DNA Mutant

mtDNA

Mitochondrial alterations, including mitochondrial DNA (mtDNA) mutations have been noted in various tumor types. It has been hypothesized that a selective advantage conferred by mtDNA mutation could in particular contribute to benign tumorigenesis of a slowly replicating tissue like the human parathyroid. Acquired mitochondrial DNA mutations were identified in a subset of parathyroid adenomas, particularly in those with an oxyphil cell phenotype.98 Oxyphil cells have a characteristic eosinophilic granular cytoplasm that is densely packed with mitochondria,4,99 as compared with the typical chief cell. While the exact mechanism remains controversial, mtDNA mutations may well contribute to the molecular pathogenesis of benign parathyroid tumors. Statistically significant differences in mutation prevalence in oxyphil vs chief cell adenomas also suggest that mtDNA mutations may contribute to the oxyphil phenotype.98

Christine Mummery, ... Bernard A.J. Roelen, in Stem Cells: Scientific Facts and Fiction, 2011

Interaction Between Cancer Cells and Their Environment

Apart from accumulating DNA mutations that may directly influence the characteristics of cancer cells, signals emanating from the close environment of a tumor probably also modify the behavior of adjacent cancer cells. These signals, which originate outside of the cell, may vary depending on the location of the cancer cell in the tumor and in the surrounding healthy tissue. We often refer to this local environment as the niche. One important environmental factor can be the supply of oxygen, an essential cell nutrient, which varies in the tumor depending on the availability of local blood vessels and influences the way cancer cells behave. At the border between the tumor and the surrounding normal tissue, cancer cells may have more opportunities to interact with other, non-tumor, cell types, like fibroblasts, blood vessel cells, or various immune cells that have been attracted to the tumor through blood vessels. Some acquired DNA mutations in the cancer cell may influence the outcome of these interactions with the cells in its environment, and together this may lead to changes in the appearance and/or properties of some cancer cells, potentially contributing to the pattern of heterogeneity.

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Nonhuman Primates as Models for Reproductive Aging and Human Infertility

Barry D. Bavister, Carol A. Brenner, in Handbook of Models for Human Aging, 2006

EVALUATION OF MITOCHONDRIAL COPY NUMBER, DELETIONS AND MUTATIONS AS MARKERS OF OOCYTE COMPETENCE

Accumulation of mtDNA mutations in the mitochondrial genome may be inherent in oocytes and in embryos derived from them, especially those derived using IVP (see above), and could contribute to impaired metabolic function and thus to developmental incompetence (Keefe et al., 1995; Brenner et al., 1998; Barritt et al., 1999, 2000). Mutations may result in diminished ATP content, leading to defects such as slow or arrested cell division, apoptosis, numerical chromosomal abnormalities such as aneuploidy, and ultimately failure to develop or establish pregnancy (Barnett et al., 1997; Van Blerkom et al., 1995, 2001). But any adverse affect of mtDNA mutations associated with respiratory function would depend upon the magnitude of the mutant population (mutant load). This load could increase with each embryo cell division. Functional defects could also result from asymmetrical mitochondrial distribution following cell division, which could lead to disproportionate mitochondrial inheritance and perhaps thereby produce cells with diminished ATP-generating capacity. This type of error could be related to mitochondrial distribution in the cell (see Migration of Mitochondria in Oocytes and Embryos) (Van Blerkom et al., 1995, 2000). Mitochondria are not only the major site of ATP production in cells but also an important source of reactive oxygen species (ROS) under certain pathological conditions. Because mitochondrial DNA (mtDNA) in the mitochondrial matrix is exposed to ROS that leak from the respiratory chain, this extranuclear genome is prone to mutations. Therefore, the mitochondrial genome is a rich site for both deletions and mutations.

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MENDELIAN GENETIC TRAITS

Ronald J Trent PhD, BSc(Med), MB BS (Sydney), DPhil (Oxon), FRACP, FRCPA, in Molecular Medicine (Third Edition), 2005

Genotypic Assays

Testing for DNA mutations has advantages over protein assays: (1) Access to DNA is unlimited, whereas an abnormal protein may not be easy to obtain; and (2) Unlike protein, DNA is not affected by physiological fluctuations. The former is not a problem in haemophilia because a blood sample is adequate. The latter is an important consideration for the reasons mentioned previously. An indirect DNA linkage approach for diagnosis has been used in haemophilia since (1) The majority of defects are point mutations; (2) The genes are large; and (3) There are many mutations (see Table 3.15). A number of DNA polymorphisms have been described, which are located within (intragenic) and in close proximity to (extragenic) the factor VIII and factor IX genes. These polymorphisms allow DNA diagnosis (prenatal or carrier) to be made in ~70–80% of families (Figure 3.19). Intragenic polymorphisms have the advantage that recombination is unlikely to occur since the markers are located within the gene. Despite the many different mutations described for haemophilia A and B, the number of polymorphisms in these genes are relatively small. Hence, options for linkage analysis are limited.

Fig. 3.19. Simple DNA linkage analysis for an X-linked disorder. The child with severe haemophilia B (→) has a factor IX level of 1%. The mother is an obligatory carrier because she has an uncle who is affected (not shown in the pedigree). Factor IX coagulant levels are given as percentages in the pedigree (normal is >50%). Factor IX levels for the mother and her daughter are within the normal range (74% and 80%, respectively), but as indicated previously, this does not exclude the carrier state because of random X-inactivation in females. Therefore, a DNA study is undertaken to determine the carrier status of the daughter. From the DNA polymorphism patterns, it is evident that the haemophilia B defect co-segregates with the 1.8 kb DNA polymorphism since this is the marker present in the haemophiliac son. The haemophiliac boy has only one polymorphic DNA marker (1.8) compared to his female relatives; i.e., he is hemizygous since he does not inherit an X chromosome from his father. Therefore, the daughter's carrier status can be determined on the basis of which DNA polymorphism she inherits from her mother; i.e., if the daughter is homozygous for the 1.8 kb marker (she will always inherit one 1.8 kb marker from her father), she is a carrier. If the daughter has both 1.8 and 1.3 kb markers, then the latter must have come from her mother; i.e., the daughter is not a carrier since the 1.3 kb polymorphism is a marker for the normal maternal X chromosome.

The disadvantages inherent in DNA linkage testing must also be considered. They include (1) Key family members are required to allow phase of the polymorphism to be determined, i.e., identify which polymorphic marker in that family is co-inherited with the disease phenotype. Key family members may be deceased or unavailable. (2) It is difficult to determine whether mutations are spontaneous events if there is no family history of haemophilia. (3) Germline mosaicism, in which an individual has two or more cell lines of different chromosomal content derived from the same fertilised ovum, cannot be excluded. This is discussed further in Chapter 4. (4) An additional problem with the DNA linkage approach, particularly in haemophilia B, is the effect that linkage disequilibrium (preferential association of linked markers) can have on the informativeness of polymorphisms. For example, five biallelic DNA polymorphisms are associated with the factor IX gene (Figure 3.20). Some of these polymorphisms are inherited in a preferential association; i.e., the XmnI and MnlI polymorphisms are in linkage disequilibrium, which means that results obtained with either are similar since one allele of the polymorphism is nearly always inherited with the same allele of the other. Therefore, not all five polymorphisms will necessarily be informative. This is a particular problem with the factor IX gene locus in Chinese and Asian Indian populations. (5) Non-paternity and its effect on DNA polymorphisms is not an issue if male offspring are studied because the father does not contribute his X chromosome to males. However, the source of the paternal X chromosome is important if a female is being assessed for carrier status.

Fig. 3.20. DNA polymorphisms in the factor IX gene. The eight exons of the factor IX gene are shown. Five polymorphic restriction enzyme sites giving restriction fragment length polymorphisms (RFLPs) are indicated by↓. Three occur within the gene (intragranic), and two (BamHI and HhaI) are extragenic. Some of these polymorphisms are inherited in a preferential association known as linkage disequilibrium (e.g., XmnI and MnlI) and are therefore less useful in DNA testing.

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Mitochondrial Medicine

Douglas C Wallace, ... Vincent Procaccio, in Emery and Rimoin's Principles and Practice of Medical Genetics, 2013

11.1.37 Movement Disorders and Dementias

In certain instances, mtDNA mutations can present with neuropsychiatric symptoms including depression, movement disorders, and dementias. Movement disorders have been most commonly associated with mtDNA polypeptide gene mutations. Examples discussed previously include the MTND6∗LDYT14459A mutations (537,538), which can present as generalized dystonia, and the report of the MTND4∗LHON11778A mutation associated with Parkinson-like symptoms (518,523). A tRNAVal T1659C mutation has also been linked to movement disorders (747).

Patients harboring mtDNA mutations may also develop progressive dementia. One patient with progressive cognitive decline, dementia, deafness, ataxia, and chorea was found to be heteroplasmic for a tRNATrp mutation, MTTW∗DEMCHO5549A. Postmortem analysis of the brain revealed diffuse and moderate neuronal loss in the cortex and basal ganglia, with gliosis present throughout the brain. RRFs and COX-negative-staining fibers were evident on skeletal muscle analysis, as were morphologically abnormal mitochondria on electron microscopy of skeletal muscle. A complex I defect was detected in mitochondrial respiration assays. Hence, this tRNATrp mutation demonstrates that respiratory defects can cause dementia (748). This has been substantiated by the identification of the tRNAGln gene mutation at np 4336, MTTQ∗ADPD4336G, which has been associated with about 5% of late-onset AD and also linked to hearing loss and migraine (633,749–751). The tRNAGln 4336C mutation has also been associated with a 16S rRNA mutation at G3196A (633).

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Mitochondrial Function

Sandra R. Bacman, ... Carlos T. Moraes, in Methods in Enzymology, 2014

11 Changing mtDNA Heteroplasmy in Cultured Cells with mito-TALENs

Cells with heteroplasmic mtDNA mutations were transfected with GenJet DNA In Vitro Transfection Reagent version II (SignaGen Laboratories) following the protocol suggested by the manufacturers (3:1 ratio of DNA/GenJet and 1–2 μg DNA for a 6-well dish transfection). We scaled up and transfected a 70–80% confluent T-75 flask with 30 μg total DNA. When the two mito-TALEN monomers were cotransfected, 15 μg of each plasmid was used for the transfection (Bacman et al., 2013). The expectation was that cells subjected to mito-TALEN would change mtDNA heteroplasmy in a predictable manner.

Fluorescent-activated cell sorting (FACs) is an excellent tool to isolate different cell populations after transfection with the mito-TALENs. This was possible due to the fact that each plasmid coding for the TALEN monomer posses a fluorescence protein, eGFP or mCherry, that can be used to sort the cells after transient transfection. We were able to cell sort the population that did not get the TALEN transfection (untransfected), the populations that incorporated only one TALEN monomer individually (green-eGFP or red-mCherry) or the population that incorporated both TALENs in the same cell. Approximately 48 h after transfection, cells were collected in DMEM with no addition of serum or antibiotics, sorted and collected in PBS, using a FACSAria IIU by gating on single-cell fluorescence using a 561-nm laser and 600LP, 610/20 filter set for mCherry and a 488-nm laser and 505LP, 530/30 filter set for eGFP (Bacman et al., 2013). Cells were separated based on having only one fluorescent marker, or having both, which were referred to as "yellow" cells (Fig. 18.6A). Sorted cells not expressing fluorescent markers were likely untransfected and were referred to as "black" cells (Fig. 18.6A). FAC-sorted cells were allowed to grow in complete medium or were directly subjected to DNA extraction.

Figure 18.6. Analyzing mtDNA heteroplasmy changes by mito-TALENs. We transfected cells harboring high levels of a pathogenic mutation (m.14459G>A). Because the two plasmids had fluorescent markers, we sorted the cells for double transfectants 48 h after transfection (panel A). Cells gated for both fluorophores were termed "yellow, Y." Control cells "black, B" showed no fluorescence. These cells had their DNA isolated and analyzed by RFLP after "last-cycle hot" PCR (panel B). Total mtDNA levels were also determined by qPCR (panel C).

Adapted from Bacman et al. (2013).

DNA from sorted cell populations was subjected to "last-cycle hot PCR" (Moraes et al., 1992), which visualizes only nascent amplicons and avoid interference from heteroduplexes formed during the final PCR cycles. Following by a digestion with appropriate diagnostic restriction endonuclease, samples were electrophoresed in 8%–12% acrylamide/polyacrylamide (BioRad) in Tris Boric EDTA. The radioactive signal was quantified using a Cyclone Phosphorimager System (Perkin Elmer; Bacman et al., 2007; Bayona-Bafaluy, Muller, & Moraes, 2005).

As an example, to determine the levels of the m.14459G > A mutation by the "last-cycle hot" PCR, total DNA extracted from FAC-sorted cells was used as template, and PCR was performed with the following mtDNA primers: F-BclI-mut-F, 5′-CCCCCATGCCTCAGGATACTCCTCAATAGTGATC-3′ and 14579B, 5′-TGATTGTTAGCGGTGTGGTCGGGTGTGT-3′. The "F" primer creates a BclI restriction site in combination with the amplified mutated mtDNA. The use of mismatch primers is useful when a specific mutation does not create or destroys a site that can be recognized by commercially available enzymes. PCR products were digested with BclI and resolved in a 12% acrylamide/polyacrylamide gel. Radioactive signal was quantified using a Cyclone Phosphorimaging System (Perkin Elmer; Bacman et al., 2013). As illustrated in Fig. 18.6B–C, the comparison of yellow and black cells showed a marked reduction in mtDNA mutation load in "yellow" cells.

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The second genome: Effects of the mitochondrial genome on cancer progression

Adam D. Scheid, ... Danny R. Welch, in Advances in Cancer Research, 2019

2.5 mtDNA mutations and haplotype predispositions

Interestingly, the mtDNA mutations that contribute to the altered, oncogenic functionality of mitochondria described above can occur throughout the mitochondrial genome. Thus, a somatic mutation in a region defining a particular haplotype can "convert" the sequence to another haplotype, thereby possibly confusing interpretation of haplotype-dependent susceptibility (Brandon et al., 2006, 2005). While incidence of the latter observation may be overestimated due to sequencing errors, as parallel sequencing of normal and tumor tissue from the same individual is not performed in most studies, its occurrence has been definitively demonstrated (Parrella et al., 2001). Large insertions or deletions that give rise to changes in conserved amino acids can have drastic impacts on mtDNA and mitochondrial function, e.g., truncation of ETC components that may be important for early tumorigenesis. Mutagenic conversions that match non-self mtDNA haplotype sequences, however, may be important for metabolic adaptations to dynamic tumor microenvironments, much like divergent mtDNA haplotypes were important for adaptations to new climates in ancient peoples.

Understanding how mtDNA haplotype variants contribute to tumorigenesis and cancer progression is important for two main reasons. One reason is that individuals with particular mtDNA haplotypes have increased predispositions for developing certain cancers relative to individuals with other mtDNA haplotypes (Brinker et al., 2017; Bussard & Siracusa, 2017; Feeley et al., 2015). Indeed, adaptive advantages that mtDNA variants confer can also resemble oncogenic mitochondrial function discussed above (Ross et al., 2001; van der Walt et al., 2003). Better understandings of these predispositions can enable more effective cancer screening and prevention. The second reason why a better understanding of how mtDNA variants contribute to cancer is critical is because it can precipitate development of therapeutic interventions that block the ability of tumors to adapt to changing microenvironments, which may halt tumor growth and prevent therapeutic resistance.

Since nDNA-encoded components are instrumental to mtDNA maintenance and mitochondrial function, querying direct contributions of mtDNA to cancer requires separating nDNA and mtDNA as isolated variables. As outlined below, there are several ways these variables can be isolated in vitro in mouse and man. However, corresponding in vivo studies in humans present major ethical barriers. These studies can be performed in vivo in mouse models, but traditional backcrossing on female genetic backgrounds to obtain conplastic mice with nDNA from one mouse strain and mtDNA from another can introduce confounding recombinations in nDNA. To address this issue, we generated MNX mice by exchanging embryonic pronuclei among mouse strains (Fetterman et al., 2013; Kesterson et al., 2016), and the resulting model has enabled and will continue to enable novel insights on direct mtDNA contributions to cancer and other complex phenotypes that can interact with the disease.

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Bioenergetics and the Mitochondrial Genome

D.C. Wallace, in Encyclopedia of Biological Chemistry (Second Edition), 2013

Disease-Causing mtDNA Mutations

A wide range of pathogenic mtDNA mutations have been described over the past 20 years. The first inherited pathogenic mtDNA mutations were base substitution mutations. These included the mtDNA missense mutation in the ND4 gene at nt 11778 that converted the arginine at codon 340 to histidine and causes Leber's hereditary optic neuropathy, a form of sudden-onset, mid-life, blindness, and the tRNALys nt 8344 base substitution protein synthesis mutation that causes myoclonic epilepsy and ragged red fiber disease. The first spontaneous mtDNA disease-causing mutations were deletions associated with mitochondrial myopathy. Deletions in the mtDNA are now known to cause chronic progressive external ophthalmplegia, the Kearns–Sayre syndrome, or the Pearson's marrow pancreas syndrome. Since these discoveries, a plethora of pathogenic mutations have been described in the mtDNA.

Mitochondrial defects primarily affect the more energetic tissues including central nervous system, heart, muscle, kidney, and endocrine systems. While the frequency of deleterious mtDNA mutations in the human population is high, in fact only the milder of all possible deleterious mutations generally appear in the population due to the filtering function of the intra-ovarian selection system. A catalogued identified mtDNA pathogenic base substitution mutations is available at the MITOWEB website.

The high mtDNA mutation rate, paired with intra-ovarian selection, permits the continuous introduction of new mtDNA nucleotide substitutions into the population. These range from deleterious to advantageous, depending on the environmental and genetic context in which the mtDNA mutations arise. Mutations that are beneficial in a particular environment will preferentially reproduce and become enriched in the region. Since the mtDNA is exclusively maternally inherited, and can only change by sequential mutations along radiating maternal lineages, this results in the development of regional groups of related mtDNA haplotypes derived from the mtDNA in which the original adaptive mutation first appeared. Such regional groups of related haplotypes are called haplogroups.

By defining the regional mtDNA haplogroups and deducing the sequence of mutation events that connect the haplogroups, it has been possible to reconstruct the history of woman (Figure 3). These studies have revealed that humans arose in Africa about 150 000–200 000 years before present (YBP). Sub-Saharan African mtDNAs radiate into four African-specific lineages: L0, L1, L2, and L3, the most ancient mtDNA lineages being found among the Khoisan Bushman. About 65 000 YBP, L3 gave rise to two new lineages in northeastern Africa, designated M and N. Only M and N left Africa to colonize all of Eurasia, with one group migrating along the southeastern Asian coast to colonize Australia. In Europe, lineage N gave rise to the European-specific lineages H, I, J, K, T, U, V, W, and X. In Asia, both M and N radiated to generate a plethora of Asian-specific mtDNA lineages, including A, B, and F from N and C, D, and G from M. Among all of the Asian mtDNA lineages, only A, C, and D successfully occupied arctic Chukotka in northeastern Siberia. When the Bering land bridge appeared, individuals baring these mtDNAs moved to the Americas about 20 000 YBP. A subsequent coastal migration brought lineage B to mix with A, C, and D about 12 000–15 000 YBP, and another migration across the arctic brought the European haplogroup X to the Great Lakes region of North America about 15 000 YBP. The sum of these migrations generated the Paleo-Indians. A migration from around the Sea of Okhostk about 7000–9000 YBP brought a modified A to find the Na-Dene. Finally, recent migrations bringing predominantly D but also A and C gave rise to the Eskimos and Aleuts.

Figure 3. Global radiation of mtDNA types showing the regional association of mtDNA types. Capital letters represent major region groups of related haplotypes (haplogroups). In sub-Saharan Africa, haplogroup L0, which arose between 150 000 and 200 000 years before present (YBP), radiated into haplogroups L1, L2, and L3. At about 65 000 YBP haplogroup L3 gave rise to haplogroups M and N in northeastern Africa. M and N then left Africa in two directions. A southeastern migration moved through Southeast Asia, ultimately giving rise to Australian mtDNAs. A northeastern migration colonized Eurasia, with haplogroup N giving rise to the European mtDNA halpogroups H, I, J, K, T, U, V, W, and X and to the Asian haplogroups A, B, F, etc. Haplogroup M radiated into Asia to give rise to Asian haplogroups C, D, G, etc. Haplogroups A, C, and D became enriched in northeastern Siberia and crossed the Bering land bridge about 20 000 YBP. European-like haplogroup X arrived in Central North America about 15 000 YBP and haplogroup B migrated along the Asian and Bering coasts into southern North America, Central America, and northern South America about 12 000–15 000 YBP. X and B mixed with the haplogroups A, C, D mtDNA to generate the Amerind paleo-Indian population. About 7000–9000 YBP derivatives of haplogroup A crossed the Bering Strait to generate the Na-Dene, and around 3000–5000 YBP derivatives of predominantly haplogroup D but also A and C crossed the Bering Strait to give rise to the Eskimos and Aleuts. The +/+, +/−, −/− represent important M and N diagnostic restriction sites. The ages were calculated using the empirically determined mtDNA sequence evolution rate of 2.2–2.9%/million years (MYR).

Adapted from Wallace DC (2007) Why do we still have a maternally inherited mitochondrial DNA? Insights from evolutionary medicine. Annual Review of Biochemistry 76: 781–821.

Since these regional haplogroups permitted individuals to adapt to local bioenergetic environments, a change in the local environment may result in a mismatch between the mtDNA genotype and the environment, predisposing the individuals to disease. The migration of indigenous people out of their traditional homelands and cultural globalization of the Northwestern high-fat European diet have contributed to an increasing mismatch between mitochondrial genotype and bioenergetic environment. This results in a marked rise in predisposition to a wide range of metabolic and degenerative diseases, including diabetes and obesity, Alzheimer's and Parkinson's disease, cardiovascular disease, predisposition to blindness, sensitivity to infections, tolerance of trauma, etc.

Further exacerbation of the negative effects of the mismatch between mtDNA genes and environment is the rise in toxic industrial and agriculture chemical toxins in the environment, many of which are potent inhibitors of mitochondrial function. These factors interact to contribute to the rapid rise in complex diseases in the industrialized countries.

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Developmental Disorders and Interventions

Kim M. Cornish, ... Nicole J. Rinehart, in Advances in Child Development and Behavior, 2010

II Genetic Profile

An emerging family of DNA mutations known as trinucleotide repeat expansions is responsible for causing a range of cognitive and clinical consequences such as Huntington disease, Friedreich's ataxia, and FXS. The fragile X mental retardation 1 (FMR1) gene is a fascinating gene located at the long arm of the X chromosome. This gene contains a cytosine, guanine, guanine (CGG) triplet repeat region that when expanded can result in a continuum of fragile X disorders. Normal CGG repeat sizes correspond to between 7 and 55 repeats, with 30 repeats being the most common. When expanded to > 200 repeats (large expansion) the FMR1 gene is turned off leading to the lack of the fragile X mental retardation protein (FMRP), and results in the neurodevelopmental disorder known as FXS. Because FMRP is involved in normal brain development, through its impact on synaptic formation and function, the absence of FMRP results in the characteristic intellectual impairment and cognitive profile associated with this disorder and represents one of the few known single gene causes of autism.

Due to genetic variation in the form of X-inactivation (when one of the two X chromosomes remains inactive and the other active) girls with FXS, compared to boys, produce a broader range of cognitive abilities and have IQ's ranging from moderate to the normal range. In contrast, this is not an issue of concern in FXS males whose impairment, without the protection of X-inactivation, shows greater severity. For this reason, we will focus on boys and girls separately.

Most recently, interest has focused on more common, medium size expansions between 55 and 200 CGG repeats (referred to "carrier status"). This research is especially important given the relative frequency of these expansions in the general population calculated as 1 in 130 to 250 females and 1 in 260 to 800 males (Song, Lee, Li, Koo, & Jung, 2003). Until recently carriers were believed to be "phenotypic free", that is without any known cognitive deficits. However, there is a now well documented subtle profile of cognitive strengths and weaknesses, notably in males, that can mirror those found in those individuals with the large CGG expansion (FXS). See Cornish, Turk, et al. (2008) for a review of these findings.

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Multidrug-Resistant Bacteria

Christopher Grace MD, FACP, in Critical Care Secrets (Fifth Edition), 2013

7 How do bacteria become multiresistant?

Bacteria become resistant to antibiotics by DNA mutation at select points or by insertions or deletions that alter microbial enzymes or the antibiotic targets. Genetic material can be transferred between bacteria by plasmids (extrachromosomal double-stranded circular DNA) via direct cell-to-cell contact. Bacteria may also acquire new resistance genes by infection with bacteriophage viruses that carry resistance genes with them when they infect bacteria. Once bacteria develop or acquire new resistance genes they have a selective advantage when antibiotics are used. As more mutations or transferred genetic material accumulates, the more classes of antibiotics the bacteria become resistant to, inducing MDR.

Dominant lethal mtDNA mutations?

Are effects of mtDNA mutations limited to respiratory deficiency? Mitochondria are involved in many other processes other than respiration. Of those, most relevant to aging are perhaps apoptosis and generation of ROS. However, absolute majority of mtDNA mutations studied so far apparently neither cause cell death (in fact, RC-deficient cells apparently survive for long periods of time), nor cause pronounced increase of ROS. This does not necessarily mean, however, that mtDNA mutations with such properties do not exist. Our knowledge about the effects of mtDNA mutations mostly comes from studies of inherited mtDNA mutations, and thus may be incomplete. Inherited mutations have to be compatible with development, i.e., be "mild" in certain sense. Somatic mutations are free from this limitation and, therefore, can potentially be more "severe" and even lethal. A demonstrative example is a mutation of the mouse mtDNA called 13885insC (an insertion of a C at position 13885). This is a "severe" mutation: it creates a translation frameshift in a complex I gene, ablates assembly of complex I,25 and increases ROS production.26 This is not a natural inherited mutation: it was isolated by in vitro selection in a cell line (this mutation confers resistance to a certain chemical that is toxic only when complex I is functional). When artificially introduced into the germ line, this severe mtDNA mutation is purged from progeny, as it causes oocyte death. Interestingly, oocytes die even if they carry as low as ~ 12% of the mutant genome. That is, the mutation is dominant, but unlike dominant mutations discussed in Section 3.4, its primary action is not merely in disabling of RC, but in killing the oocyte. It is therefore tempting to speculate that there exists a whole class of mtDNA mutations that are lethal to the cell even at low fractions. Such hypothetical mutation were termed "dominant lethal."27 Dominant lethal mutations might act, for example, by causing an increase in ROS production that would be toxic for the cell and cause apoptosis.

Dominant lethal mutations would be difficult to detect because of their trend to self-eliminate. While these mutations may be rare, they could potentially have contributed to some important aging phenomena, in particular, progressive loss of stem cells. Dominant mitochondrial mutations were implied in a model explaining early heart pathology in mice with increased rate of somatic mtDNA mutations.27 While dominant lethal mtDNA mutations are very intriguing, they are yet to be discovered.

In summary, the most common phenotypic effect of mtDNA mutations is the inactivation of the RC. To disturb RC, mutation needs to exceed a threshold of 50–90%, depending on mutation and cell type. Dominant mutations that are not subject to phenotypic threshold are less common. It is tempting to speculate that some dominant mutations are lethal, and these mutations, if they existed, might be very important for the aging process.

Mitochondrial Dysfunction and Pathophysiology

Mitochondrial DNA (mtDNA) mutations can accumulate in tissues over the life course, causing aging-related declines in energy output, cell damage and loss, and disease and dysfunction. The mitochondrial aging hypothesis is closely allied with the free radical and oxidative theories of aging: it proposes that aging results when mitochondrial function deteriorates as a result of oxidative damage to mitochondrial genes and membranes (Wallace, 2008). Mutations in genes affecting mitochondrial function or structure have been implicated in degenerative diseases in the nervous system, heart, skeletal muscle, and kidneys, as well as in some endocrine and metabolic disorders, including diabetes mellitus. mtDNA mutations also are implicated in many conditions normally associated with aging, including some cancers, ocular cataracts, and retinopathy. Nucleic-acid base substitutions in the mtDNA control region in human populations have been associated with increased risks of diabetes and cardiomyopathy (heart disease). Brains of people with Alzheimer's disease have more mitochondrial mutations on average than controls. Moreover, mitochondrial mutations in somatic tissues have been shown to accumulate in all animal tissues that have been examined, and the rate of accumulation is inversely correlated with species life span.

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Generating Mouse Models of Mitochondrial Disease

Emil Ylikallio, Henna Tyynismaa, in Movement Disorders (Second Edition), 2015

43.2.1 Modification of mtDNA

Although primary mtDNA mutations are an important cause of mitochondrial disease, many hurdles exist to the generation of mouse models with specific mtDNA mutations (Dunn et al., 2012). MtDNA cannot be directly modified because current technology does not allow efficient transfection of engineered mtDNA into mitochondria. Even if mutated mtDNA was successfully introduced into mitochondria, it would be heteroplasmic. Furthermore, deleterious mtDNA mutations may be removed during oocyte development (Fan et al., 2008; Stewart et al., 2008), which means that germ-line transmission may not be achieved. Mutations in protein-coding genes have been particularly shown to be subject to purifying selection, while tRNA gene mutation can apparently evade the selection (Stewart et al., 2008). Different strategies have been developed to overcome the inability to directly manipulate mtDNA.

Exogenous mitochondria that already contain mutated mtDNA can be inserted into mice at an early developmental stage. In one of the first efforts toward generation of such transmitochondrial mice, a mutation in the 16S rRNA gene of mtDNA, which confers resistance to chloramphenicol (CAPR), was created through chemical mutagenesis and clonal selection in a melanoma cell line (Watanabe et al., 1978). The cytoplasmic portion (cytoplast) of the melanoma clone was fused with a teratocarcinoma stem cell to create a cytoplasmic hybrid (cybrid). The cybrid cells obtained the chloramphenicol resistance trait, which they retained also after being injected into nude mice where they differentiated into diverse cell types. The injection of teratocarcinoma cybrid cells into blastocysts and their insertion into pseudopregnant females led to generation of chimeric mice that were mixtures of cells with wild-type mtDNA and CAPR mtDNA, but no germ line transmission was detected in this work (Watanabe et al., 1978). In another experiment, heteroplasmic mice were generated that carried two naturally occurring mtDNA variants, one from the NZB mouse strain and the other from the BALB mouse strain (Jenuth et al., 1996). This was achieved by fusion of cytoplasts from one mouse strain with one-cell stage embryos from the other mouse strain. The resulting mice, heteroplasmic for NZB and BALB mtDNAs, were used to study the segregation pattern of mtDNA in oocytes and in differentiated tissues (Jenuth et al., 1997).

Transmitochondrial disease model mice demonstrating germ-line transmission were first generated by recovering mitochondria from brain synaptosomes (cytoplasmic vesicles released form nerve endings) of NZB mice (Sligh et al., 2000). The synaptosomes were then fused to cells lacking mtDNA (ρ0-cells). The reason for using ρ0-cells was to prevent carryover of the endogenous mtDNA, and thus the cybrids were completely or nearly homoplasmic for the NZB mtDNA. The NZB mitochondria were then introduced into the embryonic stem (ES) through electrofusion of enucleated cybrids and mtDNA-depleted ES cells. The resulting cybrid ES cells were inserted into blastocysts, which were transplanted into pseudopregnant females, leading to chimeric mice that had homoplasmic NZB mtDNA in part of their cells. Subsequent breeding showed that the NZB mtDNA was carried through the germ-line. In the same report, CAPR mtDNA was successfully introduced into ES cells from mouse 501-1 cells, and transmitted through the germ line, which resulted in a severe phenotype with perinatal or in utero lethality (Sligh et al., 2000).

To create a transmitochondrial disease model, another group isolated synaptosomes from the brains of aged mice, fused them to ρ0 cells, and used PCR screening to identify a cybrid clone that contained an mtDNA deletion (Inoue et al., 2000). The clone was enucleated and fused to mouse pronuclear stage embryos that were then transplanted into the oviduct of pseudopregnant mice. The proportion of live births was low, but the investigators were able to identify founder females that were heteroplasmic for the deleted mtDNA, and germ-line transmission was obtained over three generations. The load of deleted mtDNA varied between 20% and 80%, and the proportion of deleted mtDNA varied with the severity of the phenotype, which replicated some, but not all, features of KSS. Most of the mice died from renal failure (Inoue et al., 2000). These mice were called "mito-mice," and the experiment proved that somatic mtDNA mutations are harmful to mitochondrial function.

Transmitochondrial mice homoplasmic for disease-associated point mutations in mtDNA have since been generated (Kasahara et al., 2006; Lin et al., 2012; Yokota et al., 2010). A model for LHON was created with a similar methodology as the mito-mice (Lin et al., 2012); UV-light and psoralen were used to mutagenize mtDNA in LMTK- cells. Then, ethidium bromide was administrated to induce mtDNA depletion, after which mtDNA was allowed to reamplify in cell clones to enrich for a specific mtDNA genotype. Clones homoplasmic for a deleterious mtDNA mutation were then identified by culture in galactose-media, and a clone that was homoplasmic for the LHON-associated c.G13997A mutation in MT–ND6 was enucleated and fused to ES cells, which again allowed creation of chimeric mice and subsequent germ-line transmission. These mice recapitulated key features of LHON. The electroretinogram showed reduced retinal function, and the optic nerve had pathological abnormalities including age-related loss of the smallest central optic nerve fibers, neuronal accumulation of abnormal mitochondria, axonal swellings, and demyelination. Bioenergetic analyses showed that the mutant mice were able to maintain ATP homeostasis even under stressed conditions, but that they had elevated production of reactive oxygen species (ROS) by CI. These results suggested that LHON is caused by oxidative stress rather than ATP deficiency (Lin et al., 2012).

A limitation of the transmitochondrial method by chemical and UV-mutagenesis is that somatic mutations are random, or occur predominantly at hotspots, and thus desired disease-associated mutations may never arise. Furthermore, some mutations may confer a growth advantage in cultured cells, leading to overrepresentation in transmitochondrial models (Lin et al., 2012; Yokota et al., 2010). Therefore, a technique with which to transform cells with engineered mtDNA remains an important goal. A recently developed possibility to obtain new mtDNA mutations are the "mtDNA Mutator" mice, which are knockin mice carrying proofreading-deficient POLG that leads to accumulation of mtDNA mutations (Trifunovic et al., 2004). By backcrossing female mice carrying the mutant polymerase to wild-type males, maternal lines transmitting different mtDNA mutations were generated (Stewart et al., 2008). A mouse line with a heteroplasmic single-base-pair deletion in tRNA-Methionine gene (MT-TM) was created using this method, but heteroplasmy levels higher than 86% could not be obtained through breeding, indicating a selective effect during transmission (Freyer et al., 2012).

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Neurogenetics, Part I

Ryan L. Davis, ... Carolyn M. Sue, in Handbook of Clinical Neurology, 2018

Mitochondrial DNA mutations and human disease

Pathogenic mtDNA mutations may be categorized into point mutations, large-scale rearrangements (single deletions, duplications, or multiple deletions), as well as mtDNA depletion (Davis and Sue, 2011). To date, over 300 mtDNA mutations and rearrangements have been reported (Vafai and Mootha, 2012; Ohtake et al., 2014) to cause mitochondrial disease and have historically been classified into syndromes (Table 10.1) (Schon et al., 2012). A database of reported mtDNA mutations and variants can be found at http://www.mitomap.org (Lott et al., 2013). Pathogenic mtDNA point mutations occur in 35 of the 37 genes encoding ribosomal RNAs (rRNA), transfer RNAs (tRNAs) and respiratory chain protein subunits. Point mutations are largely maternally transmitted, but clinical evidence of disease depends on a variety of factors such as level of heteroplasmy, functional metabolic demands of the tissue involved, and the nuclear genetic background.

Table 10.1. Mitochondrial genes associated with mitochondrial disease

Deletions in mtDNA may vary in size (often between 2 and 10 kb) and involve any part of the mitochondrial genome, with the exception of the noncoding regulatory D-loop. A "common" deletion that is 4.9 kb in length can be identified in a third of patients with progressive external ophthalmoplegia. This common deletion is flanked by a 13-bp repeat sequence and probably arises due to a polymerase "slipping" error during mtDNA replication (Schon et al., 1989). Deletions are typically sporadic, but rare familial cases have been reported (Shanske et al., 2002). Although uncommon, deletions may be associated with duplications, which can be transmitted through the maternal line (Poulton et al., 1989, 1993). Multiple deletions (multiple species of partially deleted mtDNA) can also occur (Zeviani et al., 1989, 1990) and are often found in patients with nuclear gene defects that affect the fidelity of mtDNA replication.

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Peripheral Nerve Diseases Associated with Mitochondrial Respiratory Chain Dysfunction

MICHAEL G. HANNA, PAULA CUDIA, in Peripheral Neuropathy (Fourth Edition), 2005

Acute and Subacute Axonal Neuropathy with A3234G Mutation

The A3243G mtDNA mutation most frequently causes the MELAS phenotype. A mild chronic axonal sensorimotor neuropathy may be a minor feature, as described above.24 However, occasionally the A3243G mutation is associated with more aggressive neuropathic disease. Hara et al.19 described a 33-year-old woman who developed an acute sensorimotor neuropathy with significant limb weakness and facial weakness in her second pregnancy. She was considered to have Guillain-Barré syndrome but went on to develop severe lactic acidosis and rhabdomyolysis. Genetic analysis showed that she harbored the mtDNA A3243G mutation. Electrophysiologic studies indicated a severe acute axonal neuropathy. Sural nerve biopsy showed a severe decrease in myelinated fibers. Teased nerve preparations showed myelin ovoid formation but no demyelination.

Ciafaloni et al.6 described a 21-year-old man who developed subacute distal limb weakness of moderate severity. Electrophysiologic studies showed an axonal polyneuropathy that was confirmed on sural nerve biopsy. A year after the presentation with neuropathy, he developed a strokelike episode and generalized seizures and a diagnosis of MELAS was made. Genetic analysis confirmed the A3243G mutation.

Van Domburg et al.53 reported six adults from three separate families with an unusual heredoataxic syndrome. In all cases the early phase of the disease was characterized by a severe sensory neuropathy. Sural nerve biopsies showed severe loss of myelinated axons at an early stage in the disease. The patients subsequently develop CPEO and later develop myoclonic epilepsy with a high incidence of status epilepsy and sudden death. Muscle biopsy showed RRF and CSF lactate was elevated, confirming a mitochondrial disease, although the genetic basis was not determined.

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Mitochondrial Mutagenesis in the Brain in Forensic and Pathological Research

Nicole von Wurmb-Schwark, in Oxidative Stress and Neurodegenerative Disorders, 2007

mtDNA alteration are possibly associated with neurodegenerative diseases

The discovery that mtDNA mutations are of pathological importance (82,129,130), and that mitochondria play an important role in the mechanisms of aging ((131–133), reviewed in (2)) and cell death (7,134) shows the importance of mitochondria in pathological research. The spectrum of phenotypes has expanded from rare myopathies to multiple diseases representing virtually all branches of medicine. The possibility that some of the most common and devastating degenerative diseases seems to involve mitochondria implicates the importance of investigations of mitochondrial genetics and biochemical changes in these organelles. Although mitochondrial mutations are present at low levels (usually < 2%) in the whole tissue, it could be possible that mutations clonally expand within one cell and exceed a defined threshold which could cause defects of mitochondrial oxidative metabolism and may lead to cell death.

Although cells possess an intricate network of defense mechanisms to neutralize excess ROS and reduce oxidative stress, some tissues, especially the brain, are much more vulnerable to oxidative stress because of their elevated consumption of oxygen and the consequent generation of large amounts of ROS. For the same reason, the mtDNA of brain cells is highly susceptible to structural alterations resulting in mitochondrial dysfunction (135).

There are many reports on mitochondria and mtDNA and a possible involvement with neurological degenerative diseases (136–138), e.g. Parkinson's disease (PD) or Alzheimer's disease (AD) (139–146). Still, investigations are contradictory or not yet confirmed (147). Screening for a specific substitution did not reveal any differences between brains from normal elderly persons or patients with AD or PD (140,148). However, increasingly studies report correlations between certain neurological diseases and mtDNA mutagenesis. In this section, some of the diseases that are possibly connected to mitochondrial mutagenesis and that are currently under excessive investigation will be presented briefly as an overview.

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Mitochondrial DNA: Structure, Genetics, Replication and Defects

Jan-Willem Taanman, Albert M. Kroon, in Mitochondria in Obesity and Type 2 Diabetes, 2019

5.5 Transmission of Heteroplasmic mtDNA Mutations

The transmission of heteroplasmic mtDNA mutations is an intensely studied subject because clarification of the mechanism will help to predict the recurrence risk of mtDNA mutations relevant to genetic counseling. In 1982, Hauswirth and Laipis133 reported large heteroplasmic shifts within a few generations of a single maternal lineage of Holstein cows. The authors suggested that this change could be because of a massive reduction in mtDNA content during oogenesis, leading to segregation of mtDNA variants by random genetic drift. Since this first observation, rapid shifts in heteroplasmy levels have been found in many animal models and humans.134 These findings led to the germline genetic bottleneck hypothesis of mtDNA inheritance, which proposes that during female germ line development a small number of mtDNA molecules are sampled from a larger population for amplification and transmission, resulting in a mtDNA mutation load that is highly variable between offspring. Several studies of animal models have shown that there is a marked reduction in mtDNA content during female germ line development. Single-cell deep mtDNA sequencing of early primordial germ cells (mtDNA copy number: ~ 1400) from early-gestation human female embryos has revealed rare variants that reached higher heteroplasmy levels in late primordial germ cells, consistent with the genetic bottleneck theory.135 In addition, there appeared to be selection against severely deleterious mutations, concomitant with a progressive upregulation of mtDNA transcription and replication, and linked to a change from glycolytic to oxidative metabolism. It is thought that the metabolic transition during germ line development exposes severely deleterious mutations to a filtering mechanism, averting the relentless buildup of mtDNA mutations predicted by Muller's ratchet. Mutations evading this process, however, will show rapid shifts in heteroplasmy levels, explaining the dramatic phenotypic variation seen in families with inherited mtDNA disorders (Fig. 4).

Fig. 4. Inheritance of mtDNA point mutations. MtDNA in a cell or tissue can be either homoplasmic (all copies are identical in sequence) or heteroplasmic (a mixture of wild-type (green) and mutated (red/X) mtDNA). A mtDNA mutation will result in a clinical phenotype only when the mutation load exceeds a critical threshold. A reduction in mtDNA content during female germ line development, known as the mitochondrial genetic bottleneck, and random genetic drift leads to a wide variation of heteroplasmy levels in oocytes. Therefore, the mtDNA mutation load passed on from mother to child will vary, resulting in extensive phenotypic heterogeneity among siblings.

Several studies of oocytes and embryonic tissues from females carrying mtDNA mutations have indicated that segregation of the m.3243A > G and m.8993T > G point mutations are governed by random genetic drift.136–138 This suggests that these mutations are not subjected to purifying selection.139 Analyses of human pedigrees transmitting a number of common mtDNA point mutations (m.3243A > G, m.3460G > A, m. 8344A > G, m. 8993T > C/G, m.11778G > A) confirmed the absence of selection during transmission but suggested that the segregation rate varies between different mutations.140 The predicted range of heteroplasmy levels for these mutations in offspring is wide and, consequently, genetic counseling is difficult. In contrast, other studies found evidence of purifying selection acting on deleterious mtDNA variants during germline development.135, 141, 142 Notably, there appears to be a strong selection against mtDNA deletions because the recurrence risk of a mtDNA deletion disorder is small (~ 4%).143 The nature of the filtering mechanism remains unresolved.

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Cardiovascular Toxicology

W. Lewis, in Comprehensive Toxicology, 2010

6.16.9 mtDNA Mutations and NRTI Toxicity

An important correlate is that mtDNA mutations may result from oxidative mtDNA damage, aberrant mtDNA replication, and altered mtRNA transcription. Together, these interlinked events are the cornerstones of the 'mitochondrial dysfunction hypothesis' (Lewis et al. 2001b) that we applied in the laboratory in models of AIDS CM (Lewis 1989; Lewis et al. 1991, 1992, 2000, 2001b). Additionally, the same principles are applicable to mitochondrial toxicity in other targets (Lewis et al. 1994a,b, 1996, 1997). The 'mitochondrial dysfunction hypothesis' (Lewis et al. 2001a) clarifies important pathophysiological events in NRTI toxicity. It is reviewed herein in the context of NRTI mitochondrial metabolism and AIDS CM. Similarly, the pathogenesis of mtDNA somatic mutations is incompletely understood, but severely deleterious mutations appear to be removed early in oogenesis (Fan et al. 2008), whereas less severe mtDNA mutations persist and may relate to more common diseases (Bensch et al. 2007).

It may be reasonably argued that analysis of mechanisms of NRTI-induced mitochondrial toxicity is analogous to approaches that examine defects in genetic mitochondrial illnesses in which the defective mitochondrial gene product, the oxidative stress, and the environment contribute to disease pathogenesis (Schapira and Cooper 1992). It should be noted that clinical and basic reviews of mitochondrial toxicity of NRTIs have been presented elsewhere where other aspects of the clinical and biological events are detailed (Brinkman et al. 1998, 1999; Kakuda et al. 1999; Lewis and Dalakas 1995; Lewis et al. 2001a; Morris and Carr 1999).

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Molecular Clock

M. Wang, G. Caetano-Anollés, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Controversy over the DNA Clock

With the advent of genomics, studies of DNA mutation rates among species (i.e., the DNA clock) attracted more and more attention. For example, the study of Miyata and Tasunaga on molecular evolution of mRNA triggered a long-run controversy over the existence of a global DNA clock in mammals, especially because of the claim that mammals had similar DNA mutation rates. However, follow-up studies did not support their initial viewpoint. DNA mutation rates in humans were much lower than that in some rodents. Generation-time effects, changes in DNA repair mechanisms as well as metabolic rates were used to explain this phenomenon. More recently, studies confirmed mutation rate differences within and among the main groups of mammals. For example, Kumar and Subramanian verified rate differences using a mass of sequence data. Their results suggest that replication-independent processes (e.g., DNA methylation, recombination, and repair mechanisms) play a more important role in mutation than generation times, physiological attributes, and other life-history traits.

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Mitochondrial DNA and Aging

Mikhail F. Alexeyev, ... Glenn L. Wilson, in Handbook of Models for Human Aging, 2006

MTDNA MUTATIONS ARE PATHOGENIC AND CAN INCREASE ROS PRODUCTION BY MITOCHONDRIA

The discovery, in 1988, that mtDNA mutations can be pathogenic has provided a major support for the mitochondrial theory of aging. That year, several groups reported that both mtDNA point mutations and deletions could be the underlying cause of defined human pathologies. Moreover, being heteroplasmic, these diseases have revealed that not all the mtDNA copies in a cell need to be mutated in order to achieve a disease state. The last decade produced an explosive growth of the number of mtDNA mutations implicated in human disease. The recent release of the Mitomap database lists almost 200 pathogenic point mutations, single nucleotide deletions and insertions (Brandon et al., 2005). Not only have mitochondrial diseases revealed a causative link between mtDNA mutations and pathology, but they also have displayed, in agreement with the predictions of the mitochondrial theory of aging, an increased oxidative burden in patients suffering from these diseases (Kunishige et al., 2003; Lu et al., 2003).

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