mitomap is a comprehensive database of human mtdna variation and its relation to human evolution and disease . the need for a curated and thus reliable database of mitochondrial gene sequence variation with easily used web - based query tools is becoming increasingly essential as the importance of mtdna variation in the common age - related metabolic and neurodegenerative diseases , aging and cancer is elucidated . the mtdna encompasses the 13 core polypeptide genes that define the efficiency of the mitochondrial energy generating system oxidative phosphorylation ( oxphos ) , the trnas and rrnas for their expression , and a 1121 np control region that regulates mtdna transcription and replication . the mtdna also has a unique genetics , being maternally inherited , present in thousands of copies per cell , and capable of encompassing various percentages of mutant and normal molecules ( or heteroplasmy ) ( 1 ) . hence , the mtdna imparts a quantitative genetics to the vital , systemic function of energy production ( 1,2 ) . the 16  569 nt pairs ( np ) of the closed circular mtdna are each numbered sequentially and the standard base at each position ( 3 ) is specified in the  revised cambridge reference sequence ( rcrs ) , genbank j01415.2 . this permits any new mtdna sequence to be unambiguously defined by specifying only the deviant positions and bases relative to the rcrs . the human mtdna has a very high mutation rate , and this has generated three classes of sequence variants : pathogenic , adaptive and neutral . pathogenic mutations damage conserved gene functions and as the deleterious mutation increases in its percentage heteroplasmy , it erodes systemic cellular energy output . this deficiency is acted on by natural selection and the mutation is soon eliminated from the population as hereditary disease . adaptive mtdna mutations also alter conserved functions but these alterations are advantageous in certain environments . consequently , they become established in the individual ( or homoplasmic ) and increase in the population that occupies the compatible environment . neutral mutations accumulate by chance , but those linked to advantageous mutations can become enriched in particular populations by hitchhiking . mutations in the mtdna not only arise in the female germline , but also accumulate in the post - mitotic cells of the body with age . these somatic mtdna mutations now appear to be the aging clock and may also be important in the etiology of certain cancers ( 1,2 ) . pathogenic mtdna mutations can be divided into those that alter more than one gene ( inter - gene or contiguous gene defects ) and those that affect only one gene ( intra - gene or single gene defects ) . the intra - gene mutations can either alter polypeptide coding genes or protein synthesis ( rrna and trna ) genes . the functional severity of pathogenic nucleotide substitutions can be estimated , in part , through the inter - species conservation index ( ci ) and for rrna or trna genes by the effect of the base change on the rna secondary structure . since the clinical effect of a mutation is the product of both the severity of the mutation and its percentage of heteroplasmy , the clinical phenotypes of heteroplasmic mtdna mutations can be highly variable among the maternal relatives ( 1,2 ) . since mtdna mutations can arise in both the germline and somatic tissues , they have been associated with both degenerative diseases and cancers ( 46 ) . moreover , since they are retained in the population by selection , they are frequently ancient and present at polymorphic frequencies . about 25% of the polypeptide amino acid substitution ( missense ) mutations present in the human mtdna phylogenetic tree are adaptive ( 7,8 ) , 1020% of trna mutations appear to be adaptive , as do various rrna variants ( 9 ) . many of these same mutations have also been reported as somatic mutations in cancer cells suggesting that tumors adapt to their changing energetic environment through the evolution of mtdna variation ( 10 ) . a surprisingly high proportion of cancer control region somatic mutations are also population polymorphisms ( 10 ) , suggesting that modulation of mtdna replication and transcription is an important adaptive strategy . since mtdna mutations accumulate sequentially along maternal mtdna lineages , the mtdnas of region - specific branches of the mtdna tree are founded by a common mtdna haplotype and thus share a set of common polymorphisms . from this founder , additional mutations are added sequentially over time forming groups of related haplotypes is called a haplogroup . since selection may have caused the haplogroup to flourish in the new environment , different haplogroups may be functionally different ( 8,9 ) . evidence that haplogroups can be functionally different comes from the fact that haplogroups are associated with predilection to or protection from various clinical phenotypes . for instance , haplogroup j increases the risk of developing blindness in association with the milder leber hereditary optic neuropathy ( lhon ) mutations ( 1113 ) and haplogroup t increases risk for depression ( 14 ) . however , other haplogroups are protective against alzheimer disease ( 1517 ) and parkinson disease ( 1719 ) and are associated with increased longevity ( 8,2024 ) . throughout the evolution of the symbiosis between the nuclear - cytosol and the mitochondrial organisms , sequences from the mtdna have been transferred to the ndna with the result that the vast majority of mitochondrial genes are now located in the nucleus . the transfer of mtdna sequences to the ndna continues today , but differences in the mtdna genetic code that arose prior to the origin of the fungal - animal mtdna lineage render the mtdna sequences unintelligible to the nuclear - cytosol system ( 2 ) . consequently , all recent mtdna transfers into the human ndna have resulted in nuclear - mtdna pseudogenes ( numts ) ( 25 ) . numts are potentially problematic since they can be inadvertently amplified using the polymerase chain reaction ( pcr ) , sequenced , and the pseudogene variants mistaken for pathogenic mtdna mutations ( 26,27 ) . while the mtdna retains the 13 central oxphos proton and electron carries for the oxphos circuitry , all of the structural genes necessary to assemble the mitochondrial oxphos complexes as well as all of the genes needed to create a mitochondrion are now encoded by the nucleus , a total of 1500 mitochondrial genes . therefore , many mutations that affect mitochondrial function are the result of ndna mutations ( 1,2 ) . a number of factors must be considered when evaluating the nucleotide variants observed in a new mtdna sequence relative to the rcrs . these include the association between different variants on particular branches of the mtdna tree ( the haplogroup ) , the time in human mtdna radiation that the variant appeared and the position of the variant in the protein or rna gene . all of these considerations were utilized in developing our sequential mutational phylogenetic tree of the human mtdna . our mtdna tree encompasses the information in 2959 mtdna coding region sequences , using the rcrs ( genbank j01415.2 ) as the reference . while our tree was assembled primarily using mtdna coding region single nucleotide polymorphisms ( snps ) , informative control region snps are also being incorporated when available . however , this dataset is incomplete since the control region sequences have not been reported for many of the published mtdna sequences used in building the tree . each sequence was analyzed for possible sequencing errors , identified by base changes inconsistent with the most likely haplogroup . black polymorphisms represent nonsynonymous mutations ( amino acid , position , amino acid , gene ) , red synonymous mutations ( gene ) , green trna or rrna mutations ( gene ) , blue indicates non - coding nucleotides in the coding region and underlined yellow represent pathological mutations . large blue letters indicate a haplogroup designation and large black letters allow for navigation of the tree at low magnification . for example , 4(55 ) indicates howell 's article ( 4 is the code for this article ) and 55 is the number of the sequence in the article ) . ( b ) magnification of blue box in figure1a showing details of the tree for subhaplogroup j2b . the core topology of the tree was generated by the neighbor - joining program from mega ( 28 ) using 1060 human mtdna sequences . the branching order used is highly robust as demonstrated by its consistency within 1000 bootstrap iterations . separate neighbor - joining trees were also built for each major haplogroup , again with the branching order being robust based in 1000 bootstrap iterations . then using sequence alignment and sequence comparisons , the positions of the individual nucleotide changes were incorporated into the various branches and nodes of the tree . since the core tree encompasses all of the major mtdna haplogroups , new mtdna sequences can be continually incorporated into the existing tree manually without necessity for further computer analysis . atypical variants such as insertion - deletions are also added manually and unsolved parallel mutations are resolved by network analysis . the overall branching order of the human mtdna tree has been clear for many years ( 2932 ) . however , the presence of identical mutations in multiple different parts of the phylogeny was initially puzzling . this was resolved when we concluded that many of these recurrent mutations were due to convergent evolution resulting from adaptive selection acting on specific functional variants ( 8,9 ) . the letters were assigned as the mtdna lineages became apparent . because native americans were found to have a limited number of mtdna lineages ( 33 ) , the four major native american haplogroups were designated a , b , c and d ( 34,35 ) . subsequent analysis added haplogroups in europe ( h , i , j , t , u , uk , v , w and x ) ( 36,37 ) , asia ( many derivatives of macro - haplogroups m and n which radiated out - of - africa ) ( 38,39 ) and africa ( haplogroups l0 , l1 , l2 and l3 ) ( 7,40,41 ) . a fifth native american haplogroup was also found to be related to eurasian mtdna haplogroup x ( 42 ) . the sub - branching order of the haplogroups has been further refined by incorporating subsequent nomenclatural proposals ( 43 ) and application of the information derived from complete mtdna sequences ( 7,44,45 ) . since the african mtdnas lie at the root of the mtdna phylogeny ( 2931 ) , the african haplogroups are presented at the beginning ( top ) of the phylogenetic tree ( figure 1 ) . the complete phylogeny with all of the ordered snps requires 126 pages to print . to make the tree more accessible , it was integrated and transferred into the canvas x ( acd systems , inc , the entire tree can be viewed at any resolution desired , from ultra - low resolution , where all of the branches can be observed but no detail can be perceived , to ultra - high resolution , where the branching order is lost but the individual nucleotide changes can be seen ( figure 1 ) . the nucleotide position of each change is located in the tree in the order that they arose , and the nature of the mutation , transition versus transversion , given . if the nucleotide change alters a trna or an rrna gene , the mutation is presented in green with the gene designated . if the mutation alters a polypeptide gene , but is synonymous , then it is presented in red with the gene identified . however , if the mutation alters the amino acid sequence of a polypeptide gene , the mutation is presented in black with the affected gene identified and the codon position and amino acid substitution reported . non - coding variants are shown in blue , and few representative pathogenic mutations are included in the tree in yellow . in this way , all of the common mtdna sequence changes , their relationship to each other and their potential significance can easily be observed . because of how our tree was developed , it is invaluable for analyzing the nature of the nucleotide differences found in any new mtdna sequence relative to the rcrs . by tracing the nucleotide variants in the new mtdna sequence along the branching order of our tree , the new sequence 's haplogroup is defined , the time that each variant arose ( ancient versus recent ) is determined and the effect of the sequence variant on the polypeptide or rna gene revealed . ancient functional variants associated with a new sequence may be adaptive variants and may have also arisen independently in other branches of the phylogeny . variants that do not appear in our phylogenetic tree are likely to be relatively recent mutations . if the mtdna sequence is from a potentially maternally inherited disease patient and the new mutation is potentially functionally significant , the mutation becomes a candidate for a pathogenic mutation . neutral variants can be either ancient or recent , but do not alter an important mitochondrial function . with these rules , a mtdna sequence from a patient with a potentially maternally inherited disease would be analyzed by first comparing the sequence to the rcrs , and all differences will be listed . these variants would then be used to trace down the various branches of the phylogenetic tree until you arrive at the end of a terminal branch . any variants that are left over must be recent and require additional analysis . if a residual nucleotide change alters an amino acid in a mtdna polypeptide or the sequence of a trna or rrna , calculation of the ci of the variant amino acid can give further insight into pathogenicity ( 8) . similarly , for a trna or rrna sequence change , if the variant alters a conserved nucleotide and/or affects the secondary structure of the rna then it becomes a candidate for a pathogenic mutation ( 9 ) . additional criteria suggesting pathogenicity could include the presence of heteroplasmy and a correlation between the severity of the pathogenic mutation and the percentage of mutant mtdnas present in various family members along a maternal lineage . another criterion could be the similarity between the pathological presentation of the mutation and that of a similar type of pathogenic mutation that presents with a similar phenotype . in certain cases , multiple patients from unrelated families may be found that have closely related mtdna haplotypes . in these situations , it is possible that the background mtdna haplogroup is also contributing to the pathogenicity of the deleterious mutation . this type of phenomenon has been documented for the milder pathogenic mutations at nt g11778a , t14484c , and t10663c which cause lhon , which are preferentially associated with mtdna haplogroups j and t ( 1113 ) . one strong criterion for identifying a pathogenic mtdna mutation would be if the same mutation had been identified in other patients with similar clinical presentation . to facilitate this type of comparison , the current searchable tables are divided into three major classes : ( i ) mutations associated with lhon , ( ii ) mutations that affect polypeptide sequences but present with other phenotypes and ( iii ) mutations that alter trnas and rrnas and present with a various multi - system clinical problems . these tables and the associated clinical diseases are explained in depth in our various clinical reviews ( 2,46,47 ) . as an example , the lhon disease mutations have been sub - classified into two main categories . the first category includes well characterized and causative mutations designated the  top 10 primary lhon mutations . this group includes the three most common lhon mutations ( g3460a , g11778a and t14484c ) with account for 95% of lhon patients . the second mutation category includes mutations that have been reported in a single family and thus require additional independent reports to confirm their causal association with lhon . if in the process of analyzing an mtdna sequence from a patient , one of the recent sequence differences is also present in one of the pathogenic mutational tables , then this mutation becomes a good likely candidate for the disease mutation . however , it must be remembered that the mtdna haplogroup background can be a contributing factor in some diseases such as lhon . hence , it is important that the role of the mutation be considered in the larger context of what we know about human mitochondrial genetics ( 2 ) . while the mtdna encodes 37 critical genes for oxphos ( 13 polypeptides , 22 trnas and 2 rrnas ) the ndna encodes in the order of 1500 additional mitochondrial proteins and mutations in these genes can also cause mitochondrial disease . hence , many of the more severe mitochondrial diseases can be caused by mutations in ndna encoded genes for the structural or assembly proteins of the oxphos complexes and a spectrum of disorders can result from ndna mutations that affect mtdna replication ( 48 ) . to alert mitomap users to these important classes of mitochondrial disease , mitomap now includes summary tables of the known ndna - encoded mitochondrial diseases ( 2 ) . the ndna also encodes 247 fragments of the mtdna of sufficiently recent origin to be easily mistaken for legitimate mtdna sequences . inadvertent pcr amplification of one of these sequences during a clinical study can result in the erroneous conclusion that the sequence variants within the numt pseudogene are the cause of the disease ( 26,27 ) . to address this problem , we have developed a mitomap human numt database ( 25 ) . by scanning the numt sequences that encompass the same region as that of clinical interest , the potential ndna pseudogene variants can be identified . the mitomap database currently encompasses over 5100 references . these pertain to the mtdna sequence , sequence variation and mitochondrial role in a wide variety of diseases . all of the data for mitomap is now managed in postgresql ( ) , an open - source database management system ( i.e. dbms ) with object - relational modeling capabilities . data for mitomap are initially extracted by the database curators and stored , formatted and screened in excel spreadsheets . updated versions of the curated data are then periodically uploaded to the server , where perl programs implementing the parseexcel and dbi modules are used to parse the values from the spreadsheets and populate the data structures implemented in postgresql . the mitomap servers are located at the university of california , irvine , on a dual - processor server running redhat linux as its operating system . by adding the mtdna mutational tree , updating the depth and breadth of the mtdna disease mutation tables and creating a numt pseudogene sequence database , we have now generated the informational infrastructure that will permit mitomap to evolve from a simple factual database to an intelligent analytical system . the sequential mutation mtdna phylogenetic tree places all of the factual information about mtdna sequence variation into an evolutionary and functional context . this single integrated entity thus automatically sorts mtdna variants into ancient versus recent mutations , defines the gene affected and provides preliminary information on the potential functional implications of the mutation . from this foundation , mitomap is now poised to implement our much more powerful data integration and analysis system , mitomaster . the mitomap - mitomaster system will permit the user to up - load a new mtdna sequence , and the system will automatically identify the sequence variants relative to the rcrs , determine the mtdna haplogroup , indicate the potentially functional variants , check for numt pseudogene variants and provide a list of potential disease causing mutations . hence mitomap - mitomaster will soon be an important bioinformatics tool in the analysis of human mtdna sequence variation .
the mitomap ( ) data system for the human mitochondrial genome has been greatly enhanced by the addition of a navigable mutational mitochondrial dna ( mtdna ) phylogenetic tree of 3000 mtdna coding region sequences plus expanded pathogenic mutation tables and a nuclear - mtdna pseudogene ( numt ) data base . the phylogeny reconstructs the entire mutational history of the human mtdna , thus defining the mtdna haplogroups and differentiating ancient from recent mtdna mutations . 
 pathogenic mutations are classified by both genotype and phenotype , and the numt sequences permits detection of spurious inclusion of pseudogene variants during mutation analysis . 
 these additions position mitomap for the implementation of our automated mtdna sequence analysis system , mitomaster .