Pathological Features in Paediatric Patients with TK2 Deficiency.

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.

Pediatric SMA patients with complex spinal anatomy: Implementation and evaluation of a decision-tree algorithm for administration of nusinersen.

The approval of nusinersen for the treatment of spinal muscular atrophy (SMA) has significantly changed the natural history of the disease. Nevertheless, scoliosis secondary to axial muscle weakness occurs at some point in most of patients with SMA and a conventional posterior interlaminar approach for intrathecal administration of nusinersen can be particularly challenging to perform in patients with severe scoliosis and/or previous spine fusion surgeries. We developed a protocol for the administration of nusinersen in pediatric patients, which includes a decision-tree algorithm that categorizes patients according to the estimated technical difficulty for the intrathecal administration. Complex spine patients were defined as those with a Cobb angle greater than 50° and/or a history of spinal surgery, while the rest of patients were considered non-complex. Nusinersen was successfully administered through a conventional non-CT-guided lumbar puncture in all 14 non-complex spine patients (110 out of 110 procedures; 100%). The feasibility of the intrathecal injection in the 15 complex spine patients was assessed by 3D CT. Administration was considered unfeasible in 7 out of these 15 patients according to imaging. In the 8 complex spine patients in whom the administration was considered feasible, conventional non-CT-guided lumbar punctures were successful only in 19 out of 53 procedures (36%). The remaining 34 procedures (64%) were guided by CT scan, all successful. Our work demonstrates that a cut-off point of 50° in Cobb angle and history of spinal surgery can reliably be used to anticipate the need for CT guidance in nusinersen administration.

Recurrent rhabdomyolysis and exercise intolerance: A new phenotype of late-onset thymidine kinase 2 deficiency.

A 29-year-old man developed, since the age of 18, exercise intolerance and exercise-induced rhabdomyolysis, with myoglobinuria. Muscle biopsy showed ragged-red fibers. Multiple mitochondrial DNA deletions were detected. The previously reported pathogenic homozygous mutation c.323C>T (p.Thr108Met) in TK2 was identified. This case expands the phenotypic spectrum of TK2 deficiency and indicates that it should be considered in the differential diagnosis of episodic rhabdomyolysis and exercise intolerance, along with other metabolic and mitochondrial myopathies. Since a new treatment is under development, it is essential improving knowledge of the natural history of TK2 deficiency.

The Phenotype and Genotype of Congenital Myopathies Based on a Large Pediatric Cohort.

BACKGROUND: Congenital myopathies (CMs) are a clinically and genetically heterogeneous group of hereditary muscular disorders. The distribution of genetic and histologic subtypes has been addressed in only a few cohorts, and the relationship between phenotypes and genotypes is only partially understood. METHODS: This is a retrospective cross-sectional data collection study conducted at a single center. The clinical, histopathological, and molecular characterization of 104 patients with CM is reported. RESULTS: The most common histopathological subtype was core myopathy (42%). Patients with severe endomysial fibrosis were more commonly unable to walk than patients with only a mild-grade endomysial fibrosis (56% vs 16%). Inability to walk was also more prevalent in patients with severe fatty replacement (44% vs 19%). The genetic etiology was more frequently identified among those patients with "specific" histologic findings (74% vs 62%). A definite molecular diagnosis was reached in 65 of 104 patients (62%), with RYR1 (24/104) and TTN (8/104) being the most frequent causative genes. Neonatal onset occurred in 56%. Independent ambulation was achieved by 74%. Patients who walked late were more likely to become wheelchair-dependent. Respiratory support was needed in one of three patients. Gastrostomy placement was required in 15%. Cardiac involvement was observed in 3%, scoliosis in 43%, and intellectual disability in 6%. CONCLUSIONS: This study provides an updated picture of the clinical, histopathological, and molecular landscape of CMs. Independently of the causative gene, fibrosis and fatty replacement in muscle biopsy specimens are associated with clinical severity. Mutations in TTN are responsible for a higher proportion of cases than previously thought.

Biallelic mutations in Tenascin-X cause classical-like Ehlers-Danlos syndrome with slowly progressive muscular weakness.

Tenascin-X, is an extracellular matrix glycoprotein expressed in skin, muscle, tendons, and blood vessels with an anti-adhesive function. Biallelic Tenascin-X mutations cause classical-like Ehlers-Danlos syndrome. We report a 46-year-old woman with slowly progressive weakness of the lower limbs and myalgia from age 28 years. In the past she had Raynaud's phenomenon, multiple sprains and joint dislocations, conjunctival haemorrhages and a colonic perforation during colonoscopy. Neurologic examination showed moderate asymmetric proximal and axial muscular weakness, distal amyotrophy of 4 limbs, moderate skin hyperextensibility, and hypermobility of distal joints of fingers. Whole body Magnetic Resonance Imaging showed symmetric fatty infiltration of thigh and leg muscles, with predominant atrophy of thighs. Next Generation Sequencing revealed two pathogenic TNXB variants, g.32024681C>G, c.7826-1G>C, and g.32016181dup, c.9998dupA, p.(Asn3333Lysfs*35). Western Blot and immunofluorescence studies confirmed a marked Tenascin-X reduction in both patient's serum and muscle. Here we further detail the clinical and genetic spectrum of a patient with classical-like Ehlers-Danlos syndrome and prominent muscle involvement.

Oxidative Phosphorylation Dysfunction Modifies the Cell Secretome.

Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell's secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials.

Epilepsy in LAMA2-related muscular dystrophy: An electro-clinico-radiological characterization.

OBJECTIVE: To delineate the epileptic phenotype of LAMA2-related muscular dystrophy (MD) and correlate it with the neuroradiological and muscle biopsy findings, as well as the functional motor phenotype. METHODS: Clinical, electrophysiological, neuroradiological, and histopathological data of 25 patients with diagnosis of LAMA2-related MD were analyzed. RESULTS: Epilepsy occurred in 36% of patients with LAMA2-related MD. Mean age at first seizure was 8 years. The most common presenting seizure type was focal-onset seizures with or without impaired awareness. Visual aura and autonomic signs, including vomiting, were frequently reported. Despite a certain degree of variability, bilateral occipital or temporo-occipital epileptiform abnormalities were by far the most commonly observed. Refractory epilepsy was found in 75% of these patients. Epilepsy in LAMA2-related MD was significantly more prevalent in those patients in whom the cortical malformations were more extensive. In contrast, the occurrence of epilepsy was not found to be associated with the patients' motor ability, the size of their white matter abnormalities, or the amount of residual merosin expressed on muscle. SIGNIFICANCE: The epileptic phenotype of LAMA2-related MD is characterized by focal seizures with prominent visual and autonomic features associated with EEG abnormalities that predominate in the posterior quadrants. A consistent correlation between epileptic phenotype and neuroimaging was identified, suggesting that the extension of the polymicrogyria may serve as a predictor of epilepsy occurrence.

CHRNG-related nonlethal multiple pterygium syndrome: Muscle imaging pattern and clinical, histopathological, and molecular genetic findings.

Mutations in the CHRNG gene cause autosomal recessive multiple pterygium syndrome (MPS). Herein we present a long-term follow-up of seven patients with CHRNG-related nonlethal MPS and we compare them with the 57 previously published patients. The objective is defining not only the clinical, histopathological, and molecular genetic characteristics, but also the type and degree of muscle involvement on whole-body magnetic resonance imaging (WBMRI). CHRNG mutations lead to a distinctive phenotype characterized by multiple congenital contractures, pterygium, and facial dysmorphism, with a stable clinical course over the years. Postnatal abnormalities at the neuromuscular junction were observed in the muscle biopsy of these patients. WBMRI showed distinctive features different from other arthrogryposis multiple congenita. A marked muscle bulk reduction is the predominant finding, mostly affecting the spinal erector muscles and gluteus maximus. Fatty infiltration was only observed in deep paravertebral muscles and distal lower limbs. Mutations in CHRNG are mainly located at the extracellular domain of the protein. Our study contributes to further define the phenotypic spectrum of CHRNG-related nonlethal MPS, including muscle imaging features, which may be useful in distinguishing it from other diffuse arthrogryposis entities.

Secondary coenzyme Q10 deficiencies in oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders.

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.

Activation of osmolyte pathways in inflammatory myopathy and Duchenne muscular dystrophy points to osmoregulation as a contributing pathogenic mechanism.

Alongside well-known nuclear factor κB (NFκB) and its associated cytokine networks, nuclear factor of activated T cells 5 (NFAT5), the master regulator of cellular osmoprotective programs, comes forward as an inflammatory regulator. To gain insight into its yet unexplored role in muscle disease, we studied the expression of NFAT5 target proteins involved in osmolyte accumulation: aldose reductase (AR), taurine transporter (TauT), and sodium myo-inositol co-transporter (SMIT). We analyzed idiopathic inflammatory myopathy and Duchenne muscular dystrophy muscle biopsies and myotubes in culture, using immunohistochemistry, immunofluorescence, and western blotting. We report that the level of constitutive AR was upregulated in patients, most strongly so in Duchenne muscular dystrophy. TauT and SMIT expression levels were induced in patients' muscle fibers, mostly representing regenerating and atrophic fibers. In dermatomyositis, strong staining for AR, TauT, and SMIT in atrophic perifascicular fibers was accompanied by staining for other molecular NFAT5 targets, including chaperones, chemokines, and inducible nitric oxide synthase. In these fibers, NFAT5 and NFκB p65 staining coincided, linking both transcription factors with this important pathogenic hallmark. In sporadic inclusion body myositis, SMIT localized to inclusions inside muscle fibers. In addition, SMIT was expressed by a substantial subset of muscle-infiltrating macrophages and T cells in patient biopsies. Our results indicate that osmolyte pathways may contribute to normal muscle functioning, and that activation of AR, TauT, and SMIT in muscle inflammation possibly contributes to the tissue's failing program of damage control.

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies.

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy.

BACKGROUND: Between 8% and 22% of female carriers of DMD mutations exhibit clinical symptoms of variable severity. Development of symptoms in DMD mutation carriers without chromosomal rearrangements has been attributed to skewed X-chromosome inactivation (XCI) favouring predominant expression of the DMD mutant allele. However the prognostic use of XCI analysis is controversial. We aimed to evaluate the correlation between X-chromosome inactivation and development of clinical symptoms in a series of symptomatic female carriers of dystrophinopathy. METHODS: We reviewed the clinical, pathological and genetic features of twenty-four symptomatic carriers covering a wide spectrum of clinical phenotypes. DMD gene analysis was performed using MLPA and whole gene sequencing in blood DNA and muscle cDNA. Blood and muscle DNA was used for X-chromosome inactivation (XCI) analysis thought the AR methylation assay in symptomatic carriers and their female relatives, asymptomatic carriers as well as non-carrier females. RESULTS: Symptomatic carriers exhibited 49.2% more skewed XCI profiles than asymptomatic carriers. The extent of XCI skewing in blood tended to increase in line with the severity of muscle symptoms. Skewed XCI patterns were found in at least one first-degree female relative in 78.6% of symptomatic carrier families. No mutations altering XCI in the XIST gene promoter were found. CONCLUSIONS: Skewed XCI is in many cases familial inherited. The extent of XCI skewing is related to phenotype severity. However, the assessment of XCI by means of the AR methylation assay has a poor prognostic value, probably because the methylation status of the AR gene in muscle may not reflect in all cases the methylation status of the DMD gene.

A comparative study of alpha-dystroglycan glycosylation in dystroglycanopathies suggests that the hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical severity.

Hypoglycosylation of alpha-dystroglycan underpins a subgroup of muscular dystrophies ranging from congenital onset of weakness, severe brain malformations and death in the perinatal period to mild weakness in adulthood without brain involvement. Mutations in six genes have been identified in a proportion of patients. POMT1, POMT2 and POMGnT1 encode for glycosyltransferases involved in the mannosylation of alpha-dystroglycan but the function of fukutin, FKRP and LARGE is less clear. The pathological hallmark is reduced immunolabeling of skeletal muscle with antibodies recognizing glycosylated epitopes on alpha-dystroglycan. If the common pathway of these conditions is the hypoglycosyation of alpha-dystroglycan, one would expect a correlation between clinical severity and the extent of hypoglycosylation. By studying 24 patients with mutations in these genes, we found a good correlation between reduced alpha-dystroglycan staining and clinical course in patients with mutations in POMT1, POMT2 and POMGnT1. However, this was not always the case in patients with defects in fukutin and FKRP, as we identified patients with mild limb-girdle phenotypes without brain involvement with profound depletion of alpha-dystroglycan. These data indicate that it is not always possible to correlate clinical course and alpha-dystroglycan labeling and suggest that there might be differences in alpha-dystroglycan processing in these disorders.

Antenatal and postnatal brain magnetic resonance imaging in muscle-eye-brain disease.

BACKGROUND: Muscle-eye-brain disease (MEB) is a rare autosomal recessive disorder characterized by congenital muscular dystrophy, structural eye abnormalities, and type II lissencephaly. Previous reports of brain abnormalities on magnetic resonance images (MRIs) in MEB have been in children older than 1 year. OBJECTIVE: To describe serial antenatal and postnatal brain MRIs in a child with MEB. DESIGN: Case report. PATIENT: We report a 2-year-old white boy with genetically confirmed MEB. Antenatal MRIs at 25 and 35 weeks' gestation showed posterior ventriculomegaly but no cortical dysplasia. A postnatal brain MRI at age 1 week showed frontal cortical dysplasia and abnormal signal intensity within the frontal white matter. A brain MRI at 8 months showed bilateral frontoparietal polymicrogyria. All images demonstrated flattening of the pons and mild hypoplasia of the inferior vermis. The child had no weakness, and muscle involvement was only suspected when the serum creatine kinase level was found to be elevated at age 8 months. CONCLUSION: Cortical dysplasia in MEB may not be evident until several postnatal months; therefore, if MEB is suspected, brain MRI performed in the first few months of life should be interpreted with caution.

Prenatal diagnosis of Ullrich congenital muscular dystrophy using haplotype analysis and collagen VI immunocytochemistry.

OBJECTIVES: Ullrich congenital muscular dystrophy (UCMD) is a recessively inherited condition characterised by proximal joint contractures, marked distal joint hyperextensibility, rigidity of the spine and early respiratory failure. Recently, mutations in the genes encoding the subunits of collagen VI have been identified in this disease. We undertook two prenatal diagnoses for UCMD in a consanguineous family where the disease was consistent with linkage to the COL6A3 locus and immunolabelling of collagen VI in the proband's skeletal muscle was severely reduced. METHODS: Both haplotype analysis and collagen VI immunolabelling were used to determine the status of the fetuses. RESULTS: Haplotype analysis of DNA extracted from chorionic villus samples (CVS) from the initial at-risk pregnancy with markers encompassing COL6A3 demonstrated that this fetus had inherited the same haplotypes as the affected child, and immunolabelling of the at-risk CVS demonstrated the virtual absence of collagen VI. A second latter fetus inherited neither of the at-risk haplotypes and collagen VI expression in the CVS was normal. During the second pregnancy, a homozygous G > A change in the last nucleotide of exon 27 of COL6A3 was identified in the proband, substantiating the results obtained from haplotype analysis and collagen VI immunolabelling. CONCLUSION: These findings demonstrate that haplotype analysis in combination with immunocytochemistry is a rapid and reliable method for prenatal diagnosis of UCMD, provided the family is genetically informative and reduced collagen VI expression in the proband has been demonstrated.

Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan.

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders. A new pathomechanism has recently been identified in a group of these disorders in which known or putative glycosyltransferases are defective. Common to all these conditions is the hypoglycosylation of alpha-dystroglycan. Fukuyama CMD, muscle-eye-brain disease and Walker-Warburg syndrome, each associated with eye abnormalities and neuronal migration defects, result from mutations in fukutin, POMGnT1 and POMT1, respectively, while mutations in the fukutin-related protein (FKRP) gene cause congenital muscular dystrophy 1C, typically lacking brain involvement. Another putative glycosyltransferase, Large, is mutated in the myodystrophy mouse. The human homologue of this gene is therefore a strong candidate for involvement in novel forms of muscular dystrophy. We studied 36 patients with muscular dystrophy and either mental retardation, structural brain changes or abnormal alpha-dystroglycan immunolabelling, unlinked to any reported CMD loci. Linkage analysis in seven informative families excluded involvement of LARGE but sequencing of this gene in the remaining 29 families identified one patient with a G1525A (Glu509Lys) missense mutation and a 1 bp insertion, 1999insT. This 17-year-old girl presented with congenital muscular dystrophy, profound mental retardation, white matter changes and subtle structural abnormalities on brain MRI. Her skeletal muscle biopsy showed reduced immunolabelling of alpha-dystroglycan. Immunoblotting with an antibody to a glycosylated epitope demonstrated a reduced molecular weight form of alpha-dystroglycan that retained some laminin binding activity. This is the first description of mutations in the human LARGE gene and we propose to name this new disorder MDC1D.