MtDNA

Mitochondria have their own DNA (mtDNA) and genetic continuity. This DNA only encodes 13 peptide subunits synthesized in the matrix that are components of complexes I, III, IV, and V of the respiratory chain. Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported by specific mechanisms to their specific locations in the mitochondrion (see below). 

Mitochondria are unique organelles in man and higher animals in that they contain their own genome. Mitochondrial DNA (mtDNA) in humans is a small (16.5 kb), circular genome that encodes only 13 proteins, 22 transfer RNA (tRNA), and 2 ribosomal RNA (rRNA) molecules. mtDNA is inherited only from the mother and is present in multiple copies within one mitochondrion. 

Mitochondrial DNA is transcribed as a polycistronic RNA which is subsequently cleaved to generate the various mature mRNA, tRNA, and rRNA (Clayton, 1984). The 13 proteins encoded by mtDNA are all components of the respiratory chain and are seven subunits of complex I, one subunit of complex III, three subunits of complex IV, and two subunits of complex V. 

Mitochondria are unique organelles in that they contain their own DNA (mtDNA), which, in addition to ribosomal RN A (rRNA) and transfer RN A (tRNA)-coding sequences, also encodes 13 polypeptides which are components of complexes I, III, IV, and V (Anderson et al., 1981). This fact has important implications for both the genetics and the etiology of the respiratory chain disorders. Since mtDNA is maternally-inherited, a defect of a respiratory complex due to a mtDNA deletion would be expected to show a pattern of maternal transmission. However the situation is complicated by the fact that the majority of the polypeptide subunits of complexes I, III, IV, and V, and all subunits of complex II, are encoded by nuclear DNA. A defect in a nuclear-coded subunit of one of the respiratory complexes would be expected to show classic Mendelian inheritance. A further complication exists in that it is now established that some respiratory chain disorders result from defects of communication between nuclear and mitochondrial genomes (Zeviani et al., 1989). Since many mitochondrial proteins are synthesized in the cytosol and require a sophisticated system of posttranslational processing for transport and assembly, it is apparent that a diversity of genetic errors is to be expected. 

This complex consists of at least 25 separate polypeptides, seven of which are encoded by mtDNA. Its catalytic action is to transfer electrons from NADH to ubiquinone, thus replenishing NAD concentrations. Complex I deficiency has been described in myopathic syndromes, characterized by exercise intolerance and lactic acidemia. In at least some patients it has been demonstrated that the defect is tissue specific and a defect in nuclear DNA is assumed. Muscle biopsy findings in these patients are typical of those in many respiratory chain abnormalities. Instead of the even distribution of mitochondria seen in normal muscle fibers, mitochondria are seen in dense clusters, especially at the fiber periphery, giving rise to the ragged-red fiber (Figure 10). This appearance is a hallmark of many mitochondrial myopathies. 

This complex contains 11 polypeptide subunits of which only one is encoded by mtDNA. Defects of complex III are relatively uncommon and clinical presentations vary. Fatal infantile encephalomyopathies have been described in which severe neonatal lactic acidosis and hypotonia are present along with generalized amino aciduria, a Fanconi syndrome of renal insufficiency and eventual coma and death. Muscle biopsy findings may be uninformative since abnormal mitochondrial distribution is not seen, i.e., there are no ragged-red fibers. Other patients present with pure myopathy in later life and the existence of tissue-specific subunits in complex III has been suggested since one of these patients was shown to have normal complex 111 activity in lymphocytes and fibroblasts. 

This respiratory complex consists of 13 subunits, of which the three largest are encoded on mtDNA and contain the redox centers. Complex IV is involved in a greater diversity of defects affecting human skeletal muscle than any other respiratory complex. 

Multiple deletions of mtDNA have been recorded in several families, where members show an autosomal dominant mode of inheritance of a syndrome involv-... 

Figure 13. Mosaic of cytochrome oxidase-deficient muscle fibers (asterisks) in a patient with KSS and a heteroplasmic mtDNA deletion.

Myoclonic epilepsy with ragged-red fibers (MERPF) is a rare syndrome which shows clear maternal inheritance and a variable clinical pattern including progressive myoclonus, cerebellar ataxia, dementia, and muscle weakness. It is associated with an A-to-G transition at position 8344 of the tRNA Lys gene in the mtDNA. The mutation is heteroplasmic and produces similar multicomplex deficiencies as are seen in KSS. 

The majority of the peptides in mitochondria (about 54 out of 67) are coded by nuclear genes. The rest are coded by genes found in mitochondrial (mt) DNA. Human mitochondria contain two to ten copies of a smaU circular double-stranded DNA molecule that makes up approximately 1% of total ceUular DNA. This mtDNA codes for mt ribosomal and transfer RNAs and for 13 proteins that play key roles in the respiratory chain. The linearized strucmral map of the human mitochondrial genes is shown in Figure 36-8. Some of the feamres of mtDNA are shown in Table... 

An important feamre of human mitochondrial mtDNA is that—because aU mitochondria are contributed by the ovum during zygote formation—it is transmitted by maternal nonmendefian inheritance. 

Comparisons of mtDNA sequences provide evidence about evolutionary origins of primates and other species. 

Thus, in diseases resulting from mutations of mtDNA, an affected mother would in theory pass the disease to all of her children but only her daughters would transmit the trait. However, in some cases, deletions in mtDNA occur during oogenesis and thus are not inherited from the mother. A number of diseases have now been shown to be due to mutations of mtDNA. These include a variety of myopathies, neurologic disorders, and some cases of diabetes mellitus. 

The third human DNA SRM developed by NIST was designed to meet the need for quality control when amplifying and sequencing human mitochondrial DNA (mtDNA). 

Three laboratories in addition to NIST participated in an inter-laboratory evaluation of the CHR template. All of the laboratories essentially followed the NIST protocol. Three of the four labs found essentially the same polymorphisms. Laboratory 4, who had less experience with sequencing mtDNA, did find differences that the other laboratories did not observe. The differences noted by Laboratory 4 confirm and emphasize the need for a standard reference material for sequencing mtDNA. Had Laboratory 4 had run NIST mtDNA SRM 2392 simultaneously with their unknown sample, they would have realized that they were finding an undue number of differences and could have reexamined their procedures to try to determine the reason for these differences. 

Tab. 5.2 Primer sets used for PCR amplification of human mtDNA and differences with the anderson sequence found in three templates at NIST for SRM 2392...

Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the mtDNA from the second CHR cell culture line. It is DNA from the second CHR cell culture line which is supplied in NIST SRM 3392. 

King MP and Attardi G (1989) Human cells lacking mtDNA repopulation with exogenous mitochondria by complementation. Science 246 500-503. 

Blouin, M.S., Yowell, C.A., Courtney, C.H. and Dame, J.B. (1997) Haemonchusplacei and Haemonchus contortus are distinct species based on mtDNA evidence. InternationalJournal for Parasitology 27, 1383-1387. 

Mitochondrial DNA is inherited maternally. What makes mitochondrial diseases particularly interesting from a genetic point of view is that the mitochondrion has its own DNA (mtDNA) and its own transcription and translation processes. The mtDNA encodes only 13 polypeptides nuclear DNA (nDNA) controls the synthesis of 90-95% of all mitochondrial proteins. All known mito-chondrially encoded polypeptides are located in the inner mitochondrial membrane as subunits of the respiratory chain complexes (Fig. 42-3), including seven subunits of complex I the apoprotein of cytochrome b the three larger subunits of cytochrome c oxidase, also termed complex IV and two subunits of ATPase, also termed complex V. 

In the formation of the zygote, all mitochondria are contributed by the ovum. Therefore, mtDNA is transmitted by maternal inheritance in a vertical, nonmendelian fashion. Strictly maternal transmission of mtDNA has been documented in humans by studies of restriction fragment length polymorphisms (RFLPs) in DNA from platelets. As exemplified by the disorders outlined above,... 

TABLE 42-1 Clinical features of mitochondrial diseases associated with mtDNA mutations... 

Because there are hundreds or thousands of copies of mtDNA in each cell, the phenotypic expression of a mitochondrially encoded gene depends on the relative proportions of mutant and wild-type mtDNAs within a cell this is termed the threshold effect . 

Because mitochondria replicate more often than do nuclei, the relative proportion of mutant and wild-type mtDNAs may change within a cell cycle. 

---