Late-onset thymidine kinase 2 deficiency: a review of 18 cases

BACKGROUND: TK2 gene encodes for mitochondrial thymidine kinase, which phosphorylates the pyrimidine nucleosides thymidine and deoxycytidine. Recessive mutations in the TK2 gene are responsible for the 'myopathic form' of the mitochondrial depletion/multiple deletions syndrome, with a wide spectrum of severity. METHODS: We describe 18 patients with mitochondrial myopathy due to mutations in the TK2 gene with absence of clinical symptoms until the age of 12. RESULTS: The mean age of onset was 31 years. The first symptom was muscle limb weakness in 10/18, eyelid ptosis in 6/18, and respiratory insufficiency in 2/18. All patients developed variable muscle weakness during the evolution of the disease. Half of patients presented difficulty in swallowing. All patients showed evidence of respiratory muscle weakness, with need for non-invasive Mechanical Ventilation in 12/18. Four patients had deceased, all of them due to respiratory insufficiency. We identified common radiological features in muscle magnetic resonance, where the most severely affected muscles were the gluteus maximus, semitendinosus and sartorius. On muscle biopsies typical signs of mitochondrial dysfunction were associated with dystrophic changes. All mutations identified were previously reported, being the most frequent the in-frame deletion p.Lys202del. All cases showed multiple mtDNA deletions but mtDNA depletion was present only in two patients. CONCLUSIONS: The late-onset is the less frequent form of presentation of the TK2 deficiency and its natural history is not well known. Patients with late onset TK2 deficiency have a consistent and recognizable clinical phenotype and a poor prognosis, due to the high risk of early and progressive respiratory insufficiency.Instituto de Salud Carlos III PI16-01843 PI16/00579 CP09/00011Subdirección General de Evaluación y Fomento de la Investigación Sanitaria PI16-01843 PI16/00579 CP09/00011 PI 15/00431 PMP15/0002

The myokine GDF-15 is a potential biomarker for myositis and associates with the protein aggregates of sporadic inclusion body myositis.

Background: The cytokine growth differentiation factor-15 (GDF-15) has been associated with inflammatory and mitochondrial disease, warranting exploration of its expression in myositis patients. Methods: GDF-15 protein levels are evaluated in 35 idiopathic inflammatory myopathy (IIM) serum samples using enzyme-linked immunosorbent assays, comparing with levels in samples from healthy individuals and from patients with genetically confirmed hereditary muscular dystrophies and mitochondrial disorders. Muscle tissue expression of GDF-15 protein is evaluated using immunofluorescent staining and Western blotting. Results: GDF-15 protein levels are significantly higher in IIM sera (625 +/- 358 pg/ml) than in that of healthy controls (326 +/- 204 pg/ml, p = 0.01). Western blotting confirms increased GDF-15 protein levels in IIM muscle. In skeletal muscle tissue of IIM patients, GDF-15 localizes mostly to small regenerating or denervated muscle fibres. In patients diagnosed with sporadic inclusion body myositis, GDF-15 co-localizes with the characteristic protein aggregates within affected muscle fibres. Conclusions: We describe for the first time that GDF-15 is a myokine upregulated in myositis and present the cytokine as a potential diagnostic serum biomarker

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 ± 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 ± 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 ± 0.03 to 0.36 ± 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme

Lower Limb Radiology of Distal Myopathy due to the S60F Myotilin Mutation

Distal myopathies are a clinically and genetically heterogenous group of disorders in which the distal limb musculature is selectively or disproportionately affected. Precisely defining specific categories is a challenge because of overlapping clinical phenotypes, making it difficult to decide which of the many known causative genes to screen in individual cases. In this study we define the distinguishing magnetic resonance imaging findings in myotilin myopathy by studying 8 genealogically unrelated cases due to the same point mutation in TTID. Proximally, the vastii, biceps femoris and semimembranosus were involved with sparing of gracilis and sartorius. Distally, soleus, gastrocnemius, tibialis anterior, extensor hallicus and extensor digitorum were involved. This pattern contrasts with other distal myopathies and provides further support for the role of imaging in the clinical investigation of muscle disease. Copyright (C) 2009 S. Karger AG, Base

Progression of myopathology in Kearns-Sayre syndrome

We report on the progression of myopathology by comparing two biopsies from a patient with a Kearns-Sayre-Syndrome. The first biopsy was taken in 1979 and showed 10% ragged-red fibers. Myopathic changes were slight including internal nuclei and fiber splitting in 10% of the fibers. Electron microscopy revealed typical mitochondrial abnormalities with regard to number and shape. In 1989 a second biopsy was performed for an extended analysis of mitochondrial DNA. This time less than 5% of all fibers were ragged-red. Severe myopathic changes could be detected which so far has rarely been reported in mitochondrial cytopathy

Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies

Proteolytic Processing of OPA1 Links Mitochondrial Dysfunction to Alterations in Mitochondrial Morphology

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase {gamma}. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network

Proteomics of Cytochrome c Oxidase-Negative versus -Positive Muscle Fiber Sections in Mitochondrial Myopathy

The mosaic distribution of cytochrome c oxidase(+) (COX+) and COX - muscle fibers in mitochondrial disorders allows the sampling of fibers with compensated and decompensated mitochondrial function from the same individual. We apply laser capture microdissection to excise individual COX+ and COX- fibers from the biopsies of mitochondrial myopathy patients. Using mass spectrometry-based proteomics, we quantify >4,000 proteins per patient. While COX+ fibers show a higher expression of respiratory chain components, COX- fibers display protean adaptive responses, including upregulation of mitochondrial ribosomes, translation proteins, and chaperones. Upregulated proteins include C1QBP, required for mitoribosome formation and protein synthesis, and STOML2, which organizes cardiolipin-enriched microdomains and the assembly of respiratory supercomplexes. Factoring in fast/slow fiber type, COX (-) slow fibers show a compensatory upregulation of beta-oxidation, the AAA(+) protease AFG3L1, and the OPA1-dependent cristae remodeling program. These findings reveal compensatory mechanisms in muscle fibers struggling with energy shortage and metabolic stress

Potentially Diagnostic Electron Paramagnetic Resonance Spectra Elucidate the Underlying Mechanism of Mitochondrial Dysfunction in the Deoxyguanosine Kinase Deficient Rat Model of a Genetic Mitochondrial DNA Depletion Syndrome

A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes