Detection of mutations in mitochondrial DNA by droplet digital PCR

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.     

A new method for analysis of mitochondrial DNA point mutations and assess levels of heteroplasmy

Determination of mitochondrial DNA (mtDNA) heteroplasmy for the diagnosis of patients with mitochondrial disorders is a difficult task due to the coexistence of wild-type and mutant genomes. We have developed a new method for genotyping and quantification of heteroplasmic point mutations in mtDNA based on the SNaPshot technology. We compared the data of this method with the widely used "last hot-cycle" PCR-RFLP method by studying 15 patients carrying mtDNA mutations. We showed that SNaPshot is an accurate, reproducible, and sensitive technique for the determination of heteroplasmic mtDNA mutations in different tissues from patients, and it is a promising system to be used in prenatal and postnatal diagnosis of mtDNA-associated disorders.     

Prenatal diagnosis of mitochondrial DNA8993 T----G disease.

We have previously described a family with a neurological syndrome comprising neurogenic muscle weakness, ataxia, retinitis pigmentosa, and variable sensory neuropathy, seizures, and mental retardation or dementia. This is associated with a heteroplasmic point mutation of mtDNA at bp 8993. The mother of a severely affected child underwent prenatal diagnosis in two further pregnancies. Analysis of chorionic villus samples showed a higher proportion of mutant mtDNA on both occasions, and this was reflected in the majority of fetal tissues, including brain and muscle. Prenatal diagnosis is a rational approach to the prevention of severe diseases caused by point mutations of mtDNA but is currently hampered by incomplete knowledge concerning the proportion of mutant mtDNA: its relationship to disease severity, how it may change during fetal and postnatal development, and its tissue distribution.     

Transmission of Mitochondrial DNA Diseases and Ways to Prevent Them

Recent reports of strong selection of mitochondrial DNA (mtDNA) during transmission in animal models of mtDNA disease, and of nuclear transfer in both animal models and humans, have important scientific implications. These are directly applicable to the genetic management of mtDNA disease. The risk that a mitochondrial disorder will be transmitted is difficult to estimate due to heteroplasmy—the existence of normal and mutant mtDNA in the same individual, tissue, or cell. In addition, the mtDNA bottleneck during oogenesis frequently results in dramatic and unpredictable inter-generational fluctuations in the proportions of mutant and wild-type mtDNA. Pre-implantation genetic diagnosis (PGD) for mtDNA disease enables embryos produced by in vitro fertilization (IVF) to be screened for mtDNA mutations. Embryos determined to be at low risk (i.e., those having low mutant mtDNA load) can be preferentially transferred to the uterus with the aim of initiating unaffected pregnancies. New evidence that some types of deleterious mtDNA mutations are eliminated within a few generations suggests that women undergoing PGD have a reasonable chance of generating embryos with a lower mutant load than their own. While nuclear transfer may become an alternative approach in future, there might be more difficulties, ethical as well as technical. This Review outlines the implications of recent advances for genetic management of these potentially devastating disorders.     

Relaxed replication of mtDNA: A model with implications for the expression of disease

Heteroplasmic mtDNA defects are an important cause of human disease with clinical features that primarily involve nondividing (postmitotic) tissues. Within single cells the percentage level of mutated mtDNA must exceed a critical threshold level before the genetic defect is expressed. Although the level of mutated mtDNA may alter over time, the mechanism behind the change is not understood. It currently is not possible to directly measure the level of mutant mtDNA within living cells. We therefore developed a mathematical model of human mtDNA replication, based on a solid foundation of experimentally derived parameters, and studied the dynamics of intracellular heteroplasmy in postmitotic cells. Our simulations show that the level of intracellular heteroplasmy can vary greatly over a short period of time and that a high copy number of mtDNA molecules delays the time to fixation of an allele. We made the assumption that the optimal state for a cell is to contain 100% wild-type molecules. For cells that contain pathogenic mutations, the nonselective proliferation of mutant and wild-type mtDNA molecules further delays the fixation of both alleles, but this leads to a rapid increase in the mean percentage level of mutant mtDNA within a tissue. On its own, this mechanism will lead to the appearance of a critical threshold level of mutant mtDNA that must be exceeded before a cell expresses a biochemical defect. The hypothesis that we present is in accordance with the available data and may explain the late presentation and insidious progression of mtDNA diseases.     

Molecular diagnosis and counseling in a family presenting compound heterozygosity for autosomal recessive limb-girdle muscular dystrophy.

The present report concerns two patients, male and female siblings, manifesting a different degree of severity for the same autosomal recessive limb-girdle muscular dystrophy. The index case (male sib) carried the clinical diagnosis of Becker muscular dystrophy at the time when the sister, with a much milder presentation, first sought counseling and prenatal diagnosis for a pregnancy already in course. Molecular and immunocytochemical tests then available favoured the diagnosis of an autosomal recessive myopathy, but did not enable exclusion of a dystrophinopathy The couple was counseled accordingly, although prenatal diagnosis could not be offered. Both patients were later found to carry one gamma- and two alpha-sarcoglycan gene mutations, one of the latter being new This raised a counseling dilemma: depending on which combination was the disease-causing genotype, there would be a minimal or a significant 25% risk to offspring. We describe the studies carried out and emphasise the importance of differential diagnosis and extensive molecular characterisation in this group of disorders, so as to enable correct genetic counseling and prenatal diagnosis.     

Wild-Type Mitochondrial DNA Copy Number in Urinary Cells as a Useful Marker for Diagnosing Severity of the Mitochondrial Diseases

The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.     

Diagnostic challenges of mitochondrial DNA disorders

Although mitochondrial disorders are increasingly being recognized, confirming a specific diagnosis remains a great challenge due to the genetic and clinical heterogeneity of the disease. The heteroplasmic nature of most pathogenic mitochondrial DNA mutations and the uncertainties of the clinical significance of novel mutations pose additional complications in making a diagnosis. Suspicion of mitochondrial disease among patients with multiple, seemingly unrelated neuromuscular and multi-system disorders should ideally be confirmed by the finding of deleterious mutations in genes involving mitochondrial biogenesis and functions. The genetics are complex, as the primary mutation can be either in the nuclear or the mitochondrial DNA (mtDNA). MtDNA mutations are often maternally inherited, but can also be sporadic or secondary to mutations in nuclear-encoded mitochondrial-targeted genes. Several well-defined clinical syndromes associated with specific mutations have been described, yet the genotype-phenotype correlation is often unclear and most patients do not fit within any defined syndrome and even within a family the expressivity of the disease can be extremely variable. This article describes examples representing diagnostic challenges of mitochondrial diseases that include the limitations of the mutation detection method, the occurrence of mitochondrial disease in families with another known neuromuscular disorder, atypical clinical presentation, the lack of correlation between the degree of mutant heteroplasmy and the severity of the disease, variable penetrance, and nuclear gene defects causing mtDNA depletion.     

Sequence analysis of the complete mitochondrial genome in patients with Leber's hereditary optic neuropathy lacking the three most common pathogenic DNA mutations

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by central vision loss in young adults. The majority of LHON cases around the world are associated with mutations in the mitochondrial genome at nucleotide positions (np) 3460, 11,778, and 14,484. Usually, these three mutations are screened in suspected LHON patients. The result is important not only in respect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. There are, however, a significant number of patients without any of these primary mutations. In these situations, genetic counselling of a patient and his family can be difficult. We sequenced the complete mitochondrial DNA (mtDNA) in 14 LHON patients with the typical clinical features but without a primary mtDNA mutation to evaluate the potential of extensive mutation screening for clinical purposes. Our results suggest to include the mutation at np 15,257 in a routine screening as well as the ND6 gene, a hot spot for LHON mutations. Screening for the secondary LHON mutations at np 4216 and np 13,708 may also help in making the diagnosis of LHON as these seem to modify the expression of LHON mutations. Although they do not allow to prove the clinical diagnosis, their presence increases the probability of LHON. Sequencing the complete mitochondrial genome can reveal novel and known rare disease causing mutations. However, considering the effort it adds little value for routine screening.     

The distribution of mitochondrial DNA heteroplasmy due to random genetic drift

Cells containing pathogenic mutations in mitochondrial DNA (mtDNA) generally also contain the wild-type mtDNA, a condition called heteroplasmy. The amount of mutant mtDNA in a cell, called the heteroplasmy level, is an important factor in determining the amount of mitochondrial dysfunction and therefore the disease severity. mtDNA is inherited maternally, and there are large random shifts in heteroplasmy level between mother and offspring. Understanding the distribution in heteroplasmy levels across a group of offspring is an important step in understanding the inheritance of diseases caused by mtDNA mutations. Previously, our understanding of the heteroplasmy distribution has been limited to just the mean and variance of the distribution. Here we give equations, adapted from the work of Kimura on random genetic drift, for the full mtDNA heteroplasmy distribution. We describe how to use the Kimura distribution in mitochondrial genetics, and we test the Kimura distribution against human, mouse, and Drosophila data sets.     

Germ-line deletions of mtDNA in mitochondrial myopathy.

mtDNA encodes subunits of the electron transport chain and is exclusively maternally inherited in mammals. It has been suggested that mtDNA might be the site of some of the mutations causing a group of human disorders called the "mitochondrial myopathies," because these may both be (1) accompanied by defects in the electron transport chain and (2) display a maternal pattern of inheritance. However, all of the deletions and duplications of mtDNA which occur in these patients have been sporadic, apart from families in whom affected members all carry different deletions suggesting a mutant autosomal dominantly inherited nuclear gene with de novo deletions in each individual. We present the first evidence for the presence of deleted mtDNAs in the germ line in these disorders. The patient carries a higher level of deleted mtDNAs than do his relatives, corresponding to severity of symptoms and consistent with a predicted dosage effect. "Selfishness" of deleted mtDNAs is probably one of the factors over and above random segregation of a small number of "founder" mtDNAs (the bottleneck hypothesis) which may be invoked to explain the usual distribution of mtDNAs in different tissues of patients with mtDNA deletions.     

Decrease of 3243 A-->G mtDNA mutation from blood in MELAS syndrome: a longitudinal study

It is widely held that changes in the distribution of mutant mtDNAs underlie the progressive nature of mtDNA diseases, but there are few data documenting such changes. We compared the levels of 3243 A-->G mutant mtDNA in blood at birth from Guthrie cards and at the time of diagnosis in a blood DNA sample from patients with mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome. Paired blood DNA samples separated by 9-19 years were obtained from six patients with MELAS. Quantification of mutant load, by means of a solid-phase minisequencing technique, demonstrated a decline (range 12%-29%) in the proportion of mutant mtDNA in all cases (P=.0015, paired t-test). These results suggest that mutant mtDNA is slowly selected from rapidly dividing blood cells in MELAS.     

Clinical, biochemical and molecular genetic features of Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON) has traditionally been considered a disease causing severe and permanent visual loss in young adult males. In nearly all families with LHON it is associated with one of three pathogenic mitochondrial DNA (mtDNA) mutations, at bp 11778, 3460 or 14484. The availability of mtDNA confirmation of a diagnosis of LHON has demonstrated that LHON occurs with a wider range of age at onset and more commonly in females than previously recognised. In addition, analysis of patients grouped according to mtDNA mutation has demonstrated differences both in the clinical features of visual failure and in recurrence risks to relatives associated with each of the pathogenic mtDNA mutations. Whilst pathogenic mtDNA mutations are required for the development of LHON, other factors must be reponsible for the variable penetrance and male predominance of this condition. Available data on a number of hypotheses including the role of an additional X-linked visual loss susceptibility locus, impaired mitochondrial respiratory chain activity, mtDNA heteroplasmy, environmental factors and autoimmunity are discussed. Subacute visual failure is seen in association with all three pathogenic LHON mutations. However, the clinical and experimental data reviewed suggest differences in the phenotype associated with each of the three mutations which may reflect variation in the disease mechanisms resulting in this common end-point.     

α-ketobutyrate links alterations in cystine metabolism to glucose oxidation in mtDNA mutant cells

Pathogenic mutations in the mitochondrial genome (mtDNA) impair organellar ATP production, requiring mutant cells to activate metabolic adaptations for survival. Understanding how metabolism adapts to clinically relevant mtDNA mutations may provide insight into cellular strategies for metabolic flexibility. In this study, we use ¹³C isotope tracing and metabolic flux analysis to investigate central carbon and amino acid metabolic reprogramming in isogenic cells containing mtDNA mutations. We identify alterations in glutamine and cystine transport which indirectly regulate mitochondrial metabolism and electron transport chain function. Metabolism of cystine can promote glucose oxidation through the transsulfuration pathway and the production of α-ketobutyrate. Intriguingly, activating or inhibiting α-ketobutyrate production is sufficient to modulate both glucose oxidation and mitochondrial respiration in mtDNA mutant cells. Thus, cystine-stimulated transsulfuration serves as an adaptive mechanism linking glucose oxidation and amino acid metabolism in the setting of mtDNA mutations.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations

Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR–RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful in identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

Inheritance of mitochondrial disorders

Over the last decade there have been major advances in our understanding of the genetic basis of mitochondrial disease, enabling genetic counseling for patients with autosomal dominant and autosomal recessive disorders. Genetic counseling for patients with mitochondrial DNA (mtDNA) mutations is less well established. Approximately one-third of adults with a mtDNA disorder are sporadic cases, usually due to a single deletion of mtDNA. About two-thirds of adults with mtDNA disease harbor a maternally transmitted point mutation. The recurrence risks are well documented for homoplasmic mtDNA mutations causing Leber hereditary optic neuropathy, but the situation is less clear for families with heteroplasmic mtDNA disorders. Two large studies have shown that for some heteroplasmic point mutations there appears to be a relationship between the percentage level of mutant mtDNA in a mother's blood and her risk of having clinically affected offspring. The situation is less clear for other point mutations, some of which may cause sporadic disease. Recent evidence has cast light on the general principles behind the transmission of heteroplasmic mtDNA point mutations, which may be important for genetic counseling in the future.     

The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases

In humans, mutations in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) are associated with a spectrum of disease that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. Previous attempts to correlate different types or locations of mutations with different elements of the disease phenotype have been limited by the relatively small numbers of available cases. The current article describes the molecular genetic basis for 75 new cases of HPRT deficiency, reviews 196 previously reported cases, and summarizes four main conclusions that may be derived from the entire database of 271 mutations. First, the mutations associated with human disease appear dispersed throughout the hprt gene, with some sites appearing to represent relative mutational hot spots. Second, genotype-phenotype correlations provide no indication that specific disease features associate with specific mutation locations. Third, cases with less severe clinical manifestations typically have mutations that are predicted to permit some degree of residual enzyme function. Fourth, the nature of the mutation provides only a rough guide for predicting phenotypic severity. Though mutation analysis does not provide precise information for predicting disease severity, it continues to provide a valuable tool for genetic counseling in terms of confirmation of diagnoses, for identifying potential carriers, and for prenatal diagnosis.     

Mutant copies of mitochondrial DNA in tissues and plasma of mice subjected to X-ray irradiation

Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.