Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene

BACKGROUND: Myofibrillar myopathies, often referred to as desmin-related myopathies, are a heterogeneous group of inherited or sporadic distal-onset skeletal myopathies associated with cardiomyopathy. Among the myofibrillar proteins that characteristically accumulate within the muscle fibers of affected patients, the one found most consistently is desmin, a muscle-specific intermediate-filament protein responsible for the structural integrity of the myofibrils. Skeletal and cardiac myopathy develops in mice that lack desmin, suggesting that mutations in the desmin gene may be pathogenic. METHODS: We examined 22 patients from 8 families with dominantly inherited myofibrillar or desmin-related myopathy and 2 patients with sporadic disease and analyzed the desmin gene for mutations, using complementary DNA (cDNA) amplified from muscle-biopsy specimens and genomic DNA extracted from blood lymphocytes. Restriction-enzyme analysis was used to confirm the mutations. Expression vectors containing normal or mutant desmin cDNA were introduced into cultured cells to determine whether the mutant desmin formed intermediate filaments. RESULTS: Six missense mutations in the coding region of the desmin gene that cause the substitution of an amino acid were identified in 11 patients (10 members of 4 families and 1 patient with sporadic disease); a splicing defect that resulted in the deletion of exon 3 was identified in the other patient with sporadic disease. Mutations were clustered in the carboxy-terminal part of the rod domain, which is critical for filament assembly. In transfected cells, the mutant desmin was unable to form a filamentous network. Seven of the 12 patients with mutations in the desmin gene had cardiomyopathy. CONCLUSIONS: Mutations in the desmin gene affecting intermediate filaments cause a distinct myopathy that is often associated with cardiomyopathy and is termed "desmin myopathy." The mutant desmin interferes with the normal assembly of intermediate filaments, resulting in fragility of the myofibrils and severe dysfunction of skeletal and cardiac muscles.     

Mutations in the mitochondrial tRNA Ser(AGY) gene are associated with deafness, retinal degeneration, myopathy and epilepsy

Although over 200 pathogenic mitochondrial DNA (mtDNA) mutations have been reported to date, determining the genetic aetiology of many cases of mitochondrial disease is still not straightforward. Here, we describe the investigations undertaken to uncover the underlying molecular defect(s) in two unrelated Caucasian patients with suspected mtDNA disease, who presented with similar symptoms of myopathy, deafness, neurodevelopmental delay, epilepsy, marked fatigue and, in one case, retinal degeneration. Histochemical and biochemical evidence of mitochondrial respiratory chain deficiency was observed in the patient muscle biopsies and both patients were discovered to harbour a novel heteroplasmic mitochondrial tRNA (mt-tRNA)(Ser(AGY)) (MTTS2) mutation (m.12264C>T and m.12261T>C, respectively). Clear segregation of the m.12261T>C mutation with the biochemical defect, as demonstrated by single-fibre radioactive RFLP, confirmed the pathogenicity of this novel variant in patient 2. However, unusually high levels of m.12264C>T mutation within both COX-positive (98.4 ± 1.5%) and COX-deficient (98.2 ± 2.1%) fibres in patient 1 necessitated further functional investigations to prove its pathogenicity. Northern blot analysis demonstrated the detrimental effect of the m.12264C>T mutation on mt-tRNA(Ser(AGY)) stability, ultimately resulting in decreased steady-state levels of fully assembled complexes I and IV, as shown by blue-native polyacrylamide gel electrophoresis. Our findings expand the spectrum of pathogenic mutations associated with the MTTS2 gene and highlight MTTS2 mutations as an important cause of retinal and syndromic auditory impairment.     

Hereditary mitochondrial hypertrophic cardiomyopathy with mitochondrial myopathy of skeletal muscle,congenital cataract and lactic acidosis

Summary A six day old boy died from an hereditary hypertrophic cardiomyopathy which was associated with mitochondrial myopathy of skeletal muscle, congenital cataract and lactic acidosis. In heart and skeletal muscle identical mitochondrial abnormalities were found: paucity and abnormal arrangement of cristae, formation and extrusion of vesicle-like structures and crystalline inclusions in the matrix compartment. Electron-cytochemistry revealed that only part of the mitochondria reacted positively for cytochrome oxidase activity. Morphometric analysis indicated that the cardiomegaly was due to cellular hypertrophy, which might be caused by an increase in the mitochondrial mass. The cardiac hypertrophy in this syndrome can be classified histopathologically as mitochondrial hypertrophic cardiomyopathy.     

Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.     

Novel mutations in KARS cause hypertrophic cardiomyopathy and combined mitochondrial respiratory chain defect

Mutations in KARS, which encodes for both mitochondrial and cytoplasmic lysyl‐tRNA synthetase, have been so far associated with three different phenotypes: the recessive form of Charcot–Mary–Tooth polyneuropathy, the autosomal recessive nonsyndromic hearing loss and the last recently described condition related to congenital visual impairment and progressive microcephaly. Here we report the case of a 14‐year‐old girl with severe cardiomyopathy associated to mild psychomotor delay and mild myopathy; moreover, a diffuse reduction of cytochrome C oxidase (COX, complex IV) and a combined enzymatic defect of complex I (CI) and complex IV (CIV) was evident in muscle biopsy. Using the TruSight One sequencing panel we identified two novel mutations in KARS. Both mutations, never reported previously, occur in a highly conserved region of the catalytic domain and displayed a dramatic effect on KARS stability. Structural analysis confirmed the pathogenic role of the identified variants. Our findings confirm and emphasize that mt‐aminoacyl‐tRNA synthetases (mt‐ARSs) enzymes are related to a broad clinical spectrum due to their multiple and still unknown functions.     

Novel VCP mutations in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia

Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD, OMIM 167320) has recently been attributed to eight missense mutations in valosin-containing protein (VCP). We report novel VCP mutations N387H and L198W in six individuals from two families who presented with proximal muscle weakness at a mean age of diagnosis of 40 years, most losing the ability to walk within a few years of onset. Electromyographic studies in four individuals were suggestive of 'myopathic' changes, and neuropathic pattern was identified in one individual in family 1. Muscle biopsy in four individuals showed myopathic changes characterized by variable fiber size, two individuals showing rimmed vacuoles and IBM-type cytoplasmic inclusions in muscle fibers, and electron microscopy in one individual revealing abundant intranuclear inclusions. Frontotemporal dementia associated with characteristic behavioral changes including short-term memory loss, language difficulty, and antisocial behavior was observed in three individuals at a mean age of 47 years. Detailed brain pathology in one individual showed cortical degenerative changes, most severe in the temporal lobe and hippocampus. Abundant ubiquitin-positive tau-, alpha-synuclein-, polyglutamine repeat-negative neuronal intranuclear inclusions and only rare intracytoplasmic VCP positive inclusions were seen. These new mutations may cause structural changes in VCP and provide some insight into the functional effects of pathogenic mutations.     

Somatic mitochondrial DNA mutations in human chromophobe renal cell carcinomas

We sequenced the entire mitochondrial genome in 8 chromophobe renal cell carcinomas (RCCs) and corresponding normal kidneys. Our study disclosed 68 known and 45 new sequence variations occurring 132 and 45 times, respectively. We found 6 somatic nucleotide changes in 5 out of the 8 chromophobe RCCs. One A --> T substitution occurred in the D-loop region and an insertion of a 9-bp sequence in the noncoding region of the MTNC7. One G --> A substitution and one C --> T substitution were seen in the MTRNR1 and MTRNR2 genes, respectively. One C deletion in MTND5 and one T insertion in the MTND3 gene resulted in frameshift mutations in two tumors. All somatic alterations, with the exception of the 9-bp insertion, were heteroplasmic changes. Although somatic mtDNA mutations are found in chromophobe RCCs, their role in the maintenance of tumor cell phenotype or in tumorigenesis remains to be elucidated.     

线粒体肌病患者线粒体DNA的突变分析

目的分析线粒体肌病患者线粒体DNA的突变情况，为疾病诊断提供依据。方法用常规HE、酶组化染色和电镜检查等病理形态学方法对3例线粒体肌病疑似患者进行诊断，并用聚合酶链反应-单链构象多态和DNA测序等方法对患者线粒体DNA中全部22个tRNA基因进行突变筛查。结果3例患者均被确诊为线粒体肌病，其中例1tRNA—VaI基因发生A1627G纯合突变，例2tRNA—Val基因发生A1627G／A杂合突变，例3tRNA—Trp基因发生T5554C突变、tRNA—Arg基因发生A10412C／A杂合突变。结论线粒体DNA中的tRNA基因突变是线粒体肌病的重要病因之一。     

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.     

Novel mitochondrial DNA mutations responsible for maternally inherited nonsyndromic hearing loss

Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides. Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A [p.MT-ND1:Leu28Met]), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A [p.MT-CO2:Val165Ile]), the tRNA of Serine 2 (AGU/C) (m.12236G>A), and Cytochrome B, subunit of complex III (m.15077G>A [p.MT-CYB:Glu111Lys]). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein-coding genes, can be responsible for nonsyndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology.     

In silico analysis for predicting pathogenicity of five unclassified mitochondrial DNA mutations associated with mitochondrial cytopathies' phenotypes

Mitochondrial DNA (mtDNA) mutations have been assigned as a major cause of genetic disease. When a novel sequence variation is found, it is necessary to evaluate its functional impact, usually requiring functional molecular studies. Given the fact that this approach is difficult to put in practice in a routine basis, it is possible to take advantage of the in silico tools available and predict protein/RNA structure changes and therefore pathogenicity. Here, we describe the characterization of five undescribed mtDNA variants, upon detection of 23 unclassified alterations at Laboratory of Biochemical Genetics, from 2004 to 2014. Those five sequence variations are located in protein-coding genes, in five patients with a diverse range of mitochondrial respiratory chain disease phenotypes including encephalopathy, optic neuropathy, developmental delay, deafness and epilepsy. According to the prediction established by in silico analysis using tools to predict structure and function changes (ClustalW2^®, PolyPhen-2^®, SIFT^®, MutationAssessor^®, PredictProtein^®, Provean^®, I-TASSER^®, Haplogrep^®), from the 23 variants analyzed, the five described are potentially pathogenic. This approach is inexpensive and compatible with a rapid first line response to clinical demanding, contributing to a more rationale genetic diagnosis concerning novel mutations and to clarify the mtDNA involvement in these pathologies.     

Point mutation of the mitochondrial tRNA(Leu) gene (A 3243 G) in maternally inherited hypertrophic cardiomyopathy, diabetes mellitus, renal failure, and sensorineural deafness.

The A 3243 G mutation of the mitochondrial tRNA(Leu) gene was found to segregate with maternally inherited diabetes mellitus, sensorineural deafness, hypertrophic cardiomyopathy, or renal failure in a large pedigree of 35 affected members in four generations. Presenting symptoms almost consistently involved deafness and recurrent attacks of migraine-like headaches, but the clinical course of the disease varied within and across generations. The A 3243 G mutation has been previously reported in association with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode syndrome (MELAS) and with diabetes mellitus and deafness. To our knowledge, however, hypertrophic cardiomyopathy is not a common feature in people with the A 3243 G mutation and renal failure has not been hitherto reported in association with this mutation. The present observation gives additional support to the variable clinical expression of mtDNA mutations in humans.     

Pathogenic mitochondrial mt-tRNAAla variants are uniquely associated with isolated myopathy

Pathogenic mitochondrial DNA (mtDNA) point mutations are associated with a wide range of clinical phenotypes, often involving multiple organ systems. We report two patients with isolated myopathy owing to novel mt-tRNA^Ala variants. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for both mutations (m.5631G>A and m.5610G>A) whilst single-muscle fibre segregation studies (revealing statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres), hierarchical mutation segregation within patient tissues and decreased steady-state mt-tRNA^Ala levels all provide compelling evidence of pathogenicity. Interestingly, both patients showed very high-mutation levels in all tissues, inferring that the threshold for impairment of oxidative phosphorylation, as evidenced by COX deficiency, appears to be extremely high for these mt-tRNA^Ala variants. Previously described mt-tRNA^Ala mutations are also associated with a pure myopathic phenotype and demonstrate very high mtDNA heteroplasmy thresholds, inferring at least some genotype:phenotype correlation for mutations within this particular mt-tRNA gene.     

Minisequencing mitochondrial DNA pathogenic mutations

Background  

There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA) diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations.