mtDNA has unusual properties that are important for our understanding of mitochondrial disease caused by mtDNA mutations.
Heteroplasmy and the threshold effect
While most human cells contain two copies of nuclear DNA, they contain many more copies of mtDNA (from 1000 to 100 000, depending on the cell type). These are all identical in a healthy individual at birth (homoplasmy). By contrast, patients harbouring pathogenic mtDNA defects often have a mixture of mutated and wild-type mtDNA (heteroplasmy).30 The percentage of mutated mtDNA can vary widely among different patients, and also from organ to organ, and even between cells within the same individual. In vitro studies using "transmitochondrial cytoplasmic hybrid (cybrid)" cells37 containing different amounts of mutated mtDNA have shown that most mtDNA mutations are highly recessive. In other words, the cells were able to tolerate high percentage levels of mutated mtDNA (typically 70–90%) before they developed a biochemical respiratory chain defect. The precise threshold for biochemical expression varies from mutation to mutation, and from tissue to tissue. Large retrospective studies have shown that the percentage level of mutated mtDNA in clinically relevant tissues does correlate with the severity of disease.38,39
Maternal inheritance and the genetic bottleneck
Although it has been known for some time that mtDNA is transmitted from mother to offspring,40 the mechanisms are only just becoming clear. Sperm contain around 100 mtDNAs which enter the zygote on fertilisation before being actively degraded.41 There has been a recent report of a pathogenic mtDNA microdeletion in a patient with a sporadic muscle specific mitochondrial disorder.42 The mutated mtDNA arose on a mitochondrial genome that was paternal in origin, bringing into question the traditional dogma of strict maternal inheritance. However, many families with mtDNA disease have been studied in detail over the last decade, and there are no other reports of paternal mtDNA transmission. Based upon the available evidence, paternal leakage is unlikely to be clinically significant.
One of the most remarkable features of mitochondrial disease caused by mtDNA defects is the clinical variability among siblings. This is thought to reflect the mitochondrial "genetic bottleneck".43 Our understanding of the transmission of mtDNA heteroplasmy has been greatly advanced by detailed studies of heteroplasmic mice generated by karyoplast transfer.44,45 These mice transmit heteroplasmic mtDNA polymorphisms (table 3). By measuring the variation in heteroplasmy between the offspring of a single female, and comparing this to the variation between oocytes at different stages of development, it was shown that the transmitted percentage level of heteroplasmy is determined at an early stage during oogenesis in a heteroplasmic female developing in utero.44 It is likely that there is a restriction in the number of mitochondrial genomes during early oogenesis, creating a functional "genetic bottleneck". This creates a sampling effect, akin to taking a small handful of marbles from a bag containing a large number of well mixed black and white marbles while wearing a blindfold. Each independent sample will contain different proportions of the two types, corresponding to mutated and wild-type mtDNA in the offspring. Recent work suggests that the same random mechanism operates during the transmission of pathogenic mtDNA mutations in humans.56,57 While this generates variability in the transmitted mutation load to the offspring, it occurs within a given confidence interval, explaining why retrospective family studies have shown a relation between the level of mutated mtDNA in the mother and the outcome of pregnancy.39,58
Although differences in the transmitted mutation load provide some explanation for the difference in severity between different family members, it does not explain why one sibling might present with neurological disease while another might develop heart failure. Clearly additional factors must come in to play.
The percentage level of mutated mtDNA in individual tissues may also change during development and throughout adult life, potentially influencing the phenotype within an individual. Two mechanisms contribute to this process: relaxed replication and mitotic segregation.
Relaxed replication
Unlike nuclear DNA which replicates only once during each cell cycle, mtDNA is continuously recycled, even in non-dividing tissues such as skeletal muscle and brain.59,60 mtDNA replication is therefore independent of the cell cycle (that is, it is relaxed). In a heteroplasmic cell, it is possible that mutated and wild-type mtDNA replicate at subtly different rates—either because one type was selected for destruction or replication by chance, or because of a subtle selective effect in favour of one particular type. In theory, this mechanism could lead to changes in the proportion of mutated mtDNA that have been described in patients with mtDNA disease, providing an explanation for the late onset and progression of some mtDNA disorders.61
Mitotic (vegetative) segregation
When a heteroplasmic cell divides, subtle differences in the proportion of mutated mtDNA may be passed on to the daughter cells, leading to changes in the level of mutated mtDNA within a dividing tissue.60,62 The unequal partitioning may be a purely random process, independent of any selection caused by an effect of the mutation on mitochondrial function. On the other hand, presumed shifts due to functional selection may explain why the level of some pathogenic mtDNA mutations decreases in blood during life (for example, 0.5% to 1% per annum for A3243G63).
mtDNA "background," nuclear genes, and the environment
While there are a great many different heteroplasmic mtDNA mutations, in epidemiological terms most patients with a pathogenic mtDNA defect harbour only mutated mtDNA (that is, they are homoplasmic mutated).64 The most common example is LHON (Leber hereditary optic neuropathy).64 LHON is a mitochondrial genetic disorder that is primarily caused by mutations in mtDNA complex I (ND) genes and is characterised by subacute bilateral visual failure presenting in early adult life.65 LHON is intriguing because it is essentially an organ specific disease that principally affects the retinal ganglion cells and the optic nerve.66 LHON also has a markedly reduced penetrance with a clear sex bias, with only around 50% of men and around 10% of women developing visual failure.35,67,68 Most patients with LHON are homoplasmic mutated for one of three mtDNA ND gene mutations (fig 4),69 so heteroplasmy cannot explain the varied disease penetrance, and certain unknown additional factors appear to be important.
Wild-type (normal) mtDNA can be subdivided into different genetic groups (haplogroups) based upon a characteristic pattern of polymorphism that occurs within the normal population.70 Two of the three principal LHON mtDNA mutations (T14484C in the ND6 gene and G11778A in the ND4 gene) are preferentially associated with haplogroup J, which is found in around 15% of northern Europeans.71 The reason for this association is not known, but it seems likely that haplogroup J increases the penetrance of the T14484C and G11778A mutations.72 It therefore appears that the mitochondrial genetic background can influence disease expression, but this cannot explain the sex bias in LHON.
The segregation pattern of disease in some LHON families suggests that there may be a nuclear genetic modifier locus modulating the clinical expression of the LHON mtDNA mutations. A recessive visual loss susceptibility locus on the X chromosome would explain the sex bias in LHON,73 but attempts to identify the locus have not been successful.74 Environmental factors may also play a part in LHON. There are many anecdotal reports of visual failure following alcohol intoxication, starvation, heavy smoking, and head trauma,68 but large studies have yielded conflicting results.75,76
In many ways LHON is best considered as a complex trait, where the disease phenotype arises through multiple genetic factors (both mitochondrial and nuclear) interacting with the environment. A similar mechanism might explain the variable penetrance of other homoplasmic mtDNA mutations that cause organ specific disease, such as the A1555G mtDNA mutation in the 12S rRNA gene that causes maternally inherited susceptibility to aminoglycoside induced deafness, and possibly the A4300G mtDNA mutation in tRNAIle that causes maternally inherited cardiomyopathy (see table 1). Similar nuclear–mitochondrial interactions are also likely to contribute to the varied phenotype seen in other mitochondrial disorders, be they caused by primary nDNA defects or primary mtDNA defects.
Nuclear genes and mtDNA heteroplasmy
After heteroplasmic mice were generated from laboratory strains with two different mtDNA genotypes in the mid-90s (see table 3),44 it became clear that a particular mitochondrial genome was favoured in some tissues, and the other mitochondrial genome was favoured in others.77 Detailed experiments showed that this selective effect was not a result of detectable differences in respiratory chain activity or rates of mtDNA replication, and that the selection appeared to be controlled at the level of the mtDNA molecule itself.78 Recent work has identified three specific nuclear genetic loci that influence this process.79 This has important implications for our understanding of mtDNA diseases because the equivalent genes in humans might influence the level of heteroplasmy in different tissues and organs, and therefore modulate the clinical phenotype.
Outlook
The last five years have seen major advances in our understanding of mitochondrial genetics and how mtDNA mutations cause disease. Clinical expression is influenced by heteroplasmy, mtDNA background, nuclear genes, and their interaction with the environment. Evolutionary studies are casting light on this complex relation. For example, in the char (a fish), different environments, and particularly the water temperature, have selected in favour of a particular mitochondrial genotype.80 Recent work on humans suggests that the same phenomenon may have occurred during population migrations throughout the world.81 Understanding these processes is of fundamental importance for the clinical management of patients—from genetic counselling to developing new treatments.
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