Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests

Deletions in mitochondrial DNA are a common cause of mitochondrial disorders. The molecular diagnosis of mtDNA deletions for years was based on Southern hybridization later replaced by PCR methods such as PCR with primers specific for a particular deletion (mainly the so-called common deletion of 4977bp) and long PCR. In order to evaluate the usefulness of MLPA (Multiplex Ligation-dependent Probe Amplification) in molecular diagnosis of large scale mtDNA deletions we compare four diagnostic methods: Southern hybridization, PCR, long-PCR and MLPA in a group of 16 patients with suspected deletions. Analysis was performed on blood, muscle and in one case hepatic tissue DNA. The MLPA was not able to confirm all the deletions detected by PCR methods, but due to its relative ease of processing, minimal equipment, low costs and the additional possibility to detect frequent point mtDNA mutations in one assay it is worth considering as a screening method. We recommend to always confirm MLPA results by PCR methods.     

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale hetero-plasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.     

A novel 7.4 kb mitochondrial deletion in a patient with congenital progressive external ophthalmoplegia, muscle weakness and mental retardation.

We present a patient with external ophthalmoplegia, bilateral ptosis, progressive muscle weakness with "ragged-red fibres" and mental retardation. Mitochondrial DNA analysis by Southern blot revealed heteroplasmy in muscle for a 7.4 kb deletion. In white blood cells, the deletion was only detectable by PCR. There was no evidence for duplications, nor for multiple deletions in the proband or siblings. PCR analysis did not reveal the presence of a mitochondrial DNA defect in the parents and siblings. Thus, there is no experimental support for a maternally inherited mitochondrial DNA deletion. We consider this a sporadic case with a de novo deletion. Diabetes and complaints of fatigue, also seen in this family, are probably coincidental. Mental retardation has been reported occasionally in patients with mitochondrial deletions, but is not common.     

Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle.

Several reports have shown that individual mitochondrial DNA (mtDNA) deletions accumulate with age. However, the overall extent of somatic mtDNA damage with age remains unclear. We have utilized full-length PCR to concurrently screen for multiple mtDNA rearrangements in total DNA extracted from skeletal muscle derived from physiologically normal individuals (n = 35). This revealed that both the number and variety of mtDNA rearrangements increases dramatically between young and old individuals (P < 0.0001). We further examined the mtDNA from both the younger and older subjects by Southern blot analysis and observed an age-related increase in mtDNA(s) comparable in size to mtDNA products unique to patients with known mtDNA deletions. These data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in post-mitotic tissues.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy

We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enxyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomyopathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA.     

Detection of mitochondrial DNA deletions by a screening procedure using the Polymerase Chain Reaction

We describe an accurate procedure for a rapid diagnosis of heteroplasmic mtDNA deletions based on the polymerase chain reaction (PCR). For a selective amplification of deleted mtDNA across the breakpoints of the deletion, we used seven combinations of primers surrounding the most common deleted region between the two origins of mtDNA replication. This procedure was performed on muscle biopsies of twenty patients harboring a single mtDNA deletion and one patient with multiple mtDNA deletions. The results were compared with Southern-blotting analysis. We conclude that this PCR procedure is a sensitive and convenient screening method for the detection of mtDNA deletions. (Mol Cell Biochem 174: 221–225, 1997)     

Identical mitochondrial DNA deletion in a woman with ocular myopathy and in her son with pearson syndrome

Single deletions of mitochondrial DNA (mtDNA) are associated with three major clinical conditions: Kearns-Sayre syndrome, a multisystem disorder; Pearson syndrome (PS), a disorder of the hematopoietic system; and progressive external ophthalmoplegia (PEO), primarily affecting the ocular muscles. Typically, single mtDNA deletions are sporadic events, since the mothers, siblings, and offspring of affected individuals are unaffected. We studied a woman who presented with PEO, ptosis, and weakness of pharyngeal, facial, neck, and limb muscles. She had two unaffected children, but another of her children, an infant son, had sideroblastic anemia, was diagnosed with PS, and died at age 1 year. Morphological analysis of a muscle biopsy sample from the mother showed cytochrome c oxidase-negative ragged-red fibers-a typical pattern in patients with mtDNA deletions. Southern blot analysis using multiple restriction endonucleases and probed with multiple mtDNA fragments showed that both the mother and her infant son harbored an identical 5,355-bp single deletion in mtDNA, without flanking direct repeats. The deletion was the only abnormal species of mtDNA identified in both patients, and there was no evidence for duplications. We conclude that, although the vast majority of single large-scale deletions in mtDNA are sporadic, in rare cases, single deletions can be transmitted through the germline.     

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.     

Paradoxical decrease of mitochondrial DNA deletions in epithelial cells of active ulcerative colitis patients

Ulcerative colitis (UC) is a condition characterized by chronic inflammation targeted at the epithelial layer. In addition to being involved in immune phenomena, UC epithelial cells exhibit decreased oxidation of butyrate, downregulation of oxidative pathway regulatory genes, and overexpression of mitochondrial (mt) genes. We investigated whether these events, which translate an altered energy metabolism, are associated with an abnormal pattern of mtDNA deletions. Highly purified colonocytes were isolated from surgically resected control, involved and uninvolved inflammatory bowel disease mucosa. The frequency, type, and number of mtDNA deletions were assessed by PCR amplification, Southern blot analysis, and cloning and sequencing of amplified DNA fragments. The 4977 mtDNA deletion was less frequent in UC than control and Crohn's disease (CD) epithelium, regardless of patient age. Several other deletions were detected, but all were less common in UC than control and CD cells. The frequency, variety, and number of mtDNA deletions were invariably lower in colonocytes isolated from inflamed mucosa than in autologous cells from noninflamed mucosa. In conclusion, in the absence of inflammation, UC colonocytes exhibit an mtDNA deletion pattern similar to that of control cells, indicating a normal response to physiological levels of oxidative stress. In active inflammation, when oxidative stress increases, the frequency, variety, and number of mtDNA deletions decrease. Because comparable abnormalities are absent in active CD, the mtDNA deletion pattern of active UC suggests that colonocytes respond uniquely to inflammation-associated stress in this condition.     

Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.     

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.     

Selfish Little Circles: Transmission Bias and Evolution of Large Deletion-Bearing Mitochondrial DNA in Caenorhabditis briggsae Nematodes

Selfish DNA poses a significant challenge to genome stability and organismal fitness in diverse eukaryotic lineages. Although selfish mitochondrial DNA (mtDNA) has known associations with cytoplasmic male sterility in numerous gynodioecious plant species and is manifested as petite mutants in experimental yeast lab populations, examples of selfish mtDNA in animals are less common. We analyzed the inheritance and evolution of mitochondrial DNA bearing large heteroplasmic deletions including nad5 gene sequences (nad5Δ mtDNA), in the nematode Caenorhabditis briggsae. The deletion is widespread in C. briggsae natural populations and is associated with deleterious organismal effects. We studied the inheritance patterns of nad5Δ mtDNA using eight sets of C. briggsae mutation-accumulation (MA) lines, each initiated from a different natural strain progenitor and bottlenecked as single hermaphrodites across generations. We observed a consistent and strong drive toward higher levels of deletion-bearing molecules in the heteroplasmic pool of mtDNA after ten generations of bottlenecking. Our results demonstrate a uniform transmission bias whereby nad5Δ mtDNA accumulates to higher levels relative to intact mtDNA in multiple genetically diverse natural strains of C. briggsae. We calculated an average 1% per-generation transmission bias for deletion-bearing mtDNA relative to intact genomes. Our study, coupled with known deleterious phenotypes associated with high deletion levels, shows that nad5Δ mtDNA are selfish genetic elements that have evolved in natural populations of C. briggsae, offering a powerful new system to study selfish mtDNA dynamics in metazoans.     

Mapping of heteroplasmic mitochondrial DNA deletions in Kearns-Sayre syndrome.

Kearns-Sayre syndrome (KSS) is a progressive neuromuscular disease characterised by ophtalmoplegia, cardiac bloc branch, pigmentary retinopathy associated with abnormal mitochondrial function. We have studied the mitochondrial DNA organization of patients presenting KSS and have found large deletions ranging from 3 to 8.5 kilobase pairs. DNA molecules containing deletion are accompanied by the presence of the normal sized mtDNA molecule forming heteroplasmic genomes. The deletions always map in the region which is potentially single stranded during mitochondrial DNA replication. The deletions differ in length and position between individuals but are similar within the different tissues of an individual suggesting that they arise during or before embryogenesis.     

Depletion of mitochondrial DNA in the skeletal muscle of two cirrhotic patients with severe asthenia

Qualitative and quantitative alterations of mitochondrial DNA (mtDNA) in the skeletal muscle from two patients with cirrhosis and severe asthenia have been studied. The 4977 bp (mtDNA(4977)) and the 7436 bp (mtDNA(7436)) mtDNA deletions, as well as other mtDNA deletions, revealed by long extension PCR (LX-PCR), were found in the two patients, whereas the 10,422 bp (mtDNA(10,422)) mtDNA deletion was absent. Altogether, the qualitative alterations of mtDNA in cirrhotic patients with severe asthenia were comparable to those of age-matched healthy individuals. The mtDNA content, on the contrary, was substantially decreased in both patients with respect to control. Such mtDNA depletion might be explained by an increased, disease-related, oxidative damage to mtDNA, which probably affects the replication of the mitochondrial genome as already suggested in other oxidative stress-associated diseases.     

Heteroplasmic mutation of mitochondrial DNA D-loop and 4977-bp deletion in human cancer cells during mitochondrial DNA depletion

Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various human cancers. Many cancers have high frequently of mtDNA with homoplasmic point mutations, and carry less frequently of mtDNA with large-scale deletions as compared with corresponding non-cancerous tissue. Moreover, most cancers harbor a decreased copy number of mtDNA than their corresponding non-cancerous tissue. However, it is unclear whether the process of decreasing in mtDNA content would be involved in an increase in the heteroplasmic level of somatic mtDNA point mutation, and/or involved in a decrease in the proportion of mtDNA with large-scale deletion in cancer cells. In this study, we provided evidence that the heteroplasmic levels of variations in cytidine number in np 303-309 poly C tract of mtDNA in three colon cancer cells were not changed during an ethidium bromide-induced mtDNA depleting process. In the mtDNA depleting process, the proportions of mtDNA with 4977-bp deletion in cybrid cells were not significantly altered. These results suggest that the decreasing process of mtDNA copy number per se may neither contribute to the shift of homoplasmic/heteroplasmic state of point mutation in mtDNA nor to the decrease in proportion of mtDNA with large-scale deletions in cancer cells. Mitochondrial genome instability and reduced mtDNA copy number may independently occur in human cancer.     

Maternally inherited duplication of the mitochondrial genome in a syndrome of proximal tubulopathy, diabetes mellitus, and cerebellar ataxia.

Two sisters in the first year of life presented with a proximal tubulopathy of unknown etiology. They subsequently developed a pluritissular disorder including diabetes mellitus, skin abnormalities, mitochondrial myopathy with ragged-red fibers, and cerebellar ataxia. Their mother had ptosis, ophthalmoplegia, and muscle weakness. Analysis of the mitochondrial respiratory chain showed a complex III deficiency in both skeletal muscle and lymphocytes of the second girl. Southern blot analysis provided evidence for a heteroplasmic partial duplication of the mtDNA (26 kb), involving one full-length and one partly deleted mitochondrial genome and with one single abnormal junction between the genes for ATPase 6 and cytochrome b. Using PCR amplification of lymphocyte DNA, we were able to detect minute amounts of duplicated molecules in the mother, which provided evidence for maternal inheritance of the partial duplication. While maternal transmission of point mutations have been reported in Leber disease, retinitis pigmentosa, and MERRF disease, this observation is, to our knowledge, the first example of a maternally inherited duplication of the mitochondrial genome in man.     

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.