mitochondrial DNA

A feature unique to mitochondrial DNA (mtDNA) is maternal inheritance. This peculiarity results from the fact that ova contain mitochondria within their abundant cytoplasm, whereas spermatozoa contain few, if any, mitochondria.

Hence, the mtDNA complement of the zygote is derived entirely from the ovum. Thus, mothers transmit mtDNA to all their offspring, male and female; however, daughters but not sons transmit the DNA further to their progeny. Several other features apply to mitochondrial inheritance.

Human mtDNA contains 37 genes, of which 24 are needed for mtDNA translation and 13 encode subunits of the respiratory chain enzymes. Because mtDNA encodes enzymes involved in oxidative phosphorylation, mutations affecting these genes exert their deleterious effects primarily on the organs most dependent on oxidative phosphorylation. These include the central nervous system, skeletal muscle, cardiac muscle, liver, and kidneys.

Comparison of mtDNA with mitochondrial protein sequences revealed deviations from the universal genetic code: for example, the mtDNA codon TGA is used as a tryptophan codon rather than as a termination codon. This was followed by the observation that codon usage could vary in mitochondria from different species.

Another surprising feature of the mitochondrial genetic system was that it uses a simplified decoding mechanism, which allows translation of all codons with only 22 transfer RNAs rather than the 31 predicted by Crick’s wobble hypothesis.

Mitochondrial DNA is present in 103-104 identical copies in each cell, with the exception of sperm and mature oocytes, in which mtDNA copy numbers are 102 and 105, respectively.

In general, there are believed to be two to ten copies of DNA per mitochondrion. The sequences of mtDNAs from unrelated individuals in human populations typically differ by about 50 nucleotides, or 0.3%.

Most individuals, however, have a single mtDNA sequence variant in all their cells (homoplasmy). Under normal conditions, mtDNA transmission is exclusively through the maternal lineage. Although sperm mitochondria contribute to the zygote at fertilization, they are selectively eliminated in intraspecies crosses. Failure to eliminate paternal mtDNA, however, has been observed recently in abnormal embryos.

Several mechanisms to explain paternal mtDNA elimination, such as simple dilution, destruction of paternal mtDNA, and metabolic differences between the oocytes and sperm, have not explained satisfactorily this interesting phenomenon.

In summary, there are four fundamental differences between mitochondrial genetics and Mendelian genetics that have to be considered when studying human OXPHOS diseases: maternal inheritance, polyplasmy, heteroplasmy and the threshold effect, whereby a critical number of mutated mtDNAs must be present for the OXPHOS system to malfunction.

Replication and transcription of mtDNA

Replication and transcription of mtDNA involves the activity of nuclear-encoded factors or enzymes. The transcription products of mtDNA include 2 ribosomal RNA species (12S and 16S rRNA), 13 messenger RNAs and 22 transfer RNAs. The rRNAs, together with nuclear-encoded ribosomal proteins, form mitoribosomes.

The mitochondrial tRNAs are charged by the corresponding amino acids through the activity of nuclear-encoded amino-acyl tRNA synthetases. The mitochondrial ribosomes, with the participation of the amino-acyl-tRNA complexes and of nuclear-encoded initiation (MTIF2) and elongation (MTEFTU, TS, G) factors, synthesize 13 polypeptides of the OXPHOS system.

Mitochondrial DNA mutations

In 1988, seven years after the complete sequence of human mtDNA was published, the first pathogenic mutations were discovered. In 2005, more than 100 point mutations and innumerable rearrangements have been associated with human mitochondrial disease.

As stated above, sperm mtDNA is degraded or eliminated after fertilization of the oocyte, and so mt DNA mutations (such as point mutations) are transmitted maternally. However, deleted mtDNA molecules are rarely, if ever, transmitted from affected women to their children. In such cases, the causative mutation probably originates in the germ line.

Three relatively frequently observed point mutations are A3243G in the tRNA(Leu)(UUR) gene, A8344G in the tRNA(Lys) gene and T8993G in the ATPase 6 gene45. In this context, it is worth mentioning, however, that we still lack comprehensive and unbiased epidemiological data about the frequency of known mt DNA mutations.

Although tRNA genes as a whole represent 10% of the mtDNA, mutations in these genes account for 75% of mtDNA-related diseases. These mutations have been extensively studied over the past ten years in an effort to understand their pathogenetic mechanisms. Although initially associated with multisystem syndromes, mt DNA mutations have also been observed in patients with tissue-specific disorders.

One goal of this research is to understand why one mutation, such as the A3243G, can cause such a broad spectrum of clinical phenotypes.

Simple differences in mutational load and tissue distribution alone cannot explain this phenomenon, and other factors must contribute.

Conversely, different mutations can be associated with the same clinical phenotype. A possible explanation has recently been proposed for the tRNA(Leu)(UUR) 3243 and 3271 mutations, both of which are associated with MELAS. Both of the mutant tRNA molecules are deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification might lead to the mistranslation of leucine and/or a decrease in the rate of mitochondrial protein synthesis.

Much of the molecular analysis of the various mutations has been done in CYBRID cells. Different RHO 0 CELL lines that are used to generate cybrids have included osteosarcoma, lung carcinoma or HeLa cells, which has led to different conclusions about the molecular effects of the A3243G mutation.

Börner et al. determined the relative amounts and states of aminoacylation of mutant and wild-type tRNAs in tissue samples from patients with MELAS and MERRF. Aminoacylation levels varied from null to decreased to normal. This led Jacobs and Holt to conclude that the cellular phenotype varies in cell-culture models no more than it does in vivo, and is therefore not a consequence of cell-culture artefacts. The authors suggest further that genetic background (nuclear or mitochondrial) and epigenetic effects will need to be investigated to gain insight into the phenotypic consequences of the A3243G mutation.

Giuseppe Attardi and colleagues recently proposed the following model to explain the effects of the A3243G mutation on mitochondrial protein synthesis and OXPHOS dysfunction. The A3243G mutation alters the tertiary structure of the tRNA(Leu)(UUR), impairs methylation of the G at nucleotide 3239, and impedes modification of uridine at the first position of the anticodon. As a consequence, the mutant tRNA(Leu)(UUR) is metabolically less stable and is less efficiently charged by the leucyl-tRNA synthetase.

The reduced level of charged tRNA, or the reduced ratio of charged to uncharged tRNA, then affects mRNA association with ribosomes, ultimately causing a general reduction in mitochondrial protein synthesis. In severely affected cells, it is also possible to detect the degradation of mRNAs that are not associated with ribosomes, which will exacerbate the phenotype.

Mutations have also been identified in mtDNA genes that encode proteins of the OXPHOS system, such as the cytochrome b gene and the mitochondrial complex I genes.

A prominent example of the latter group of mtDNA protein-coding gene mutations is Leber’s hereditary optic neuropathy (LHON). LHON is a common cause of subacute bilateral optic neuropathy that usually presents in early adult life and that predominantly affects males. Most LHON patients harbour one of three point mutations that affect mtDNA complex I, or the NADH:ubiquinone oxidoreductase (ND) genes: G3460A in ND1, G11778A in ND4 and T14484C in ND6.

Recently, Patrick Chinnery and colleagues showed that the mitochondrial ND6 gene is a hot spot for LHON mutations and suggested that the ND6 gene should be sequenced in all LHON patients who do not harbour one of the three common LHON mutations. It is estimated that mt DNA mutations are responsible for 20% of OXPHOS-deficient patients. The remaining patients must therefore harbour mutations nuclear gene coding for mitochondrial proteins.

Doubly uniparental inheritance in animals

The discovery of ’doubly uniparental inheritance’ (DUI) of mtDNA in some bivalves has challenged the paradigm of strict maternal inheritance (SMI). (17681397)

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