Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.

Hereditary inclusion-body myopathies.

The term hereditary inclusion-body myopathies (HIBMs) defines a group of rare muscle disorders with autosomal recessive or dominant inheritance and presence of muscle fibers with rimmed vacuoles and collection of cytoplasmic or nuclear 15-21 nm diameter tubulofilaments as revealed by muscle biopsy. The most common form of HIBM is due to mutations of the GNE gene that codes for a rate-limiting enzyme in the sialic acid biosynthetic pathway. This results in abnormal sialylation of glycoproteins that possibly leads to muscle fiber degeneration. Mutations of the valosin containing protein are instead responsible for hereditary inclusion-body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), with these three phenotypic features having a variable penetrance. IBMPFD probably represents a disorder of abnormal cellular trafficking of proteins and maturation of the autophagosome. HIBM with congenital joint contractures and external ophthalmoplegia is due to mutations of the Myosin Heavy Chain IIa gene that exerts a pathogenic effect through interference with filament assembly or functional defects in ATPase activity. This review illustrates the clinical and pathologic characteristics of HIBMs and the main clues available to date concerning the possible pathogenic mechanisms and therapeutic perspectives of these disorders. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects.

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.

Mitochondrial DNA analysis in ocular myopathy. Observations in 29 Portuguese patients.

We analyzed 29 patients with progressive external ophthalmoparesis (PEO) either alone or as part of a multisystem disorder. Ragged-red fibers were very abundant (10-20%) in 15 patients, and many of them were also cytochrome c oxidase-negative. Biochemical analysis of the respiratory chain showed partial defects of single or multiple complexes in 18 patients (64%). Eleven PEO patients (38%) harbored single large-scale mtDNA deletions in muscle, which averaged 5.4 kb in size and 47% in relative abundance. One PEO patient harbored the A3243G mutation (MELAS mutation) in muscle (63%). Our findings, the first reported in Portuguese patients, confirm that single large-scale mtDNA deletions are a significant cause of PEO. Although ophthalmoparesis was the main clinical feature in the majority of patients, the clinical spectrum is broad, ranging from severe encephalopathy of childhood to a milder, though disabling, muscle weakness in adults.

A novel mitochondrial tRNA isoleucine gene mutation causing chronic progressive external ophthalmoplegia.

We report a sporadic case of chronic progressive external ophthalmoplegia that developed during childhood and was associated with ragged-red and cytochrome c oxidase (COX)-negative fibers in skeletal muscle. Sequencing of all the mitochondrial transfer RNA (tRNA) genes identified a single potentially pathogenic mutation--a T to C transition at position 4274 in the tRNA(Ile) gene. This mutation was not present in skeletal muscle from 79 controls, and the level of the mutation in COX-negative fibers was significantly greater than the level in COX-positive fibers.

Efficient and specific amplification of identified partial duplications of human mitochondrial DNA by long PCR.

The use of PCR to identify mtDNAs containing a partial duplication (dup-mtDNA) in the presence of a heteroplasmic population of mtDNAs harboring the corresponding deletion (delta-mtDNA) leads to ambiguous results: when the primers anneal in the duplicated portion of the dup-mtDNA (which is also the non-deleted region of the delta-mtDNA) and point towards the abnormal breakpoint junction, both templates are amplified indiscriminately. We have developed two different 'long PCR' approaches to amplify dup-mtDNA even in the presence of delta-mtDNA and wild-type mtDNA (wt-mtDNA). Long PCR with two primers annealing in the non-duplicated region in dup-mtDNA (equivalent to the region missing in delta-mtDNA) and whose 3' ends pointed towards the duplicated area amplified both dup-mtDNA and coexisting wt-mtDNA. We observed, however, a preferential amplification of the wt-mtDNA over that of the longer dup-mtDNAs. This problem was partly overcome by modifying the PCR conditions (extension time, amplicon length, amount of template). In order to overcome the problem of co-amplification, we developed a novel PCR method to amplify specifically dup-mtDNAs. A forward primer annealing across the breakpoint junction was used in conjunction with a backward primer annealing in the non-duplicated region. For those duplication breakpoints flanked by direct repeats, we designed a 'breakpoint loop-out' primer whose sequence omitted the repeated region, in order to avoid the annealing of this primer to wt-mtDNA. This second approach was able to amplify specifically and efficiently the dup-mtDNA in all samples analyzed, irrespective of the size of the duplication or its proportion in the samples.

Cytochrome c oxidase activity in single muscle fibers: assay techniques and diagnostic applications.

Microphotometric enzyme assay was used to study cytochrome c oxidase activity in single human skeletal muscle fibers. The assay techniques combine the precise localization of enzyme activity provided by histochemical methodology with the precise quantitation of a sensitive assay system. Abnormalities of cytochrome c oxidase were investigated using microphotometric enzyme assay in 12 patients with Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia, or Leigh's syndrome. Control values were obtained using muscle biopsy specimens from 20 juvenile and 18 adult subjects with no evidence of neuromuscular disease. In the patients with Leigh's syndrome due to cytochrome c oxidase deficiency, the abnormality was found to be expressed uniformly throughout the muscle fiber population. In contrast, patients with Kearns-Sayre syndrome or chronic progressive external ophthalmoplegia showed abnormal heterogeneity of cytochrome c oxidase activity. In many cases, extreme degrees of variability were seen, with fibers containing high activity adjacent to fibers with no detectable activity. Mitochondrial DNA analysis showed that most of the patients with Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia had major rearrangements of mitochondrial DNA. It was concluded that the extreme variability of cytochrome c oxidase activity detected using microphotometric enzyme assay was an indicator of a probable abnormality of mitochondrial DNA. Conversely, cytochrome c oxidase defects in muscle which show a homogeneous distribution are more likely to be associated with defects of the nuclear genome.

Chronic progressive external ophthalmoplegia with NADH-CoQ reductase deficiency: report of a case.

A thirty-two year old female had chronic progressive external ophthalmoplegia (CPEO), exertional fatigue, dysarthria, dysphagia, and bilateral hearing impairment. Histochemical stains, obtained from the right vastus lateralis, showed ragged-red fibers and wide-spread abnormalities in the number, size, and the structure of mitochondria under electronomicroscopic examination. A biochemical analysis showed a low activity of NADH-cytochrome C reductase, NADH dehydrogenase and a normal activity of succinate cytochrome C reductase and cytochrome C oxidase. This data suggests a specific defect in the NADH dehydrogenase of complex I (NADH CoQ reductase). We believe that this is the first biochemically defined mitochondrial myopathy reported in Taiwan and provides additional evidence for the existence of biochemical heterogeneity in mitochondrial disorders of CPEO.

Mitochondrial DNA deletions and cytochrome c oxidase deficiency in muscle fibres.

We have studied cytochrome c oxidase (COX) deficient muscle fibre segments in 6 patients with mitochondrial myopathy and deletions of mitochondrial DNA (mtDNA). The distribution of transcripts of normal and mutated mtDNA in skeletal muscle sections was studied by in situ hybridization. The results were compared with the enzyme histochemical activity of COX and the immunohistochemical distribution of mtDNA encoded and nuclear DNA encoded subunits of COX. In all cases a proportion of the muscle fibres (less than 1-30% of the fibres in cross-sections) had low COX activity and high activity of succinate dehydrogenase (COX deficient muscle fibres). Transcripts of normal and deleted mtDNA showed the same distribution within the tissue as the corresponding mtDNA, indicating that the deleted mtDNA is transcribed. The COX deficient muscle fibres showed accumulation of transcripts of deleted mtDNA, which had a similar distribution as the accumulated mitochondria within these fibres. With few exceptions, there was a low level of transcripts of normal mtDNA in these COX deficient fibres. Immunohistochemical analysis revealed low levels of immunoreactive material using antiserum to the mtDNA encoded subunits II/III as well as the nuclear DNA encoded subunit IV of COX in all COX deficient muscle fibres. The fraction of deleted mtDNA in muscle ranged from 43 to 87%. There was no correlation between the proportion of COX deficient muscle fibres and the fraction of deleted mtDNA. In 2 cases the deletion did not involve any COX gene. One of these cases had 87% deleted mtDNA but less than 1% COX deficient muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Segmental cytochrome c-oxidase deficiency in CPEO: teased muscle fiber analysis.

In an attempt to elucidate the pathogenesis of focal cytochrome c-oxidase (COX) deficiency in skeletal muscle from patients with chronic progressive external ophthalmoplegia (CPEO), we examined the longitudinal distribution of COX activity in single muscle fibers from 6 CPEO patients with muscle mitochondrial DNA (mtDNA) deletions. A new method for teasing single muscle fibers, recently developed in our laboratory, revealed fibers with COX-positive and -negative segments in all 6 patients. The borders between the enzyme-positive and -negative segments in these fibers were sharply delineated, so that the length of each COX-negative segments could be accurately measured. The proportion of the sum of the lengths of the enzyme-negative segments to the total length of the muscle fibers correlated well with the proportion of deleted mtDNA, suggesting that abnormal mitochondria harboring mutant mtDNA may be responsible for the focal loss of COX activity.

Mitochondrial creatine kinase containing crystals, creatine content and mitochondrial creatine kinase activity in chronic progressive external ophthalmoplegia.

Mitochondrial crystals containing mitochondrial creatine kinase (Mi-CK) protein were described recently. From in vitro studies it has been suggested that alterations in creatine concentration are connected to the occurrence of these crystals. In the present study free, phosphorylated and total creatine concentrations as well as Mi-CK activity were determined in muscle samples of six patients with chronic progressive external ophthalmoplegia (CPEO). Two of them showed Mi-CK containing mitochondrial crystals. The activity of Mi-CK was found to be clearly enhanced in those muscle samples in which mitochondrial crystals were present. No relationship was found between the concentration of total, free or phosphorylated creatine and the occurrence of mitochondrial crystals. An up to now unknown mechanism seems to cause the formation of Mi-CK containing crystals in human muscle mitochondria.

An adult onset hexosaminidase A deficiency syndrome with sensory neuropathy and internuclear ophthalmoplegia.

A 42 year old man presented with a slowly progressive gait disturbance, generalised weakness, dysarthria, clumsiness and tremor of his hands, and involuntary jerks. Hexosaminidase A activity in plasma, leucocytes and fibroblasts was considerably reduced, establishing the diagnosis of GM2 gangliosidosis. Clinical examination showed two previously unreported features, a clinically evident sensory neuropathy and internuclear ophthalmoplegia.

[Expression of a defect in the respiratory chain in cultured human cells]. / Espressione dei deficit della catena respiratoria nelle colture cellulari umane.

Large scale deletions of mitochondrial DNA (mtDNA) or altered inter-genomic regulation in skeletal muscle have been demonstrated in patients with mitochondrial encephalomyopathies due to Cytochrome C oxidase (COx) deficiency. We have analyzed by Southern blotting and Polymerase Chain Reaction (PCR) the mtDNA in primary muscle cultures (myoblast-myotube stages and at clonal densities) and in fibrogenic subclones obtained from 9 patients with partial COx deficiency who had in their muscle biopsy a subpopulation of mtDNA showing deletions of variable size (between 2.1 and 6.5 Kb). Only in the cultures from one patient, southern analysis revealed in myoblasts and myotubes a mtDNA almost identical to that found in the original muscle biopsy and persistence of deletion in muscle cells grown at clonal densities. The deletion was detectable in fibrogenic lineage only by PCR amplification. The deleted mtDNA molecules were detectable in myogenic or fibrogenic cultures from other patients only by PCR amplification. The different amounts of deleted mtDNA in the various tissues could be due either to an unequal distribution of the altered mtDNA during embryogenesis with amplification of deleted molecules in myogenic lineage or could result from negative selection against the altered mtDNA in rapidly proliferating cells, such as fibroblasts.

Histological, enzymatic and mitochondrial DNA studies in patients with Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia.

Kearns-Sayre syndrome has been associated with large heteroplasmic mitochondrial DNA deletions and morphological alterations at the cytological level. We have measured the activities of different respiratory chain complexes in 3 patients presenting mitochondrial DNA deletions and found no close correlation between gene deletions and enzymatic activities. These data, therefore, point out the importance of analyses at the mitochondrial DNA level in such mitochondrial disorders because gross biochemistry may miss any defect.

Chronic progressive external ophthalmoplegia in patients with large heteroplasmic mitochondrial DNA deletions: an immunocytochemical study.

Immunocytochemical studies with a holocomplex antibody battery in patients with chronic progressive external ophthalmoplegia, with and without large mitochondrial DNA deletions, revealed positive (and often increased) immunoreactivity for all complexes studied in histochemically cytochrome oxidase (COX)-negative areas, suggesting a compensatory up-regulation of these components. Similar findings were observed with subunit-specific probes directed against both nuclear- and mitochondrially encoded gene products. Comparison of staining intensities between the different complexes revealed significantly more variability in COX-negative than COX-positive fibres, suggesting disordered stoichiometric control during up-regulation. These differences were confirmed using statistical models. This data challenges the view that COX-negative fibre segments have little or no mitochondrially coded protein translation.

Antimitochondrial autoantibodies of primary biliary cirrhosis as a novel probe in the study of 2-oxo acid dehydrogenases in patients with mitochondrial myopathies.

Autoantibodies present in the autoimmune disease primary biliary cirrhosis react by immunoblotting with four human skeletal muscle mitochondrial antigens of 70 kDa, 52 kDa, 50 kDa and 45 kDa, identified as the lipoate acetyl transferases (E2) of the pyruvate dehydrogenase, component X of E2 pyruvate dehydrogenase, E2 of 2-oxo glutarate dehydrogenase and E2 of branched-chain 2-oxo acid dehydrogenase complexes respectively. These autoantibodies have been employed as a novel probe to study whether there is a defect in the synthesis of the 2-oxo acid dehydrogenase complexes in patients with mitochondrial respiratory chain disorders. The reactive antigens are present normally in four patients with oculomyopathy in whom partial deletions of the mtDNA have been detected, and in two patients with MERRF and MELAS encephalomyopathy. Thus, unlike in the yeast Saccharomyces cerevisiae, there appear to be no regulatory interactions which coordinate the assembly of the mitochondrial respiratory chain with the development of the pyruvate dehydrogenase complex, which plays an important role in regulating the flow of metabolic intermediates to oxidative energy metabolism.

[Mitochondrial encephalomyopathy]. / Le encefalomiopatie mitocondriali.

Modern concepts regarding mitochondrial encephalomyopathies (ME) are summarized. Utilizing recent techniques of molecular biology we studied some cases of ME referred to the Institute of Clinical Neurology of Milan University. With these techniques we demonstrated different mitochondrial DNA deletions either in patients' muscle or in culture.