mtDNA Duplications Research Articles

Absence of both MGME1 and POLG EXO abolishes mtDNA whereas absence of either creates unique mtDNA duplications

Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an exonuclease activity (3’→5’) that corrects for the misincorporation of bases. MGME1 serves as an exonuclease (5’→3’), producing ligatable DNA ends. Although both have a critical role in mtDNA replication and elimination of linear fragments, these mechanisms are still not fully understood. Using digital PCR to evaluate and compare mtDNA integrity, we show that Mgme1 knock out (Mgme1 KK) tissue mtDNA is more fragmented than POLG exonuclease deficient “Mutator” (Polg MM) or WT tissue. In addition, next generation sequencing of mutant hearts showed abundant duplications in/nearby the D-loop region and unique 100bp duplications evenly spaced throughout the genome only in Mgme1 KK hearts. However, despite these unique mtDNA features at steady-state, we observed a similar delay in the degradation of mtDNA after an induced double strand DNA break in both Mgme1 KK and Polg MM models. Lastly, we characterized double mutant (Polg MM/Mgme1 KK) cells and show that mtDNA cannot be maintained without at least one of these enzymatic activities. We propose a model for the generation of these genomic abnormalities which suggests a role for MGME1 outside of nascent mtDNA end ligation. Our results highlight the role of MGME1 in and outside of the D-loop region during replication, support the involvement of MGME1 in dsDNA degradation and demonstrate that POLG EXO and MGME1 can partially compensate for each other in maintaining mtDNA.

Differential mitochondrial genome in patients with Rheumatoid Arthritis

Background Mitochondria play an important role in cell survival, function and lineage differentiation. Changes in mitochondrial DNA (mtDNA) may control mitochondrial functions and thus may impart an alternative cellular state thereby leading to a disease condition in the body. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease wherein immune cells become self-reactive causing joint inflammation, swelling and pain in patients. The changes in mtDNA may alter cellular functions thereby directing the immune cells towards an inflammatory phenotype in RA. Therefore, it becomes pertinent to identify changes in mtDNA sequence in immune cells of RA patients to understand the pathogenesis and progression of RA. Methods mtDNA from peripheral blood mono-nuclear cells (PBMCs) of 23 RA patients and 17 healthy controls (HCs) were sequenced using next-generation sequencing (NGS). Further, single nucleotide polymorphisms (SNPs) and other variable changes in mtDNA hypervariable and coding regions, amino acid changes with a putative impact on disease, levels of heteroplasmy, copy number variations and haplogroup analysis in RA patients and HCs were analysed and compared to identify any association of mtDNA changes and RA disease. Results A total of 382 single nucleotide mtDNA variants were observed, 91 (23.82%) were present in hypervariable region and 291 (76.18%) in coding region of patients and HC. The variant 513 GCA > ACA, with G present in HVR-III, known to control the mitochondrial translation function, was significantly present in RA patients. The CYTB gene had larger number of SNPs in HC samples while RNR2 was more variable in RA patients. A non-synonymous heteroplasmy in ND1 gene was found at a single nucleotide position 3533 in an increased number of RA patients as compared to the controls. A significant increase in mtDNA duplication and a higher frequency of the haplogroup U was also characteristic of RA. Also, the presence of SNPs in mitochondrial tRNA genes at two positions 12308 A > G and 15924 A > G were found to be pathogenic. Conclusion We herein observed an altered mtDNA sequence in immune cells of RA patients and thus a possible role of mitochondrial genome in the development of RA. The observed nucleotide changes in mtDNA control region, RNR2 gene, increased heteroplasmy and mtDNA duplication in RA patients may alter sites for transcription factor binding thereby influencing mtDNA gene expression, as well as copy numbers thereby affecting the mitochondrial proteins and their functions. These changes in mtDNA could be one of the probable reasons among many leading to the progression of RA.

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

A new duplication in the mitochondrially encoded tRNA proline gene in a patient with dilated cardiomyopathy

Mitochondria provide an environment conducive to mutations in DNA molecules (mtDNA). Analyses of mtDNA have shown mutations potentially leading to many cardiovascular traits. Here, we describe a patient with dilated cardiomyopathy and new mtDNA duplication. The patient presented symptoms of heart failure New York Heart Association functional class III and was diagnosed with non-familial dilated cardiomyopathy with important left ventricular systolic dysfunction. Sequencing of mtDNA control region was done, and a 15 bp duplication was observed between nucleotides 16,018 and 16,032. Part of this duplication is localized within the tRNA proline gene (tRNAPro) that has an important role in cell protection against oxidative stress and is considered an important regulatory factor for cellular reactive oxygen species balance. This duplication could alter the stability or secondary structure of tRNAPro, affecting mt-protein synthesis. In turn, the presence of duplication in tRNAPro could cause some oxidative stress imbalance and, so, mitochondrial dysfunction could result in the pathogenicity.

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

Mitochondrial DNA instability in malignant melanoma of the skin is mostly restricted to nodular and metastatic stages

Ultraviolet (UV) radiation is thought to be a major contributor to the development of sporadic malignant melanoma of the skin. It may induce alterations in genomic or mitochondrial DNA (mtDNA), especially C to T or CC to TT changes. Mutations or other alterations in mtDNA have been reported in a variety of human cancers and may be due to different mechanisms. In this study, we have attempted to elucidate whether aberrations in the mtDNA of melanoma are due to UV radiation or other factors by investigating two parts of the mitochondrial D-loop and two mitochondrial genes, as well as looking for the delta4977 mtDNA deletion and mtDNA duplications, in 61 primary malignant melanomas and neighbouring normal skin tissue (in 70% of primary tumours; otherwise, corresponding blood samples). Point mutations were a rare feature, occurring in only seven tumour samples and never as a C to T change, whereas mtDNA instability in the D-loop (mtMSI) was found in 13% of primary nodular tumours and 20% of metastases. A de novo delta4977 mtDNA deletion was demonstrated in 10% of melanomas; in 20% of patients, mtDNA duplications and/or the delta4977 mtDNA deletion was detectable. Our data indicate that mtDNA alterations in malignant melanoma are not induced by UV radiation. In addition, point mutations and mtMSI were mostly a feature of nodular and metastatic melanoma samples.

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions.

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination.

Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: duplication and nonrandom loss.

We determined the complete mitochondrial DNA (mtDNA) sequences of the millipedes Narceus annularus and Thyropygus sp. (Arthropoda: Diplopoda) and identified, in both genomes, all 37 genes typical for metazoan mtDNA. The arrangement of these genes is identical in the two millipedes, but differs from others found in arthropod mtDNAs in the location of at least four genes or gene blocks. This novel gene arrangement is unusual for animal mtDNA in that genes with identical transcriptional polarities are clustered in the genome, and the two clusters are separated by two noncoding regions. The only exception to this pattern is the gene for cysteine tRNA, which is located in the part of the genome that otherwise contains all genes with the opposite transcriptional polarity. We suggest that a mechanism involving complete mtDNA duplication followed by the loss of genes, predetermined by their transcriptional polarity and location in the genome, could generate this gene arrangement from the one ancestral for arthropods. The proposed mechanism has important implications for phylogenetic inferences that are drawn on the basis of gene arrangement comparisons.

Mitochondrial DNA and disease

Mitochondrial DNA (mtDNA) defects are a relatively common cause of inherited disease and have been implicated in both ageing and cancer. MtDNA encodes essential subunits of the mitochondrial respiratory chain and defects result in impaired oxidative phosphorylation (OXPHOS). Similar OXPHOS defects have been shown to be present in a number of neurodegenerative conditions, including Parkinson’s disease, as well as in normal ageing human tissues. Additionally, a number of tumours have been shown to contain mtDNA mutations and an altered metabolic phenotype. In this review we outline the unique characteristics of mitochondrial genetics before detailing important pathological features of mtDNA diseases, focusing on adult neurological disease as well as the role of mtDNA mutations in neurodegenerative diseases, ageing and cancer. Copyright  2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mitochondrial disorders.

Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.

Functional and Structural Features of a Tandem Duplication of the Human mtDNA Promoter Region

An approximately 260-bp tandem duplication of the human mtDNA regulatory region has been identified in patients with mitochondrial disorders and in a specific Caucasian haplogroup. The functional significance of this mtDNA duplication was difficult to assess, because it was present at very low levels in human tissues. We have isolated several transmitochondrial cybrid lines harboring this mutation, one of which (clone CA17.1) was essentially homoplasmic for the duplication. Oxidative-phosphorylation function was not impaired in clone CA17.1, suggesting that this mtDNA alteration is not pathogenic. mtDNA copy number and steady-state levels of heavy- and light-strand transcripts were unaltered in clone CA 17.1. The steady-state levels of RNAs made from the two promoters (either from the heavy-strand or from the light-strand) were also similar, indicating that oppositely oriented promoters did not interfere with each other.

Tandem duplications and large-scale deletions of mitochondrial DNA are early molecular events of human aging process.

Large-scale deletions and tandem duplications of mtDNA, which were originally identified in the patients with KSS or CPEO, have recently been found, although with lower abundance, in various tissues of aged individuals. By use of PCR techniques with back-to-back primers, we demonstrated for the first time that small tandem duplications occur in the D-loop of mtDNA in an age-dependent manner in human tissues. A total of 10 types of such tandem duplications were identified and confirmed by primer-shift PCR and DNA sequencing. Based on the sequence characteristics of the junction sites, we classified these small tandem duplications into 3 groups. Most of the tandem duplications were found to occur at hot spots containing poly C runs, and the number of C residues exhibited wide variations in type I, II, V, VI, VII, and VIII duplications. These observations suggest that these tandem duplications may be generated through similar recombination mechanisms. Among them, type I, II, III, IV and IX duplications were found to occur more frequently and abundantly in aging human tissues. They were not detectable in muscle, testis or skin tissues of young subjects or blood cells from subjects of any age. On the other hand, we also analyzed the samples for the aging-associated 4,977-bp and 7,436-bp mtDNA deletions. The results showed that these two deletions and some of the small tandem duplications could occur alone or in different combinations in human tissues in the aging process. From our data, no clear association between tandem duplications and large-scale deletions of mtDNA could be established. However, one common and important observation is that the incidence and abundance of some of the tandem duplications as well as the large-scale deletions were increased in an age-dependent manner. On the basis of these findings and data reported from this and other laboratories, we propose that tandem duplications and large-scale deletions of mtDNA are early molecular events of the human aging process.

Insertions and duplications of mtDNA in the nuclear genomes of Old World monkeys and hominoids.

Using oligonucleotide primers designed to match conserved regions of mammalian mitochondrial DNA (mtDNA), we have amplified and sequenced two divergent cytochrome b nuclear pseudogenes from orangutan cellular DNA. Evolutionary analysis suggests that a nuclear transfer occurred about 30 million years ago on the lineage leading to the catarrhines (Old World monkeys and hominoids), and involved a long (at least 3 kilobases), probably damaged, piece of mtDNA. After this transfer, the pseudogene duplicated, giving rise to the two copies that are probably present in all hominoids, including humans. More recent transfers involving the entire cytochrome b gene have also occurred in the Old World monkeys. Such nuclear copies of mtDNA can confound phylogenetic and population genetic studies, and be an insidious source of DNA contamination of 'ancient' and forensic DNA. Indeed, contamination with these anciently transferred human pseudogenes is almost certainly the source of the cytochrome b sequences recently reported from 'dinosaur bone DNA'.

Photoageing-associated mitochondrial DNA length mutations in human skin.

It has recently been suggested that mitochondrial DNA (mtDNA) mutations are important contributors to human ageing and degenerative diseases. Using PCR techniques, we demonstrated three types of mtDNA length mutations, a 4977 bp deletion, a 7436 bp deletion and tandem duplications, in normal human skin tissues. We found that these mutations started to appear in the third decade of life, and the age at which the mutations could be detected in sun-exposed skin was usually younger than in non-exposed skin. Moreover, the incidences of these deletions and tandem duplications of mtDNA in sun-exposed skin were all significantly higher than those in non-exposed skin (P < 0.05). The 4977 bp deletion was the most prevalant mtDNA mutation in human skin, and the 7436 bp deletion was the least frequent among the three types of mtDNA mutations examined. We first demonstrated the existence of tandem duplications with sizes of about 260 bp, 200 bp and 150 bp in the D-loop region of mtDNA in the skin of elderly individuals. Among the three tandem duplications, the 200-bp duplication was found to occur most frequently in ageing skin. The tandem duplications were found to coexist with either or both of the deletions in some elderly individuals. The frequency of occurrence of mtDNA deletions and tandem duplications in skin was found to increase in an age-dependent manner. However, the incidence of tandem duplications was not well correlated with the age of the subject.(ABSTRACT TRUNCATED AT 250 WORDS)

Duplications of mitochondrial DNA in Kearns-Sayre syndrome.

mtDNA duplications were detectable in 10 of 10 patients with mtDNA deletions and Kearns-Sayre syndrome (KSS) and in none of 8 patients with chronic progressive external ophthalmoplegia (CPEO). Thus, duplications of mtDNA seem to be a distinctive feature of KSS, including patients where Pearson's syndrome is the first manifestation. Diabetes mellitus was identified in 4 of 7 patients with high or moderate levels of mtDNA duplications. The balance of mtDNA rearrangements may be central to the pathogenesis of this unique group of disorders.

Parallel origins of duplications and the formation of pseudogenes in mitochondrial DNA from parthenogenetic lizards (Heteronotia binoei; Gekkonidae)

Analysis of mitochondrial DNAs (mtDNAs) from parthenogenetic lizards of the Heteronotia binoei complex with restriction enzymes revealed an approximately 5-kb addition present in all 77 individuals. Cleavage site mapping suggested the presence of a direct tandem duplication spanning the 16S and 12S rRNA genes, the control region and most, if not all, of the gene for the subunit 1 of NADH dehydrogenase (ND1). The location of the duplication was confirmed by Southern hybridization. A restriction enzyme survey provided evidence for modifications to each copy of the duplicated sequence, including four large deletions. Each gene affected by a deletion was complemented by an intact version in the other copy of the sequence, although for one gene the functional copy was heteroplasmic for another deletion. Sequencing of a fragment from one copy of the duplication which encompassed the tRNA(leu)(UUR) and parts of the 16S rRNA and ND1 genes, revealed mutations expected to disrupt function. Thus, evolution subsequent to the duplication event has resulted in mitochondrial pseudogenes. The presence of duplications in all of these parthenogens, but not among representatives of their maternal sexual ancestors, suggests that the duplications arose in the parthenogenetic form. This provides the second instance in H. binoei of mtDNA duplication associated with the transition from sexual to parthenogenetic reproduction. The increased incidence of duplications in parthenogenetic lizards may be caused by errors in mtDNA replication due to either polyploidy or hybridity of their nuclear genomes.