DNA Mutation

DNA mutations leading to activation of proto-oncogenes or inactivation of tumor suppressor genes can potentially lead to the unregulated growth seen in cancer.

From: Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2012

Related terms:

Mitochondrion

Oxidative Phosphorylation

Mitochondrial DNA

Electron Transport Chain

Oxidative Stress

Nested Gene

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Hyperparathyroidism

Jessica Costa-Guda, Andrew Arnold, in Genetics of Bone Biology and Skeletal Disease, 2013

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

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mtDNA mutations in cancer

Giulia Girolimetti, ... Ivana Kurelac, in The Human Mitochondrial Genome, 2020

Abstract

Mitochondrial DNA (mtDNA) mutations have been described in virtually all cancer types. However, due to the peculiarities of mitochondrial genetics and cancer heterogeneity, it has been difficult to assess their role in tumorigenesis and cancer progression. The advent of massive sequencing and large public data repositories are allowing to gain insight about the evolution of mtDNA variants and somewhat predict their functional effects. Here, the current knowledge of mtDNA mutation landscape in cancer is described, which generally implies to a negative selection of severely pathogenic lesions. The interplay between mtDNA mutations and different stages of progressing solid tumors is discussed, together with the potential of mtDNA variants to be used as diagnostic markers in certain cancer contexts.

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The Mitochondrion in Aging and Disease

Konstantin Khrapko, Doug Turnbull, in Progress in Molecular Biology and Translational Science, 2014

5.5 The "Vicious cycle"

Speaking about the effect of mtDNA mutations in aging, we can't set aside the famous "vicious cycle" concept, which has been influencing the field of mitochondrial mutations for many years. The concept is based on an assumption57 that mtDNA mutations may cause an increase in ROS production. Unlike the "dominant lethal" mutations model (Section 3.5), "vicious cycle" hypothesis postulates that such increase of ROS does not kill the cell per se but instead causes increased generation of mutations, which further increases ROS, resulting in a positive feedback loop. The concept predicts that mtDNA mutations should accumulate exponentially, eventually culminating in an "error catastrophe" identifiable with cellular aging/death. However, the vicious cycle hypothesis is not supported by the data. Most importantly, vicious cycle predicts that a substantial proportion of mutations should be nonclonal, because vicious cycle boosts de novo mutation generation rather than expansion of pre-existing mutations. In reality, the fraction of nonexpanded mutations appears to be rather low, at least in case of deletions.58 Also, a great majority of mutations appear not to increase ROS levels: indeed, there was no ROS increase in heavily mutated tissues of mtDNA mutator mouse.59 There is no indication that mtDNA mutations increase mutation rates. So if "vicious cycle mutations" (i.e., mtDNA mutations causing increased mtDNA mutation rate) exist, they should be rare, but if so, then a great majority of extra mutations generated because of the presence of the vicious cycle mutations will be of the nonvicious cycle type, and thus, the ability of the cycle to sustain itself is questionable. The vicious cycle concept that dominated in the field for many years is apparently being put to rest.52,60

In conclusion, the effects of somatic mtDNA mutations are maximal in limited areas such as pigmented neurons of substantia nigra, respiration-deficient zones of muscle fibers and colonic crypts. It is likely that if mtDNA mutations are somehow involved in the aging process, they act through these critical areas, rather than entire tissues or the whole body.

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Stem Cells in Cancer and Cancer Stem Cells

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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Mitochondrial DNA-related diseases associated with single large-scale deletions and point mutations

Robert D.S. Pitceathly, Shamima Rahman, in The Human Mitochondrial Genome, 2020

14.3.3 Skeletal muscle histochemistry, electron microscopy, and respiratory chain biochemistry

If mtDNA mutations are undetectable in blood, muscle biopsy may be a helpful diagnostic test to investigate further the possibility of mtDNA-related mitochondrial disease.

Histochemical techniques include Gomori trichrome, the red staining of which highlights subsarcolemmal accumulation of mitochondria, so-called RRFs, while succinate dehydrogenase (SDH) enzyme staining highlights a similar phenomenon, so-called ragged blue fibers. Combined staining with COX and SDH (COX/SDH) is a useful tool to highlight COX-negative fibers, which are highly suggestive of an underlying mtDNA mutation, particularly when a mosaic pattern is present indicative of heteroplasmy. Fig. 14.2 illustrates histochemical staining of skeletal muscle sections and demonstrates findings consistent with mitochondrial myopathy.

Figure 14.2. Histochemical staining of skeletal muscle sections demonstrating findings consistent with mitochondrial myopathy.

(A) Prominent internal mitochondria including subsarcolemmal mitochondrial aggregates. These fibers are referred to as "ragged red" fibers and are usually most clearly visible with Gomori trichrome histochemistry (B). On oxidative stains, particularly succinate dehydrogenase (SDH) staining, these fibers show "ragged blue" changes (C). Deficiency of cytochrome c oxidase (COX) is an important diagnostic feature (D) that is further highlighted using combined COX-SDH staining (E). The fibers may also show excess internal lipid droplets as seen on Oil red-O stain (F).

In our hands, electron microscopy (EM) of muscle tissue does not add significantly to diagnostic yield when histochemical, biochemical, and genetic studies are combined. However, EM may reveal subsarcolemmal and intermyofibrillar proliferation of mitochondria and the presence of abnormal mitochondria in muscle fibers. Enlarged, elongated, irregular, and dumbbell-shaped mitochondria with hypoplastic and dystrophic cristae and paracrystalline inclusions suggest mitochondrial dysfunction. However, these findings are nonspecific and can be present in other neuromuscular disorders.

Muscle homogenate (fresh or frozen) can be used to undertake enzymatic assays of specific respiratory chain complexes. This is particularly useful in children with mtDNA-related disease in whom muscle histochemistry may appear normal. Multiple respiratory chain enzyme deficiencies are seen with mtDNA tRNA mutations (e.g., m.3243A>G), while isolated defects suggest dysfunction of protein-coding genes, although these rules are not universally adhered to.

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Mechanisms of discordance in monozygotic twins: why and when?

Alexandra Matias, ... Isaac Blickstein, in Developmental and Fetal Origins of Differences in Monozygotic Twins, 2020

Asymmetrical mitochondrial DNA division

Mitochondrial DNA (mtDNA) mutations have been currently held responsible for the phenotypic variability of common Mendelian disorders (Machin, 2009). In the mitochondrial encephalomyopathies, mutated mtDNA usually coexists with wild-type mtDNA. This phenomenon is referred to as heteroplasmy. Clinical phenotype alters with the ratio and tissue distribution of the mutation (Kato et al., 2005). De novo heteroplasmic mutations of mtDNA reportedly caused the discordance of Leber's disease in MZ twins. An MZ twin pair discordant for chronic progressive external ophthalmoplegia was also reported. Discordance for neurofibromatosis type I, adrenoleukodystrophy and obesity have also been investigated. Discordant phenotypes due to uneven amount of heteroplasmic mutations were also found in DZ twins with myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and myoclonic epilepsy. This kind of genetic difference may happen at or after the twinning event.

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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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Methods and models for functional studies on mtDNA mutations

Luisa Iommarini, ... Francisca Diaz, in The Human Mitochondrial Genome, 2020

13.4.6 Blue Native Polyacrylamide Gel Electrophoresis

One of the methods that have gained popularity in the last decade to study samples of patients with mitochondrial disorders and to complement other analysis such as mitochondrial respiration and enzymatic activity is Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE). BN-PAGE is used to determine the steady-state level of respiratory complexes, their assembly, enzymatic activity, and their interactions to form supercomplexes. The technique was initially described by Shägger and von Jagow to separate respiratory complexes on the basis of their native molecular weight by gel electrophoresis [193]. Proteins are extracted with mild detergents and exposed to Coomassie Blue dye, which binds proteins conferring a negative charge and in that way, proteins can migrate through an electric field without changes in their native conformation. Depending on the detergent used, one can detect individual respiratory complexes or their association into supercomplexes. Lauryl maltoside is commonly used to detect individual complexes, while digitonin is used when it is desired to preserve supercomplex structures. Lauryl maltoside is a non-ionic glycosidic detergent efficient for hydrophobic protein solubilization that allows to solubilize and disrupt the interaction between the respiratory complexes, but mild enough to maintain their multimeric composition. Digitonin is a non-ionic steroidal glycoside detergent that does not cause denaturation of proteins during solubilization and is known to interact with cholesterol in membranes. Digitonin is a much milder detergent than lauryl maltoside and does not disrupt the interaction between respiratory complexes, leaving supercomplexes intact. The respiratory complexes can be detected by either staining gels with Coomassie or other protein stain, by western blot if proteins are transferred onto a nitrocellulose or PVDF membranes or by In-Gel Activity (IGA) stain. Because the complexes retain their native conformation after electrophoresis, gels can be incubated with appropriate substrates and dye to detect the enzymatic activity by the formation of a color precipitate. IGA assays have been developed to measure CI, CII, CIV, and CV, but these stains are semiquantitative [194]. Individual components of each complex can be analyzed running a second dimension in an SDS-PAGE. For the first dimension, BN-PAGE is run, then each gel lane is cut, incubated with SDS and reducing agents to denature native proteins, placed on top of a SDS-polyacrylamide gel and stacking gel poured around it. In the second dimension, proteins are separated under denaturing conditions and individual subunit of complexes analyzed by western blot. One of the advantages of BN-PAGE is that it allows to screen for defects on various respiratory complexes at the same time and can provide information if an enzymatic activity defect detected by other methods is due to decreased steady-state levels of the respiratory complex. Detailed protocols for BN-PAGE can be found in the following references [194–197] and an example of results is shown in Fig. 13.3.

Figure 13.3. Examples of results of BN-PAGE and 2D-PAGE for isolated OXPHOS complexes and supercomplexes. (A) Analysis of CIV deficiency using BN-PAGE in skeletal muscle from wild type (WT) and Cox10KO (KO) mouse models [198]. Cox10 is a CIV assembly factor required for maturation and stability of Cox1. Muscle mitochondria were isolated from WT and KO mice and then treated with lauryl maltoside to extract respiratory complexes. Samples were separated by BN-PAGE in a 3%–13% acrylamide native gel and proteins were stained with Coomassie blue or for CIV activity (CIV-IGA) as reported in Ref. [194]. Note the absence of fully assembled CIV in the KO compared to WT. (B) Mitochondria isolated from the same WT and KO mouse skin fibroblasts extracted with lauryl maltoside and then separated by BN-PAGE as in (A) followed by a second dimension. The second dimension was performed by cutting gel strips of BN-PAGE and separating proteins in a denaturing SDS-PAGE. Proteins were transferred onto a PVDF membrane and immunodetected with antibodies against various subunits of OXPHOS complexes. Signals were developed by chemiluminescence. For CI, we used anti-NDUFA9, for CII anti-SDHA, for CIII anti-Core2, for CV anti-ATPaseβ, and for CIV anti-COXI and anti-COXIV antibodies, respectively. Antibodies were added sequentially to the same blots. Signals of blots were color-coded and merged to create the figure. Note the absence of COXI signal and decreased levels of COXIV (in blue) in the KO sample. (C) Analysis of supercomplexes using BN-PAGE in human cybrids carrying the homoplasmic frameshift mutation m.3571insC/MT-ND1 (MUT) and corresponding control (WT). The mutation generates a premature stop codon in ND1 causing a lack of the protein [199]. Mitochondria were isolated from WT and MUT cells and then treated with digitonin to extract respiratory complexes and supercomplexes (CI+III2+IV; CI+III2; CIII2+IV). Samples were separated by BN-PAGE in a 3%–13% acrylamide native gel and proteins were stained for CI activity (CI-IGA) or transferred onto a PVDF membrane and immunodetected by using antibodies against COXI (CIV) and Core2 (CIII), which are able to highlight all the supercomplexes and isolated CIII2 and CIV. Note the absence of supercomplexes containing CI in the mutant cell line (MUT). (D) Mitochondria isolated from the same WT and MUT cybrids as in (C). Respiratory supercomplexes were separated BN-PAGE followed by a denaturing SDS-PAGE. Proteins were transferred onto a PVDF membrane and immunodetected with antibodies against various subunits of OXPHOS complexes. Signals were developed by chemiluminescence. For CI, we used anti-NDUFS3, for CIII anti-Core2, and for CIV anti-COXI antibodies, respectively. Antibodies were added sequentially to the same blots. Note the absence of supercomplexes containing CI and the presence of low molecular weight staining for NDUFS3 (subcomplexes) in the MUT sample.

Research perspectives

The role of mtDNA mutations in cell biology is still not completely understood. Several issues remain unsolved such as the regulation of heteroplasmic shift and the cellular metabolic reprogramming induced by mtDNA mutations. Understanding these mechanisms would provide novel knowledge of fundamental biological process and would enhance our ability to discover new therapies for diseases in which mtDNA mutations are involved. In this scenario, iPSCs technology may strongly contribute to shed light on the involvement and the impact of mtDNA mutations during the different phases of cell differentiation. Furthermore, the increasing ability to obtain organoids will pave the way to understand the specific function/disfunction of mitochondria in organs specially affected by mtDNA mutations. Lastly, novel approaches in gene editing are still necessary to manipulate mtDNA with the aim to generate more faithful animal models and to test gene therapy strategies for mitochondrial disorders.

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