Mitochondrial DNA Functional Map

The entire human mitochondrial DNA (mtDNA) sequence has been determined (1a, 1b) . Functions and gene products have been assigned to all mitochondrial genes including 13 protein-coding, 2 rRNA, and 22 tRNA genes (Figure 1, mtDNA Transcripts, Table 1, Table 2). The two strands of the circular mtDNA chromosome have an asymmetric distribution of Gs and Cs generating heavy (H)- and light (L)-strands. In Figure 1 the gene products encoded by the L-strand are shown in the inner complete circle and the gene products of the H-strand in the outer complete circle. Each strand is transcribed from one predominant promoter, PL and PH1, located in the control region which includes the displacement (D)-loop. The D-loop is a triple-stranded region generated by the synthesis of a short piece of H-strand DNA, the 7S DNA. While PL predominantly transcribes the L-strand and PH1 the H-strand, such that RNA synthesis proceeds around the circle in both directions, both PL and PH1 are bi-directional (34) and associated with upstream binding sites (*) for the bi-directional mitochondrial transcription factor, mtTF1 (60) . MtTF1 is a high mobility group DNA-binding protein with two DNA binding domains and a carboxyl-terminal tail essential for transcription (50, 65, 87, 120, 156) . A bi-directional attenuator sequence (MTTER) within the MTTL1 gene (Leu(UUR)) limits L-strand synthesis and maintains a high ratio of rRNA to mRNA transcripts from the H-strand (38, 39) . The mature RNAs, 1 to 17 (Figure 1), are generated by cleavage of the polycistronic transcript at the tRNAs (4, 5, 125, 144, 145) . Additional transcript processing intermediates have been observed in selected tissues (138) and in association with various pathogenic mtDNA mutations (73, 74, 98) . H-strand DNA replication is initiated within the D-loop 7S DNA at four major and three minor sites. Three of these correspond to L-strand transcription stop sequences at the conserved sequence blocks (CSB) I to III. The most prevalent 7S DNA start is at CSB-II (MTCSB2). Primers for 7S DNA synthesis at this site are thought to be generated by the cleavage of the L-strand transcript by RNAse MRP which includes a nuclear encoded RNA that appears to guide the cleavage of the RNA at the CSB-II (31-33, 40) . The mtTF1 binds throughout the D-loop with a 40 to 50 base periodicity, with MTCSB2 and MTCSB3 being unbound and MTCSB1 being strongly bound. The mtTF1 phasing downstream from MTCSB1 corresponds to DNA synthesis initiation sites suggesting the mtTF1 may play a role in defining the transition from RNA to DNA (66) . All 7S DNA molecules end at nucleotides just past the termination associated sequence (TAS) (55) which interacts with sequence-specific binding factor(s) (110) . H-strand replication starts at the 7S DNA and proceeds around the L-strand, displacing the single-stranded, parental H-strand. After traversing 2/3 of the genome, the L-strand origin is exposed. L-strand replication is then initiated with a specific primase containing the cytosol 5.8S rRNA (241) and proceeds back along the displaced H-strand template. The polypeptides of the mtDNA (Table 2) are all subunits of the mitochondrial energy-generating pathway, oxidative phosphorylation (OXPHOS). Seven of the genes (MTND1, MTND2, MTND4L, MTND4, MTND5, and MTND6) encode subunits of respiratory Complex I (NADH dehydrogenase or NADH:ubiquinone oxidoreductase); one gene (MTCYB) encodes a component of Complex III (ubiquinol:cytochrome c oxidoreductase); three genes (MTCO1, MTCO2 and MTCO3) encode constituents of Complex IV (cytochrome c oxidase or COX); and two genes (MTATP6 and MTATP8) encode subunits of respiratory Complex V (ATP synthase). The mitochondrial mRNAs are translated within the mitochondrion on chloramphenicol-sensitive ribosomes using mtDNA-encoded rRNAs and tRNAs. The mammalian mtDNAs share a unique genetic code where UGA = tryptophan, AGA and AGG = stop, and AUA = methionine (1, 2, 8, 12, 225) .

Mitochondrial DNA Variation

The mtDNA nucleotide sequence evolves 6 to 17 times faster than comparable nuclear DNA gene sequences (26, 27, 57, 122, 140, 238) . This has resulted in multiple restriction fragment length polymorphisms (RFLPs) (Tables 3, 4, 5), control region (Table 6) and coding region nucleotide variants (Tables 7, 8, 9), conformational variants (204, 222) , and length variants (Tables 10, 11, 12, 13). Polymorphic variants correlate with ethnic and geographic origin of the samples, presumably because mtDNA mutations have accumulated along radiating maternal lineages as women migrated out of Africa and into different continents (28, 91, 119, 214, 226) . Prominent continent-specific polymorphic restriction sites and their approximate frequencies are listed in Table 5. With these variants, the continental origin of roughly 70% of the African, European and Asian + Native American mtDNAs can be determined. Extensive mtDNA sequence variation has also been observed in multiple partial and complete human mtDNA sequences (Tables 6, 7). In cultured human cells, mtDNA nucleotide substitution mutants have been identified in the 16S rRNA gene which impart chloramphenicol resistance (15, 96) , and in the MTND4 and MTND5 genes where a single C or A, insertion, respectively, results in rotenone resistance and respiratory deficiency (75, 76) . Cultured human cells which lack mtDNA (rho0) have also been isolated by growth in ethidium bromide (97) . These rho0 cells, in conjunction with the cybrid mitochondrial transfer technique employing fusion with enucleated cells (223, 224, 232) , has permitted studying the pathophysiology of clinically relevant mtDNA mutations. MtDNA variation may also provide antigenic variation, since the first 17 amino acids of the mouse mtDNA MTND1 gene have been found to code for a polymorphic cell surface antigen (108) .

Mitochondrial DNA Mutations and Disease

A broad spectrum of degenerative diseases involving the central nervous system, heart, muscle, endocrine system, kidney and liver have been associated with systemic mtDNA mutations (Figure 2, Morbid mtDNA Map), either base substitutions (Tables 8, 9) or insertion-deletions (Tables 10, 11, 12, 13). Diseases resulting from base substitutions are generally maternally transmitted, consistent with the maternal inheritance of the mtDNA (30, 67, 85b) . They can either alter polypeptide genes, missense mutations, or structural RNAs, protein synthesis mutations and are designated by the gene name, an asterisk, a clinical phenotype designator, the nucleotide position, and the mutant base, e.g., MTND6*LDYT14459A (Tables 8, 9). Insertion-deletion mutations (165) can be spontaneous (77, 104, 247) , maternally inherited (6, 7, 11, 164-166, 175) , or mendelianly inherited due to predisposing nuclear mutations (42, 246, 248) . They are described by the size of the insertion-deletion, the nucleotides at the junction, the nature and size of any flanking repeat, and the locations of the repeats (Tables 10, 11, 12, 13).

Missense Mutations

Among the missense mutation diseases, the two best studied are Leber's Hereditary Optic Neuropathy (LHON) and Leigh Syndrome together with Neurogenic Muscle Weakness, Ataxia, and Retinitus Pigmentosum (NARP) and Familial Bilateral Striatal Necrosis (FBSN) (Tables 8, 9). LHON involves mid-life acute or subacute central vision loss resulting in scotoma and blindness. Eighteen mutations in mtDNA electron transport genes have been associated with this phenotype, four of which are generally felt to play a significant role in the etiology of LHON. In order of decreasing severity, these four "primary" LHON mutations are MTND6*LDYT14459 (93, 192) , MTND4*LHON11778A (236) , MTND1* LHON3460A (80, 82, 83) , and MTND6*LHON14484C (90, 109) . Together these account for over 80% of Caucasian patients, with the MTND6*LDYT14459A mutation being rare (192) , the MTND4*LHON11778A mutation accounting for 50% of cases, and the MTND1*LHON3460A and MTND6*LHON14484C mutations accounting for 15% of cases (25) . The MTND4*LHON11778A mutation accounts for 95% of Asian cases (114) . The severity of the primary LHON mutations has been assessed by the presence and severity of their additional clinical manifestations, the frequency at which they arise de novo and thus are associated with different mtDNA haplotypes (23) , their co-occurrence with secondary LHON mutations, their frequency of heteroplasmy, their penetrance, and their potential for spontaneous recovery (24, 82, 89, 141, 230, 233) . The MTND6*LDYT14459A mutation is the most severe of the LHON mutations. In addition to LHON this mutation also presents with generalized dystonia associated with bilateral striatal necrosis (LDYT). In each of the three pedigrees studied, the mutation arose on a different mtDNA haplotype and all three pedigrees encompass heteroplasmic individuals. About 61% of maternal relatives are affected, 58% of which are male, and there is no record of visual recovery for this mutation (93, 143, 192) . The MTND4*LHON11778A mutation is the next most severe of the LHON mutations. It is also occasionally associated with other neurological and cardiac manifestations (102, 149, 221) and arises in most families as a new mutation associated with a distinctive different mtDNA haplotype (23) . It only occasionally co-occurs with secondary LHON mutations, it changes a highly conserved amino acid, and is heteroplasmic in some families. It is about 82% penetrant in males and undergoes spontaneous recovery in about 4% of cases (231) . The MTND1*LHON3460A mutations is next most severe LHON mutation. It is also found on a variety of mtDNA haplotypes and generally not in association with secondary LHON mutations. It changes a moderately conserved amino acid and is occasionally heteroplasmic. It is 69% penetrant in males and exhibits a 22% spontaneous recovery rate (231) . The MTND6*LHON14484C mutation is the least severe primary mutation. This mutation is generally confined to a specific Caucasian mtDNA background defined by three "secondary" markers: MTND5*LHON13708A, MTND1*4216C and MTCYB*LHON15257A (23, 81) . The mutation changes a weakly conserved amino acid and is consistently homoplasmic. It is about 72% penetrant in males, and undergoes spontaneous recovery in 28% of cases (231) . All four of these primary LHON mutations alter Complex I (NADH dehydrogenase) subunits, and would be expected to inhibit this enzyme. This has been confirmed by demonstrating a Complex I defect in patient cells and in some cases showing that the defect can be transferred along with the mutant mtDNA in cybrid experiments. The MTND6*LDYT14459A mutation causes a 50% reduction in Complex I specific activity as well as coenzyme Q substrate-product inhibition (94) . The MTND4*LHON11778A is associated with a reduction in respiration of NADH-linked substrates (111) and a partial reduction in Complex I activity (206, 221) . It is also associated with altered coenzyme Q and rotenone binding (52) . The MTND1*LHON3460A mutation is associated with a 67-80% reduction in Complex I activity (80, 111, 206) , while the MTND6*LHON14484C mutation is associated with a 60% reduction in Complex I and a 20% reduction in Complex I-linked ATP synthesis (148) . The Leigh Syndrome missense mutations, MTATP6*NARP8993G (78, 182, 194, 211) , MTATP6*NARP8993C (51, 181) , and MTATP6*FBSN9176C (213) alter the MTATP6 gene. These mutations are invariably heteroplasmic and result in a broad range of clinical manifestations from mild peripheral retinitis pigmentosa to severe neurological disease depending on the percentage of mutant mtDNAs. Children which inherit close to 100% mutant mtDNAs can present with Leigh Syndrome, a frequently lethal disease associated with basal ganglia degeneration (51, 194, 211) . The MTATP6*NARP8993G mutation has been linked to the inhibition of proton translocation of the ATP synthase through cybrid transfer experiments (217) .

Protein Synthesis Mutations

Base substitutions which alter rRNA and tRNA genes have been identified in patients with a wide range of clinical presentations (Tables 8, 9). The mildest mutations tend to be homoplasmic, occur in specific mtDNA lineages, and are associated with late-onset diseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD), and neurosensory hearing loss. Approximately 5% of Caucasian AD patients harbor a MTTQ*ADPD4336C mutation (84, 193) . This mutation appears to define a mtDNA lineage with a predilection to AD and PD. Like mtDNA lineages prone to LHON, patients in this lineage can harbor additional contributory mutations such as the MTND1*ADPD3397G missense mutation and a five nucleotide insertion in MTRNR1 between nucleotide pairs 956 and 965 (193) . A homoplasmic mutation in the 12S rRNA gene, MTRNR1*DEAF1555G, has been found to correlate with neurosensory hearing loss. Additional factors may precipitate deafness in individuals harboring this mutation such as exposure to aminoglycoside antibiotics or other environmental or nuclear genetic factors (85, 169) . Patients harboring moderately severe tRNA mutations are generally the result of recent mutations. Hence, they are heteroplasmic and can exhibit a wide range of clinical manifestations depending on the percentage of mutant and normal mtDNAs the individual inherits. Certain of these mutations cause relatively consistent phenotypes with the severity being determined by the proportion of mutant and normal mtDNAs in affected tissues and the age of the individual. The age effect has been hypothesized to be the result of the exacerbation of the inherited respiratory deficiency by the natural age-related decline of mitochondrial OXPHOS due to the accumulation of somatic mtDNA mutations (see below). The best example of such a moderately severe tRNA mutation is MTTK*MERRF8344G, which consistently results in neurosensory hearing loss, mitochondrial myopathy and ragged red fibers (RRF), and, when the percentage of mutant is high, can also cause myoclonic epilepsy (hence the acronym for Myoclonic Epilepsy and Ragged-Red Fiber disease, MERRF) (196, 197, 229, 234, 239) . Other moderately severe tRNA mutations give several distinctive clinical presentations, the most notable of these is MTTL1*MELAS 3243G mutation. This mutation has been observed in patients with Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes (MELAS) and Kearns-Sayre Syndrome (KSS) (68, 69) when present at a high percentage of mutant. However, patients with a low percentage of mutant mtDNAs can present with adult-onset diabetes mellitus (Type I diabetes) with or without deafness (DMDF) (209, 218, 219) . Approximately 1.4% of all diabetes mellitus patients have been found to harbor this mutation (64, 231) . Correlation between genotype and phenotype is further complicated by the fact that for some tRNAs such as MTTK, different mutations (MTTK*MERRF8344G and MTTK*MERRF8356C) give similar phenotypes, while for other tRNAs such as MTTL1, different mutations give different phenotypes. Of eleven mutations reported for MTTL1 (tRNALeu(UUR)), the phenotypes range from diabetes and deafness, through mitochondrial myopathy and stroke-like episodes to hypertrophic cardiomyopathy, Figure 3, tRNA Leucine with mutations. Mitochondrial protein synthesis defects have been associated with the MTTK*MERRF8344G and MTTK*MERRF8356C mutations and have been shown to be caused by these substitutions through cybrid transfer (19, 37, 115, 142, 190, 237) . For the MTTK*MERRF8344G mutation, this defect has been correlated with a 50-60% reduction in tRNALys aminoacylation (56) . A similar assignment of protein synthesis defects has been made to the MTTL1*MELAS3243G and MTTL1*MELAS3271C mutations (36, 98, 100) . Certain more severe tRNA mutations can cause pediatric disease as they approach homoplasmy. Representative examples are MTTI*FICP4269G, MTTL1*MM3302G, MTTP*MM15990A, and MTTL1*PEM3271. The MTTI*FICP4269G alters the nucleotide pair at the base of the amino acid acceptor stem and results in early-onset multisystem disease leading to cardiac failure at age 18 (210) . The MTTL1*MM3302G changes the penultimate nucleotide of the amino acid acceptor stem, possibly inhibiting the processing of MTTL1 and causing muscle weakness in both pediatric and adult cases (14, 195) . The MTTP*MM15990A changes the proline tRNA anticodon (UGG) to serine (UGA) and causes pediatric mitochondrial myopathy (126) . The MTTL1*PEM3271 deletes one base from the stem of the anticodon loop, causing hearing loss at 5 years which progressed over 23 years to seizures, degenerative eye disease, renal failure and cerebral calcifications (Fahr disease) (191) .

Rearrangement Mutations

Spontaneous rearrangements in the mtDNA have been associated with ocular myopathies including chronic progressive ophthalmoplegia (CPEO) and KSS (127) , Pearson's Marrow/Pancreas Syndrome (177, 178) , and maternally inherited adult-onset diabetes and deafness (6, 7) (Tables 10-13). The ocular myopathies, KSS and CPEO, are multisystem disorders regularly presenting with mitochondrial myopathy, ophthalmoplegia and ptosis. KSS is the more severe, with an age of onset prior to age 20, and also manifesting at least one of the following: cardiac conduction defects, cerebellar ataxia, or elevated cerebral spinal fluid protein (166, 179) . The pediatric-onset Pearson's Syndrome manifests as bone marrow failure and pancytopenia, frequently associated with exocrine pancreatic insufficiency, hepatic and renal failure, and other neuromuscular problems. Pearson's patients generally die young due to complications of bone marrow dysfunction or transfusions (160, 198, 205) . More than half of all KSS and CPEO cases are due to spontaneous mtDNA rearrangements (189) , with KSS patients frequently harboring normal, duplicated, and deleted mtDNAs. In these cases the duplicated and deleted mtDNAs share a common breakpoint and the deleted molecules frequently occur as dimers. The milder CPEO patients generally harbor normal and deleted mtDNAs (163, 166, 167) . In addition, patients with duplications are more likely to also manifest diabetes mellitus (166) . Pearson's Syndrome patients can result from either deletion or combined duplication - deletion mutations, and some Pearson's patients spontaneously recover from their childhood sideroblastic anemia, and ultimately progress to a KSS-like phenotype (117, 166, 176) . These observations suggest that Pearson's Syndrome, KSS, CPEO, and diabetes mellitus with deafness represent a continuum of clinical severity possibly reflecting the pathogenicity of the rearranged mtDNAs and the extent of their distribution in the body (231) . Histological analysis of CPEO and KSS muscle has revealed that the deleted mtDNAs become regionally enriched within the muscle fibers, causing alternating bands of COX deficiency and accumulation of abnormal mitochondria which contribute to ragged-red muscle fibers (RRFs) (121, 128, 200, 201) . This may involve a secondary amplification of the mutant mtDNAs causing progression of the disease (58, 103, 166) .

Mitochondrial DNA Depletion

Certain pediatric myopathies have been associated with depletion of the mtDNA in individual tissues (16, 59, 116, 129, 150, 155, 168, 212, 215) . This appears to be due to a nuclear gene defect affecting mtDNA replication (16, 17) . Recently, a deficiency in DNA polymerase gamma has been implicated in mtDNA depletion associated with Alpers' Syndrome (139) .