   N       ATP6   9055   G   A   A   T       ATP6   9077   T   C   I   T       ATP6   9103   T   C   F   L       ATP6   9136   A   G   I   V       ATP6   9151   A   G   I   V       COIII   9237   G   A   V   M       COIII   9325   T   C   M   T       COIII   9355   A   G   N   S       COIII   9456   A   G   I   V       COIII   9402   A   C   T   P       COIII   9477   G   A   V   I       COIII   9559   C   G   P   R       COIII   9591   G   A   V   I       COIII   9667   A   G   N   S       COIII   9682   T   C   M   T       COIII   9822   C   A   L   I       COIII   9957   T   C   F   L       COIII   9966   G   A   V   I       ND3   10086   A   G   N   D       ND3   10086   A   C   N   H       ND3   10152   G   C   E   Q       ND3   10182   G   C   D   H       ND3   10197   G   A   A   T       ND3   10321   T   C   V   A       ND3   10398   A   G   T   A       ND4L   10609   T   C   C   R       ND4   10816   A   T   K   N       ND4   10920   C   T   P   L       ND4   11016   G   A   S   N       ND4   11078   A   G   I   V       ND4   11150   G   A   A   T       ND4   11172   A   G   N   S       ND4   11177   C   T   P   S       ND4   11654   A   G   T   A       ND4   11909   A   G   T   A       ND4   11963   G   A   V   I       ND4   11969   G   A   A   T       ND4   12083   T   G   S   A       ND4   12134   T   C   S   P       ND5   12346   C   T   H   Y       ND5   12358   A   G   T   A       ND5   12361   A   G   T   A       ND5   12373   A   G   T   A       ND5   12397   A   G   T   A       ND5   12406   T   A   V   I       ND5   12635   T   C   I   T       ND5   12850   A   G   I   V       ND5   12940   G   A   A   T       ND5   12967   A   C   T   P       ND5   13104   A   G   I   V       ND5   13105   A   G   I   V       ND5   13135   G   A   A   T       ND5   13145   G   A   S   N       ND5   13276   A   G   M   V       ND5   13477   G   A   A   T       ND5   13651   A   G   T   A       ND5   13660   A   G   N   D       ND5   13708   G   A   A   T       ND5   13759   G   A   A   T       ND5   13780   A   G   I   V       ND5   13789   T   C   Y   H       ND5   13819   T   C   F   L       ND5   13880   C   A   S   Y       ND5   13886   T   C   L   P       ND5   13924   C   T   P   S       ND5   13927   A   T   S   C       ND5   13928   G   C   S   T       ND5   13958   G   C   G   A       ND5   13966   A   G   T   A       ND5   14000   T   A   L   Q       ND5   14059   A   G   I   V       ND5   14128   A   G   T   A       ND6   14178   T   C   I   V       ND6   14272   C   G   L   F       ND6   14318   T   C   N   S       ND6   14319   T   C   N   D       ND6   14384   G   C   A   G       ND6   14459   G   A   A   V       ND6   14484   T   C   M   V       ND6   14502   T   C   I   V       ND6   14571   T   A   S   C       CytB   14766   C   T   T   I       CytB   14769   A   G   N   S       CytB   14793   A   G   H   R       CytB   14798   T   C   F   L       CytB   14861   G   A   A   T       CytB   14862   C   T   A   V       CytB   14979   T   C   I   T       CytB   15110   G   A   A   T       CytB   15113   A   G   T   A       CytB   15204   T   C   I   T       CytB   15218   A   C   T   P       CytB   15218   A   G   T   A       CytB   15238   C   G   I   M       CytB   15257   G   A   D   N       CytB   15261   G   A   S   N       CytB   15317   G   A   A   T       CytB   15318   C   T   A   V       CytB   15323   G   A   A   T       CytB   15326   A   G   T   A       CytB   15431   G   A   A   T       CytB   15452   C   A   L   I       CytB   15497   G   A   G   S       CytB   15519   T   C   L   P       CytB   15663   T   C   I   T       CytB   15731   G   A   A   T       CytB   15746   A   G   I   V       CytB   15803   G   A   V   M       CytB   15806   G   A   A   T       CytB   15812   G   A   V   M       CytB   15824   A   G   T   A       CytB   15849   C   T   T   I       CytB   15884   G   C   A   P                  
 
      A subset of the alleles in Table 14 that are associated with predispositions to physiological conditions using the methods of this invention is listed in Table 15.  
               TABLE 15                          Amino Acid Alleles Associated with Physiological       Conditions in this Invention                                         Nucleotide   Amino Acid                   Alleles Useful   Alleles Useful   Haplogroups           Genome   for Diagnosing   for Diagnosing   Diagnosable       Gene   Location   Haplogroups   Haplogroups   by Alleles                                         ND1   3796   C   P   (L1b2)       ND2   4833   G   A   G       ND2   4917   G   D   T       ND2   5046   A   I   W       ND2   5178   A   M   D       ND2   5442   C   L   L0       ND2   5460   A   T   W       COI   7146   G   A   L0, L1       COI   7389   C   H   L1       ATP8   8414   T   F   D       ATP6   8584   A   T   C       ATP6   8618   C   T   (L3b)       ATP6   8701   A   T   A, I, W, X, B,                       F, Y, U, J, T,                       V, H       ATP6   9055   A   T   (Uk)       COIII   9402   C   P   L0       ND3   10086   C   H   (L3c)       ND3   10398   A   T   (L3d)       ND4   11078   G   V   Z       ND5   12406   A   I   F       ND5   13104   G   V   (U1)       ND5   13105   G   V   L0, L1       ND5   13276   G   V   L0       ND5   13708   A   T   J       ND5   13789   C   H   L1       ND5   13958   C   A   (L2c)       ND6   14178   C   V   L1       ND6   14318   C   S   C       CytB   14766   C   T   V, H       CytB   15452   A   I   J, T       CytB   15884   C   P   W                  
 
     Example 14  
      Continent-Specific Amino Acid Substitutions in ATP6  
      To further investigate the biological significance of the human continent-specific ATP6 amino acid substitutions, the amino acid conservation for each variable human position using 39 animal species mtDNAs (12 primates, 22 other mammals, four non-mammalian vertebrates, and  Drosophila ) was analyzed. This revealed that many of the ATP6 substitutions that are associated with particular mtDNA haplogroups alter evolutionarily conserved, and hence potentially functionally important, amino acids.  
      A threonine to alanine substitution at codon 59 (T59A, nucleotide location 8701-8703) in ATP6 separates the mtDNAs of macro-haplogroup N from the rest of the World. The polar threonine at position 59 is conserved in all great apes and some old-world monkeys.  
      Among the haplogroups of macro-haplogroup M, the related Siberian-Native American haplogroups C and Z are delineated by an A20T (nucleotide location 8584-8586) variant. A non-polar amino acid found in this position occurs in all animal species except for  Macaca, Papio, Balaenoptera  and  Drosophila.    
      Among the haplogroups of macro-haplogroup N, the non-R lineage N1b harbors two distinctive amino acid substitutions M104V (nucleotide location 8836-8838) and T146A. (nucleotides location 8962-8964) The methionine at position 104 is conserved in all mammals, and the thereon at position  146  is conserved throughout all animal mtDNAs. Moreover, the T146A substitution is within the same transmembrane α-helix as the pathogenic mutation L156R that alters the coupling efficiency of the ATP synthase and causes the NARP and Leigh syndromes (I. Trounce, S. Neill, D. C. Wallace, Proceedings of the National Academy of Sciences of the United States of America 91, 8334-8338 (1994)).  
      Also in macro-haplogroup A mtDNAs harbor a H90Y (nucleotide location  8794 - 8796 ) amino acid substitution. The histidine in this position is conserved in all placental mammals except  Pongo, Cebus  and  Loxodonta  and occurs within a highly conserved region. Furthermore, among the heterogeneous group of mtDNAs carrying the tRNA Lys -CoII 9bp deletion and arbitrarily assigned to haplogroup B, one mtDNA harbored a F193L (nucleotide location  9103 - 9105 ) substitution. This position is conserved in all mammals except  Pongo, Papio, Cebus  and  Erinaceus.    
      Since each of the MyDNA sequences used in this comparison of different species is derived from only one or two individuals, it is possible that the rare deviant cases are due to the accumulation of environmentally adaptive mutations in those species that parallel those in humans. Thus, the above ATP6 amino acid polymorphisms have the characteristics expected for evolutionary adaptive mutations.  
                       TABLE 16                       Nucleotide   Nucleotide           Locus   Alleles   WIPO code                                            64   CT   y       72   TC   y       73   AG   r       89   TC   y       93   AG   r       95   AC   m       114   CT   y       143   GA   r       146   TC   y       150   CT   y       151   CT   y       152   TC   y       153   AG   r       171   GA   r       180   TC   y       182   CT   y       183   AG   r       185   GAT   d       185   GAT   d       186   CA   m       189   ACG   v       189   ACG   v       194   CT   y       195   TAC   h       195   TAC   h       198   CT   y       199   TC   y       200   AG   r       204   TC   y       207   GA   r       208   TC   y       210   AG   r       212   TC   y       215   AG   r       217   TC   y       225   GA   r       227   AG   r       228   GA   r       235   AG   r       236   TC   y       247   GA   r       250   TC   y       252   TC   y       263   AG   r       291   AG   r       295   CT   y       297   AG   r       316   GA   r       317   CAG   v       317   CAG   v       320   CT   y       325   CT   y       340   CT   y       357   AG   r       373   AG   r       400   TG   k       408   TA   w       418   CT   y       456   CT   y       462   CT   y       465   CT   y       467   CT   y       471   TC   y       480   TC   y       482   TC   y       489   TC   y       493   AG   r       499   GA   r       508   AG   r       593   TC   y       597   CT   y       663   AG   r       678   TC   y       680   TC   y       709   GA   r       710   TC   y       721   TC   y       750   AG   r       769   GA   r       825   TA   w       827   AG   r       850   TC   y       921   TC   y       930   GA   r       961   TCG   b       961   TCG   b       1018   GA   r       1041   AG   r       1048   CT   y       1119   TC   y       1189   TC   y       1243   TC   y       1290   CT   y       1382   AC   m       1406   TC   y       1415   GA   r       1420   TC   y       1438   AG   r       1442   GA   r       1503   GA   r       1598   GA   r       1700   TC   y       1703   CT   y       1706   CT   y       1709   GA   r       1715   CT   y       1719   GA   r       1736   AG   r       1738   TC   y       1780   TC   y       1811   AG   r       1888   GA   r       1927   GA   r       2000   CT   y       2060   AG   r       2092   CT   y       2245   ACG   v       2245   ACG   v       2263   CA   m       2308   AG   r       2332   CT   y       2352   TC   y       2358   AG   r       2380   CT   y       2416   TC   y       2483   TC   y       2581   AG   r       2639   CT   y       2650   CT   y       2706   AG   r       2755   AG   r       2758   GA   r       2768   AG   r       2789   CT   y       2792   AG   r       2834   CT   y       2836   CA   m       2857   TC   y       2863   TC   y       2885   TC   y       3010   GA   r       3083   TC   y       3197   TC   y       3200   TA   w       3202   TC   y       3204   CT   y       3206   CT   y       3221   AG   r       3290   TC   y       3308   TC   y       3316   GA   r       3372   TC   y       3394   TC   y       3438   GA   r       3450   CT   y       3480   AG   r       3505   AG   r       3513   CT   y       3516   CGA   v       3516   CGA   v       3547   AG   r       3549   CT   y       3552   TCA   h       3552   TCA   h       3565   AG   r       3594   CT   y       3644   TC   y       3666   GA   r       3693   GA   r       3699   CT   y       3720   AG   r       3756   AG   r       3796   AGTC   n       3796   ACGT   n       3808   AG   r       3816   AG   r       3834   GA   r       3843   AG   r       3847   TC   y       3866   TC   y       3918   GA   r       3921   CA   m       3927   AG   r       3970   CT   y       3981   AG   r       4025   CT   y       4040   CT   y       4044   AG   r       4048   GA   r       4086   CT   y       4104   AG   r       4117   TC   y       4122   AG   r       4123   AG   r       4158   AG   r       4203   AG   r       4216   TC   y       4221   CT   y       4225   AG   r       4232   TC   y       4248   TC   y       4312   CT   y       4336   TC   y       4370   TC   y       4388   AG   r       4454   TA   w       4491   GA   r       4506   AG   r       4508   CT   y       4512   GA   r       4529   ATC   h       4529   ATC   h       4541   GA   r       4580   GA   r       4586   TC   y       4596   GA   r       4646   TC   y       4655   GA   r       4688   TC   y       4695   TC   y       4715   AG   r       4742   TC   y       4767   AG   r       4769   AG   r       4820   GA   r       4824   AG   r       4833   AG   r       4841   GA   r       4883   CT   y       4907   TC   y       4917   AG   r       4960   CT   y       4977   TC   y       4994   AG   r       5004   TC   y       5027   CT   y       5036   AG   r       5043   GT   k       5046   GA   r       5063   TC   y       5096   TC   y       5108   TC   y       5147   GA   r       5153   AG   r       5178   CA   m       5231   GA   r       5237   GA   r       5255   CT   y       5262   GA   r       5263   CT   y       5285   AG   r       5300   CT   y       5330   CA   m       5331   CA   m       5390   AG   r       5393   TC   y       5417   GA   r       5426   TC   y       5442   TC   y       5460   GA   r       5465   TC   y       5471   GA   r       5492   TC   y       5495   TC   y       5580   TC   y       5581   AG   r       5601   CT   y       5603   CT   y       5606   CT   y       5633   CT   y       5655   TC   y       5711   AG   r       5773   GA   r       5811   AG   r       5814   TC   y       5821   GA   r       5826   TC   y       5843   AG   r       5951   AG   r       5984   AG   r       5987   CT   y       6026   GA   r       6029   CT   y       6045   CT   y       6071   TC   y       6077   CT   y       6104   CT   y       6150   GA   r       6152   TC   y       6164   CT   y       6167   TC   y       6182   GA   r       6185   TC   y       6221   TC   y       6227   TC   y       6253   TC   y       6257   GA   r       6324   GA   r       6366   GA   r       6371   CT   y       6392   TC   y       6473   CT   y       6491   CA   m       6524   TC   y       6548   CT   y       6587   CT   y       6607   TC   y       6680   TC   y       6713   CT   y       6719   TC   y       6734   GA   r       6752   AG   r       6770   AG   r       6776   TC   y       6815   TC   y       6827   TC   y       6875   CA   m       6938   CT   y       6962   GA   r       6989   AG   r       7028   CT   y       7052   AG   r       7055   AG   r       7058   TA   w       7076   AG   r       7146   AG   r       7154   AG   r       7175   TC   y       7196   CA   m       7202   AG   r       7226   GA   r       7256   CT   y       7257   AG   r       7271   AG   r       7274   CT   y       7319   TC   y       7337   GA   r       7347   GA   r       7389   TC   y       7403   AG   r       7424   AG   r       7444   GA   r       7476   CT   y       7493   CT   y       7521   GA   r       7561   TC   y       7571   AG   r       7600   GA   r       7624   TA   w       7645   TC   y       7648   CT   y       7660   TC   y       7664   GA   r       7673   AG   r       7675   CT   y       7693   CT   y       7694   CT   y       7697   GA   r       7744   TC   y       7765   AG   r       7768   AG   r       7771   AG   r       7858   CT   y       7861   TC   y       7864   CT   y       7867   CT   y       7933   AG   r       7948   CT   y       7999   TC   y       8014   AG   r       8020   GA   r       8027   GA   r       8080   CT   y       8087   TC   y       8113   CA   m       8142   CT   y       8149   AG   r       8152   GA   r       8155   GA   r       8185   TC   y       8200   TC   y       8206   GA   r       8248   AG   r       8251   GA   r       8260   TC   y       8269   GA   r       8271-8279   accccctct/-       8286   TC   y       8292   GA   r       8298   TC   y       8344   AG   r       8387   GA   r       8389   AG   r       8392   GA   r       8404   TC   y       8414   CT   y       8428   CT   y       8448   TC   y       8460   AG   r       8468   CT   y       8472   CT   y       8473   TC   y       8485   GA   r       8545   GA   r       8553   CT   y       8563   AG   r       8566   AG   r       8577   AG   r       8584   GA   r       8618   TC   y       8655   CT   y       8697   GA   r       8701   AG   r       8703   CT   y       8705   TC   y       8709   CT   y       8721   AG   r       8733   TC   y       8764   GA   r       8781   CA   m       8784   AG   r       8790   GA   r       8793   TC   y       8794   CT   y       8805   AG   r       8836   AG   r       8838   GA   r       8856   GA   r       8860   AG   r       8875   TC   y       8877   TC   y       8911   TC   y       8913   AG   r       8928   TC   y       8943   CT   y       8962   AG   r       8994   GA   r       9042   CT   y       9053   GA   r       9055   GA   r       9072   AG   r       9077   TC   y       9090   TC   y       9093   AC   m       9103   TC   y       9114   AG   r       9120   AG   r       9123   GA   r       9136   AG   r       9151   AG   r       9156   AG   r       9174   TC   y       9221   AG   r       9237   GA   r       9242   AG   r       9248   CT   y       9263   AG   r       9272   CT   y       9296   CT   y       9311   TC   y       9325   TC   y       9335   CT   y       9347   AG   r       9355   AG   r       9356   CT   y       9377   AG   r       9402   AC   m       9449   CT   y       9456   AG   r       9477   GA   r       9509   TC   y       9536   CT   y       9540   TC   y       9545   AG   r       9548   GA   r       9554   GA   r       9559   CG   s       9575   GA   r       9591   GA   r       9599   CT   y       9632   AG   r       9647   TC   y       9667   AG   r       9682   TC   y       9698   TC   y       9755   GA   r       9818   CT   y       9822   CA   m       9824   TA   w       9911   CT   y       9932   GA   r       9950   TC   y       9957   TC   y       9966   GA   r       9977   TC   y       10034   TC   y       10086   ACG   v       10086   ACG   v       10115   TC   y       10118   TC   y       10142   CT   y       10151   AG   r       10152   GC   s       10172   GA   r       10182   GC   s       10197   GA   r       10238   TC   y       10253   TC   y       10256   TC   y       10310   GA   r       10313   AG   r       10321   TC   y       10325   GA   r       10358   AG   r       10370   TC   y       10398   AG   r       10400   CT   y       10410   TC   y       10414   GT   k       10427   GA   r       10463   TC   y       10499   AG   r       10505   TC   y       10550   AG   r       10586   GA   r       10589   GA   r       10609   TC   y       10637   CT   y       10640   TC   y       10646   GA   r       10659   CT   y       10664   CT   y       10667   TC   y       10688   GA   r       10736   CT   y       10790   TC   y       10792   AG   r       10793   CT   y       10804   AG   r       10810   TC   y       10819   AG   r       10828   TC   y       10873   TC   y       10876   AG   r       10894   CT   y       10915   TC   y       10920   CT   y       10939   CT   y       10966   TC   y       10984   CG   s       11002   AG   r       11016   GA   r       11017   TC   y       11023   AG   r       11078   AG   r       11092   AG   r       11147   TC   y       11150   GA   r       11167   AG   r       11172   AG   r       11176   GA   r       11177   CT   y       11215   CT   y       11251   AG   r       11257   CT   y       11296   CT   y       11299   TC   y       11332   CT   y       11362   AG   r       11365   TC   y       11377   GA   r       11467   AG   r       11476   CT   y       11536   CT   y       11590   AG   r       11611   GA   r       11641   AG   r       11653   AG   r       11654   AG   r       11674   CT   y       11701   TC   y       11719   GA   r       11722   TC   y       11767   CT   y       11812   AG   r       11854   TC   y       11884   AG   r       11887   GA   r       11893   AG   r       11899   TC   y       11909   AG   r       11914   GA   r       11944   TC   y       11947   AG   r       11959   AG   r       11963   GA   r       11969   GA   r       12007   GA   r       12049   CT   y       12070   GA   r       12083   TG   k       12121   TC   y       12134   TC   y       12153   CT   y       12172   AG   r       12175   TC   y       12234   AG   r       12236   GA   r       12239   CT   y       12248   AG   r       12308   AG   r       12346   CT   y       12358   AG   r       12361   AG   r       12372   GA   r       12373   AG   r       12397   AG   r       12406   GA   r       12414   TC   y       12477   TC   y       12501   GA   r       12507   AG   r       12519   TC   y       12528   GA   r       12540   AG   r       12612   AG   r       12630   GA   r       12633   CT   y       12635   TC   y       12669   CT   y       12672   AG   r       12693   AG   r       12705   CT   y       12720   AG   r       12738   TC   y       12768   AG   r       12771   GA   r       12810   AG   r       12822   AG   r       12850   AG   r       12879   TC   y       12882   CT   y       12930   AT   w       12940   GA   r       12948   AG   r       12967   AC   m       12972   AG   r       12999   AG   r       13020   TC   y       13059   CT   y       13068   AG   r       13101   AC   m       13104   AG   r       13105   AG   r       13135   GA   r       13143   TC   y       13145   GA   r       13149   AG   r       13194   GA   r       13197   CT   y       13212   CT   y       13221   AG   r       13263   AG   r       13276   AG   r       13281   TC   y       13368   GA   r       13440   CG   s       13477   GA   r       13485   AG   r       13494   CT   y       13500   TCG   b       13500   TCG   b       13506   CT   y       13512   AG   r       13563   AG   r       13590   GA   r       13594   AG   r       13602   TC   y       13611   AG   r       13617   TC   y       13641   TC   y       13650   CT   y       13651   AG   r       13660   AG   r       13708   GA   r       13722   AG   r       13734   TC   y       13759   GA   r       13780   AG   r       13789   TC   y       13803   AG   r       13812   TC   y       13818   TC   y       13819   TC   y       13827   AG   r       13880   CA   m       13886   TC   y       13914   CA   m       13924   CT   y       13927   AT   w       13928   GC   s       13958   GC   s       13965   TC   y       13966   AG   r       13980   GA   r       14000   TA   w       14016   GA   r       14020   TC   y       14022   AG   r       14025   TC   y       14034   TC   y       14059   AG   r       14070   AGT   d       14070   AGT   d       14088   TC   y       14094   TC   y       14097   CT   y       14118   AG   r       14128   AG   r       14148   AG   r       14152   AG   r       14167   CT   y       14178   TC   y       14182   TC   y       14200   TC   y       14203   AG   r       14209   AG   r       14212   TC   y       14215   TC   y       14221   TC   y       14233   AG   r       14272   CG   s       14284   CT   y       14308   TC   y       14311   TC   y       14318   TC   y       14319   TC   y       14371   TC   y       14374   TC   y       14384   GC   s       14455   CT   y       14459   GA   r       14470   TC   y       14484   TC   y       14488   TC   y       14502   TC   y       14560   GA   r       14566   AG   r       14569   GA   r       14571   TA   w       14580   AG   r       14587   AG   r       14605   AG   r       14668   CT   y       14693   AG   r       14766   CT   y       14769   AG   r       14783   TC   y       14793   AG   r       14798   TC   y       14812   CT   y       14836   AG   r       14861   GA   r       14862   CT   y       14905   GA   r       14911   CT   y       14971   TC   y       14974   CG   s       14979   TC   y       15016   CT   y       15034   AG   r       15043   GA   r       15110   GA   r       15113   AG   r       15115   TC   y       15136   CT   y       15172   GA   r       15204   TC   y       15217   GA   r       15218   AC   m       15229   TC   y       15238   CG   s       15244   AG   r       15257   GA   r       15261   GA   r       15301   GA   r       15317   GA   r       15318   CT   y       15323   GA   r       15326   AG   r       15346   GA   r       15358   AG   r       15431   GA   r       15442   AG   r       15452   CA   m       15466   GA   r       15470   TC   y       15487   AT   w       15497   GA   r       15514   TC   y       15519   TC   y       15535   CT   y       15607   AG   r       15626   CT   y       15629   TC   y       15646   CT   y       15661   CT   y       15663   TC   y       15670   TC   y       15724   AG   r       15731   GA   r       15746   AG   r       15766   AG   r       15784   TC   y       15793   CT   y       15803   GA   r       15806   GA   r       15812   GA   r       15824   AG   r       15833   CT   y       15849   CT   y       15884   GC   s       15900   TC   y       15904   CT   y       15907   AG   r       15924   AG   r       15927   GA   r       15928   GA   r       15930   GA   r       15932   TC   y       15939   CT   y       15941   TC   y       15942   TC   y       15968   TC   y       16017   TC   y       16038   AG   r       16051   AG   r       16069   CT   y       16071   CT   y       16075   TC   y       16086   TC   y       16093   TC   y       16108   CT   y       16111   CT   y       16114   CA   m       16124   TC   y       16126   TC   y       16129   GCA   v       16129   GCA   v       16140   TC   y       16144   TC   y       16145   GA   r       16147   CT   y       16148   CT   y       16153   GA   r       16162   AG   r       16163   AC   m       16166   AC   m       16167   CT   y       16168   CT   y       16169   CT   y       16171   AG   r       16172   TC   y       16175   AG   r       16176   CT   y       16182   AC   m       16183   AC   m       16184   CT   y       16185   CT   y       16186   CT   y       16187   CT   y       16188   CAG   v       16188   CAG   v       16189   TC   y       16192   CT   y       16193   CT   y       16207   AG   r       16209   TC   y       16212   AG   r       16213   GA   r       16214   CT   y       16217   TC   y       16219   AG   r       16223   CT   y       16224   TC   y       16227   AG   r       16229   TC   y       16230   AG   r       16231   TC   y       16232   CT   y       16234   CT   y       16235   AG   r       16239   CT   y       16241   AG   r       16242   CT   y       16243   TC   y       16245   CT   y       16247   AG   r       16249   TC   y       16254   AC   m       16255   GA   r       16256   CT   y       16257   CT   y       16258   AG   r       16260   CT   y       16261   CT   y       16264   CT   y       16265   AC   m       16266   CT   y       16268   CT   y       16270   CT   y       16271   TC   y       16274   GA   r       16278   CT   y       16284   AG   r       16286   CG   s       16287   CT   y       16288   TC   y       16290   CT   y       16291   CT   y       16292   CT   y       16293   AG   r       16294   CT   y       16296   CT   y       16298   TC   y       16304   TC   y       16309   AG   r       16311   TC   y       16316   AG   r       16317   AT   w       16318   AT   w       16319   GA   r       16320   CT   y       16324   TC   y       16325   TC   y       16326   AG   r       16327   CT   y       16343   AG   r       16344   CT   y       16354   CT   y       16355   CT   y       16356   TC   y       16357   TC   y       16360   CT   y       16362   TC   y       16366   CT   y       16368   TC   y       16390   GA   r       16391   GA   r       16399   AG   r       16438   GA   r       16439   CA   m       16483   GA   r       16519   TC   y       16527   CT   y                  
 
     Reference to Sequence Listings  
      SEQ ID NO:1 is a theoretical human mtDNA genome sequence containing the nucleotide alleles of this invention as listed in Table 3.  
      SEQ ID NO:2 is the human mtDNA reference sequence called the Cambridge Sequence (Genbank Accession No. J01415).