Analysis of multiple mitochondrial DNA deletions in inclusion body myositis

Inclusion body myositis (IBM) is a sporadic progressive myopathy, which is morphologically characterized by inflammatory cell infiltrates and rimmed vacuoles in muscle fibers. Mitochondrial changes are regularly present with ragged-red fibers showing deficiency of cytochrome c oxidase. In these muscle fiber segments, there is accumulation of mitochondria with mitochondrial DNA (mtDNA) deletions. There are different deletions in different muscle fibers. In this study, we have sequenced for the first time the multiple mtDNA deletions in muscle from four patients with IBM. The deletion breakpoints were sequenced from cloned polymerase chain reaction (PCR)-amplified mtDNA fragments. The sequencing was performed directly from the bacterial colonies used for cloning. Of 122 analyzed clones, 33 different deletions were identified. The majority of these have not previously been described. There was a marked predominance of deletion breakpoints in certain regions of mtDNA. These predominant breakpoint regions are similar to those described in other conditions with multiple deletions, such as autosomal dominant progressive external ophthalmoplegia (adPEO) and normal aging, but different from those described in diseases due to single deletions such as Kearns-Sayre syndrome and sporadic PEO. These findings indicate that common factors are involved in the development of multiple mtDNA deletions in IBM, adPEO, and aging. Hum Mutat 10:381–386, 1997. © 1997 Wiley-Liss, Inc.     

ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder associated with depletion, multiple deletions and site-specific point mutations of mitochondrial DNA (mtDNA). MNGIE is caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP; endothelial cell growth factor 1). Deficiency of TP leads to dramatically elevated levels of circulating thymidine and deoxyuridine. The alterations of pyrimidine nucleoside metabolism are hypothesized to cause imbalances of mitochondrial nucleotide pools that, in turn, may cause somatic alterations of mtDNA. We have now identified five major forms of mtDNA deletions in the skeletal muscle of MNGIE patients. While direct repeats and imperfectly homologous sequences appear to mediate the formation of mtDNA deletions, the nicotinamide adenine dinucleotide dehydrogenase 5 gene is a hot-spot for these rearrangements. A novel aspect of the mtDNA deletions in MNGIE is the presence of microdeletions at the imperfectly homologous breakpoints.     

Importance of searching for associated mitochondrial DNA alterations in patients with multiple deletions

Multiple mitochondrial DNA (mtDNA) deletions have been reported in patients with autosomal dominant and recessive disorders. We studied several affected and one non-affected individuals belonging to a pedigree in which the inheritance of the pathological trait was compatible with an autosomique dominant transmission. Affected members had late-onset multisystem disorders with multiple mtDNA deletions in skeletal muscle. But this family presented a striking difference from previously described cases, because none of the patients had progressive external ophthalmoplegia (PEO). We also studied one young boy with a no contributary family history. He had a cerebellar ataxia with PEO and multiple mtDNA deletions in muscle. Molecular analysis revealed that in the first family, repeated sequences were present at the breakpoint junctions, whereas such motifs were not found in the young patient's case. In the first family, we evidenced mtDNA point mutations in clones containing breakpoint junctions and a 9-bp motif triplication in the intergenic COII/tRNA(Lys) region, whereas this sequence is repeated twice in the wild type mtDNA. Our results suggest that multiple deletions observed in the two pedigrees result from different molecular mechanisms and point out the role of repeated sequences in the first pedigree. No mtDNA repair system has been described in mammals so far, but the molecular abnormalities found in the first family suggest that a defect in an mtDNA repair system, homologous to the E. coli MutHLS pathway, could be responsible for such a phenotype.     

Autosomal dominant psychiatric disorders and mitochondrial DNA multiple deletions: report of a family

BACKGROUND: Psychiatric problems, including bipolar affective disorder (BD) and schizophrenia, are common in mitochondrial diseases (MD) and frequently precede the diagnosis of mitochondrial dysfunction. However, they are rarely the only persistent manifestation of a MD and they are usually associated with other neurological or non-neurological features. CASE REPORT: Here, we describe an Italian family with multiple deletions of mtDNA in muscle, in which BD, schizophrenia, and depression recurred over several generations in the absence of other major signs of mitochondrial dysfunction. CONCLUSION: In patients with positive family history of psychiatric problems, the possibility of MD should be kept in mind, even in absence of other canonical features of mitochondrial encephalomyopathies.     

Aging-associated deletions of human diaphragmatic mitochondrial DNA.

It is known that respiratory function deteriorates with age. Endogenous damage to DNA is thought to contribute to the aging process. The mitochondrial oxidative phosphorylation system, a bio-engine, consists of five complexes, and 13 subunits of those complexes are biosynthesized from information encoded in mitochondrial DNA. Mitochondrial DNA is shown to have a much higher mutation rate than nuclear DNA. We examined the diaphragms obtained at autopsy from 34 humans, 23 men and 11 women, ranging in age from 25 to 85 yr, for mitochondrial DNA deletions using the polymerase chain reaction method. Multiple mitochondrial DNA deletions were detected particularly among the elderly; the number of deletions in those over age 70 was significantly higher than in those under age 40. The occurrence of a 3.4-kbp deletion of mitochondrial DNA increased with age, i.e., 0% of those under age 30, 20.0% of those in their forties, 25.0% of those in their fifties, 28.6% of those in their sixties, 72.7% of those in their seventies, and in all of those over age 80. The mutation was based on the directly repeated sequence, 5'-TCACCCC-3', which exists in both the CO3 gene and the ND5 gene. Replication impairment occurred at that directly repeated sequence, which caused the elimination of a genome between the CO3 gene and the ND5 gene, and information for biosynthesis of four subunits in complex I (ND3, ND4L, ND4, and ND5), one in complex IV (CO3), and five transfer RNA genes was missing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.     

Method for in situ investigation of mitochondrial DNA deletions

A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells. Hum Mutat 10:489–495, 1997. © 1997 Wiley-Liss, Inc.     

Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma

Mitochondrial DNA (mtDNA) large deletions and mtDNA mutations have been demonstrated in various types of human cancer. The relationship between the occurrence of such alterations and the nuclear microsatellite instability (MSI) status of the neoplastic cells remains controversial. In an attempt to clarify the situation in gastric carcinoma, we studied, by PCR/SSCP and sequencing, five mitochondrial genes and two D-loop regions in 32 gastric carcinomas that had been previously screened for MSI and mitochondrial common deletion. MtDNA alterations were detected in 26 carcinomas (81%). All the mtDNA mutations, which occurred mainly in the D-loop and ND1 and ND5 genes, were transitions. D-loop alterations (insertions and/or deletions) were not significantly associated with mutations in the coding regions. There was a trend towards an inverse relationship between the occurrence of mitochondrial common deletion and mtDNA mutations. No significant relationship was observed between MSI status and mtDNA mutations, whereas the mitochondrial common deletion appeared to be almost exclusively restricted to MSI-negative tumors. The latter finding--almost no gastric carcinoma with MSI-positive phenotype has large deletions of mtDNA--needs to be confirmed in a larger series and in tumors from other organs.     

Age related somatic mitochondrial DNA deletions in bone.

BACKGROUND: It has been suggested that the accumulation of damage to mitochondrial DNA is a major cause of age related, degenerative disease. Aging is known to cause bone loss leading to a fall in bone mineral density and disruption of bone microarchitecture. However, despite the evidence of age related bone loss, no attempt has been made to detect specific deletions of mitochondrial DNA in the bone of aged individuals. AIMS: To detect bone specific, age related deletions in mitochondrial DNA. METHOD: Blood leucocytes and bone biopsies from patients who had undergone orthopaedic surgery were used as a source of mitochondrial DNA and screened for deletions using the polymerase chain reaction. RESULTS: Although no deletions were detected in the blood mitochondrial DNA, specific deletions in bone mitochondrial DNA were found in three of five elderly subjects. CONCLUSION: The findings of this study suggest that there could be a link between mitochondrial DNA deletions and free radical induced apoptosis of bone cells in the development of age related bone loss.     

Gastrointestinal dysmotility in mitochondrial neurogastrointestinal encephalomyopathy is caused by mitochondrial DNA depletion

Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall.     

POLG mutations in sporadic mitochondrial disorders with multiple mtDNA deletions

The accumulation of multiple mitochondrial DNA (mtDNA) deletions in stable tissues is a distinctive feature of several autosomal disorders, characterized by Progressive External Ophthalmoplegia (PEO), ptosis, and proximal myopathy. At least three nuclear genes are responsible for these disorders: ANT1 and C10orf2 cause autosomal dominant PEO, while mutations of DNA polymerase gammaA (POLG1 or POLG) gene on chromosome 15q25 causes both autosomal dominant and recessive forms of PEO. To investigate the contribution of these genes to the sporadic cases of PEO with multiple mtDNA deletions, we studied 31 mitochondrial myopathy patients without any family history for the disorder: 23 had PEO with myopathy, with or without the additional features of pigmentary retinopathy, ataxia, neurosensorial hypoacusia and diabetes mellitus, 7 presented isolated myopathy and one a peripheral neuropathy with ptosis. In all patients Southern blot of muscle DNA showed multiple mtDNA deletions; screening for ANT1 and C10ORF2 genes was negative. POLG analysis revealed mutations in eight patients; in six of them the mutations were allelic, while two patients were heterozygous. Five mutations were new, namely one stop codon (c.2407C>T/p.R709X) and four missense mutations (c.1085G>C/p.G268A; c.1967G>A/p.R562Q; c.2702G>C/p.R807P; c.3076C>T/p.H932W). A high degree of conservation was observed for all the new missense mutations. Only patients presenting PEO as part of their clinical phenotype had POLG mutations, in seven of them together with myopathic signs and in one with a sensori-motor peripheral neuropathy.     

Men with oligoasthenoteratozoospermia harbour higher numbers of multiple mitochondrial DNA deletions in their spermatozoa, but individual deletions are not indicative of overall aetiology

It is believed that one cause of sperm dysfunction might arise through multiple mitochondrial DNA deletions (Delta mtDNA) resulting in the formation of an incomplete electron transport chain. This study investigates the incidence of multiple Delta mtDNA in human spermatozoa prepared on Percoll gradients. Firstly, we investigated for the presence of two frequently analysed Delta mtDNA, the 4977 and 7.4 kb deletions, using conventional polymerase chain reaction (PCR). These two deletions are characteristically flanked by direct repeats. We further analysed the incidence of one other deletion, the 15 bp deletion in the cytochrome c oxidase subunit III (COX III) of complex IV to determine whether other deletions flanked by direct repeats could be equally predictive. The incidence of these three deletions was not clearly associated with the diagnostic categorization of male infertility. However, the use of long PCR showed that samples harbouring high numbers of Delta mtDNA were associated with the diagnostic categorization of male infertility. We propose that these deletions could arise through a free radical-driven event occurring at the spermatogonial cell stage resulting in the replication of Delta mtDNA molecules at the expense of wild-type molecules. These anomalies in ejaculated sperm mtDNA could account for reproductive failure in some men.     

The generation of mitochondrial DNA large-scale deletions in human cells

Large-scale deletions of human mitochondrial DNA (mtDNA) are a common cause of mitochondrial diseases. In order to prevent and treat these mitochondrial diseases, it is important and necessary to understand the mechanisms behind the generation of these deletions. Generally, there exist three kinds of large-scale deletions: deletions almost occur within two direct repeats with identical sequences (class I deletions), deletions are flanked by imperfect repeats (class II deletions) and by no direct repeats (class III deletions). Two major hypotheses are suggested to generate these deletions: replication for class I/II deletions through slipped mispairing between two repeats, and repair mainly for class II/III deletions mediated by mtDNA double-strand breaks. It seems possible that these two mechanisms work together as a powerful and complementary system to compensate for their defects in the generation of all these deletions, not respectively.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay

ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.     

Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements

A patient who wished to be treated for infertility by intracytoplasmic sperm injection (ICSI) was referred to our group for assessment. Upon clinical examination, a ptosis (partial closure of the eyelid) was noted, and histology revealed ragged red fibres in the skeletal muscle. Southern blot analysis of spermatozoa and skeletal muscle revealed the presence of multiple mitochondrial DNA deletions. This kind of rearrangement may be of nuclear origin since three nuclear loci have been ascribed to multiple mitochondrial DNA deletions in humans. Since mitochondrial DNA is maternally transmitted, the use of ICSI was feasible. However, an alteration of nuclear gene product affecting the integrity of mitochondrial DNA, and thus sperm mobility, might be transmitted to the offspring with the risk of developing a mitochondrial DNA disease.      

Detection of mitochondrial DNA deletions in blood using the polymerase chain reaction: non-invasive diagnosis of mitochondrial myopathy

Southern hybridisation demonstrates deleted mitochondrial DNAs (mtDNAs) in muscle but not in blood in a subgroup of patients with mitochondrial myopathy. The polymerase chain reaction (PCR) was used to search for low levels of rearranged mitochondrial DNAs in blood in 24 patients with mitochondrial myopathy, and 15 asymptomatic relatives, all of whom have no detectable abnormality on restriction enzyme analysis of blood mitochondrial DNA. In eight patients and two of their relatives, PCR products were obtained consistent with deletions of mitochondrial DNA. The presence or absence of a deletion was correctly predicted in 10 out of 11 patients from whom information was available from muscle DNA. No false positives were obtained in 43 controls. PCR analysis of blood may be applicable as a non-invasive screening test of affected individuals and in carrier detection.     

Genotype and phenotype analyses in 136 patients with single large-scale mitochondrial DNA deletions

We examined 136 patients with mitochondrial DNA (mtDNA) deletion. Clinical diagnoses included chronic progressive external ophthalmoplegia (94 patients); Kearns-Sayre syndrome (KSS; 33 patients); Pearson's marrow-pancreas syndrome (six patients); and Leigh syndrome, Reye-like syndrome, and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (one patient). The length and location of deletion were highly variable. Only one patient had deletion within the so-called shorter arc between the two origins of mtDNA replication. The length of deletion and the number of deleted transfer ribonucleic acid (tRNAs) showed a significant relationship with age at onset. Furthermore, KSS patients had longer and larger numbers of deleted tRNAs, which could be risk factors for the systemic involvement of single mtDNA deletion diseases. We found 81 patterns of deletion. Direct repeats of 4 bp or longer flanking the breakpoints were found in 96 patients (70.5%) and those of 10 bp or longer in 49 patients (36.0%). We found two other common deletions besides the most common deletion (34 patients: 25.0%): the 2,310-bp deletion from nt 12113 to nt 14421 (11 patients: 8.0%) and the 7,664-bp deletion from nt 6330 to nt 13993 (ten patients: 7.3%). These deletions had incomplete direct repeats longer than 13 bp with one base mismatch.     

Solitary neurocysticercosis case caused by Asian genotype of Taenia solium confirmed by mitochondrial DNA analysis

A Japanese woman presenting with neurologic symptoms was presumptively diagnosed with neurocysticercosis based on imaging findings. Hooklets in the scolex of the resected lesion were not confirmed through histopathological observation. However, the illness was confirmed by mitochondrial DNA analysis to be a solitary neurocysticercosis case caused by the Asian genotype of Taenia solium.