Proteomic profiling of polyglucosan bodies associated with glycogenin-1 deficiency in skeletal muscle.

AIMS: Polyglucosan storage disorders represent an emerging field within neurodegenerative and neuromuscular conditions, including Lafora disease (EPM2A, EPM2B), adult polyglucosan body disease (APBD, GBE1), polyglucosan body myopathies associated with RBCK1 deficiency (PGBM1, RBCK1) or glycogenin-1 deficiency (PGBM2, GYG1). While the storage material primarily comprises glycans, this study aimed to gain deeper insights into the protein components by proteomic profiling of the storage material in glycogenin-1 deficiency. METHODS: We employed molecular genetic analyses, quantitative mass spectrometry of laser micro-dissected polyglucosan bodies and muscle homogenate, immunohistochemistry and western blot analyses in muscle tissue from a 45-year-old patient with proximal muscle weakness from late teenage years due to polyglucosan storage myopathy. RESULTS: The muscle tissue exhibited a complete absence of glycogenin-1 due to a novel homozygous deep intronic variant in GYG1 (c.7+992T>G), introducing a pseudo-exon causing frameshift and a premature stop codon. Accumulated proteins in the polyglucosan bodies constituted components of glycogen metabolism, protein quality control pathways and desmin. Muscle fibres containing polyglucosan bodies frequently exhibited depletion of normal glycogen. CONCLUSIONS: The absence of glycogenin-1, a protein important for glycogen synthesis initiation, causes storage of polyglucosan that displays accumulation of several proteins, including those essential for glycogen synthesis, sequestosome 1/p62 and desmin, mirroring findings in RBCK1 deficiency. These results suggest shared pathogenic pathways across different diseases exhibiting polyglucosan storage. Such insights have implications for therapy in these rare yet devastating and presently untreatable disorders.

Inclusion body myositis with early onset: a population-based study.

INTRODUCTION: Inclusion body myositis (IBM), an inflammatory myopathy with progressive weakness without efficient treatment, typically presents after 45 years of age and younger patients are sparsely studied. METHODS: In a population-based study during a 33-year period, 142 patients with IBM were identified in western Sweden. Six patients fell outside the European Neuromuscular Centre 2011 criteria for IBM due to young age at symptom onset, verified by a muscle biopsy < 50 years of age. These were defined as early-onset IBM and included in this study. Medical records, muscle strength, comorbidities, muscle biopsies, and nuclear- and mitochondrial DNA were examined and compared with patients with IBM and age matched controls from the same population. RESULTS: The median age at symptom onset was 36 (range 34-45) years and at diagnosis 43 (range 38-58) years. Four patients were deceased at a median age of 59 (range 50-75) years. The median survival from diagnosis was 14 (range 10-18) years. The prevalence December 31 2017 was 1.2 per million inhabitants and the mean incidence 0.12 patients per million inhabitants and year. The mean decline in quadriceps strength ± 1 standard deviation was 1.21 ± 0.2 Newton or 0.91 ± 0.2% per month and correlated to time from diagnosis (p < 0.001). Five patients had swallowing difficulties. All patients displayed mitochondrial changes in muscle including cytochrome c oxidase deficiency and the mitochondrial DNA mutation load was high. CONCLUSIONS: Early-onset IBM is a severe disease, causing progressive muscle weakness, high muscle mitochondrial DNA mutation load and a reduced cumulative survival in young and middle-aged individuals.

Ribonuclease inhibitor 1 (RNH1) deficiency cause congenital cataracts and global developmental delay with infection-induced psychomotor regression and anemia.

Ribonuclease inhibitor 1, also known as angiogenin inhibitor 1, encoded by RNH1, is a ubiquitously expressed leucine-rich repeat protein, which is highly conserved in mammalian species. Inactivation of rnh1 in mice causes an embryonically lethal anemia, but the exact biological function of RNH1 in humans remains unknown and no human genetic disease has so far been associated with RNH1. Here, we describe a family with two out of seven siblings affected by a disease characterized by congenital cataract, global developmental delay, myopathy and psychomotor deterioration, seizures and periodic anemia associated with upper respiratory tract infections. A homozygous splice-site variant (c.615-2A > C) in RNH1 segregated with the disease. Sequencing of RNA derived from patient fibroblasts and cDNA analysis of skeletal muscle mRNA showed aberrant splicing with skipping of exon 7. Western blot analysis revealed a total lack of the RNH1 protein. Functional analysis revealed that patient fibroblasts were more sensitive to RNase A exposure, and this phenotype was reversed by transduction with a lentivirus expressing RNH1 to complement patient cells. Our results demonstrate that loss-of-function of RNH1 in humans is associated with a multiorgan developmental disease with recessive inheritance. It may be speculated that the infection-induced deterioration resulted from an increased susceptibility toward extracellular RNases and/or other inflammatory responses normally kept in place by the RNase inhibitor RNH1.

The Swedish COG6-CDG experience and a comprehensive literature review.

Here, we present the first two Swedish cases of Conserved Oligomeric Golgi complex subunit 6-congenital disorders of glycosylation (COG6-CDG). Their clinical symptoms include intellectual disability, Attention Deficit/Hyperactivity Disorder (ADHD), delayed brain myelinization, progressive microcephaly, joint laxity, hyperkeratosis, frequent infections, and enamel hypoplasia. In one family, compound heterozygous variants in COG6 were identified, where one (c.785A>G; p.Tyr262Cys) has previously been described in patients of Moroccan descent, whereas the other (c.238G>A; p.Glu80Lys) is undescribed. On the other hand, a previously undescribed homozygous duplication (c.1793_1795dup) was deemed the cause of the disease. To confirm the pathogenicity of the variants, we treated patient and control fibroblasts with the ER-Golgi transport inhibitor Brefeldin-A and show that patient cells manifest a significantly slower anterograde and retrograde ER-Golgi transport.

Dominantly inherited myosin IIa myopathy caused by aberrant splicing of MYH2.

BACKGROUND: Myosin heavy chain (MyHC) isoforms define the three major muscle fiber types in human extremity muscles. Slow beta/cardiac MyHC (MYH7) is expressed in type 1 muscle fibers. MyHC IIa (MYH2) and MyHC IIx (MYH1) are expressed in type 2A and 2B fibers, respectively. Whereas recessive MyHC IIa myopathy has been described in many cases, myopathy caused by dominant MYH2 variants is rare and has been described with clinical manifestations and muscle pathology in only one family and two sporadic cases. METHODS: We investigated three patients from one family with a dominantly inherited myopathy by clinical investigation, whole-genome sequencing, muscle biopsy, and magnetic resonance imaging (MRI). RESULTS: Three siblings, one woman and two men now 54, 56 and 66 years old, had experienced muscle weakness initially affecting the lower limbs from young adulthood. They have now generalized proximal muscle weakness affecting ambulation, but no ophthalmoplegia. Whole-genome sequencing identified a heterozygous MYH2 variant, segregating with the disease in the three affected individuals: c.5673 + 1G > C. Analysis of cDNA confirmed the predicted splicing defect with skipping of exon 39 and loss of residues 1860-1891 in the distal tail of the MyHC IIa, largely overlapping with the filament assembly region (aa1877-1905). Muscle biopsy in two of the affected individuals showed prominent type 1 muscle fiber predominance with only a few very small, scattered type 2A fibers and no type 2B fibers. The small type 2A fibers were frequently hybrid fibers with either slow MyHC or embryonic MyHC expression. The type 1 fibers showed variation in fiber size, internal nuclei and some structural alterations. There was fatty infiltration, which was also demonstrated by MRI. CONCLUSION: Dominantly inherited MyHC IIa myopathy due to a splice defect causing loss of amino acids 1860-1891 in the distal tail of the MyHC IIa protein including part of the assembly competence domain. The myopathy is manifesting with slowly progressive muscle weakness without overt ophthalmoplegia and markedly reduced number and size of type 2 fibers.

Respiratory chain dysfunction in perifascicular muscle fibres in patients with dermatomyositis is associated with mitochondrial DNA depletion.

AIMS: Patients with dermatomyositis (DM) suffer from reduced aerobic metabolism contributing to impaired muscle function, which has been linked to cytochrome c oxidase (COX) deficiency in muscle tissue. This mitochondrial respiratory chain dysfunction is typically seen in perifascicular regions, which also show the most intense inflammatory reaction along with capillary loss and muscle fibre atrophy. The objective of this study was to investigate the pathobiology of the oxidative phosphorylation deficiency in DM. METHODS: Muscle biopsy specimens with perifascicular COX deficiency from five juveniles and seven adults with DM were investigated. We combined immunohistochemical analyses of subunits in the respiratory chain including complex I (subunit NDUFB8), complex II (succinate dehydrogenase, subunit SDHB) and complex IV (COX, subunit MTCO1) with in situ hybridisation, next generation deep sequencing and quantitative polymerase chain reaction (PCR). RESULTS: There was a profound deficiency of complexes I and IV in the perifascicular regions with enzyme histochemical COX deficiency, whereas succinate dehydrogenase activity and complex II were preserved. In situ hybridisation of mitochondrial RNA showed depletion of mitochondrial DNA (mtDNA) transcripts in the perifascicular regions. Analysis of mtDNA by next generation deep sequencing and quantitative PCR in affected muscle regions showed an overall reduction of mtDNA copy number particularly in the perifascicular regions. CONCLUSION: The respiratory chain dysfunction in DM muscle is associated with mtDNA depletion causing deficiency of complexes I and IV, which are partially encoded by mtDNA, whereas complex II, which is entirely encoded by nuclear DNA, is preserved. The depletion of mtDNA indicates a perturbed replication of mtDNA explaining the muscle pathology and the disturbed aerobic metabolism.

Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

Four Swedish cases of CSF1R-related leukoencephalopathy: Visualization of clinical phenotypes.

Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is a rare, genetic disease caused by heterozygous mutations in the CSF1R gene with rapidly progressive neurodegeneration, behavioral, cognitive, motor disturbances. OBJECTIVE: To describe four cases of CSF1R-related leukoencephalopathy from three families with two different pathogenic mutations in the tyrosine kinase domain of CSF1R and to develop an integrated presentation of inter-individual diversity of clinical presentations. METHODS: This is an observational study of a case series. Patients diagnosed with CSF1R encephalopathy were evaluated with standardized functional estimation scores and subject to analysis of cerebrospinal fluid biomarkers. Brain computed tomography (CT) and magnetic resonance imaging (MRI) were evaluated. We performed a functional phosphorylation assay to confirm the dysfunction of mutated CSF1R protein. RESULTS: Two heterozygous missense mutations in the CSF1R gene were identified, c.2344C>T; p.Arg777Trp and c.2329C>T; p.Arg782Cys. A phosphorylation assay in vitro showed markedly reduced autophosphorylation in cells expressing mutations. According to ACMG criteria, both mutations were pathogenic. A radiological investigation revealed typical white matter lesions in all cases. There was inter-individual diversity in the loss of cognitive, motor-neuronal, and extrapyramidal functions. CONCLUSIONS: Including the present cases, currently three CSF1R mutations are known in Sweden. We present a visualization tool to describe the clinical diversity, with potential use for longitudinal follow-up for this and other leukoencephalopathies.

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant.

Two homoplasmic variants in tRNAGlu (m.14674T>C/G) are associated with reversible infantile respiratory chain deficiency. This study sought to further characterize the expression of the individual mitochondrial respiratory chain complexes and to describe the natural history of the disease. Seven patients from four families with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant were investigated. All patients underwent skeletal muscle biopsy and mtDNA sequencing. Whole-genome sequencing was performed in one family. Western blot and immunohistochemical analyses were used to characterize the expression of the individual respiratory chain complexes. Patients presented with hypotonia and feeding difficulties within the first weeks or months of life, except for one patient who first showed symptoms at 4 years of age. Histopathological findings in muscle included lipid accumulation, numerous COX-deficient fibers, and mitochondrial proliferation. Ultrastructural abnormalities included enlarged mitochondria with concentric cristae and dense mitochondrial matrix. The m.14674T>C variant in MT-TE was identified in all patients. Immunohistochemistry and immunoblotting demonstrated pronounced deficiency of the complex I subunit NDUFB8. The expression of MTCO1, a complex IV subunit, was also decreased, but not to the same extent as NDUFB8. Longitudinal follow-up data demonstrated that not all features of the disorder are entirely transient, that the disease may be progressive, and that signs and symptoms of myopathy may develop during childhood. This study sheds new light on the involvement of complex I in reversible infantile respiratory chain deficiency, it shows that the disorder may be progressive, and that myopathy can develop without an infantile episode.

Functional analysis of a novel POLγA mutation associated with a severe perinatal mitochondrial encephalomyopathy.

Mutations in the mitochondrial DNA polymerase gamma catalytic subunit (POLγA) compromise the stability of mitochondrial DNA (mtDNA) by leading to mutations, deletions and depletions in mtDNA. Patients with mutations in POLγA often differ remarkably in disease severity and age of onset. In this work we have studied the functional consequence of POLγA mutations in a patient with an uncommon and a very severe disease phenotype characterized by prenatal onset with intrauterine growth restriction, lactic acidosis from birth, encephalopathy, hepatopathy, myopathy, and early death. Muscle biopsy identified scattered COX-deficient muscle fibers, respiratory chain dysfunction and mtDNA depletion. We identified a novel POLγA mutation (p.His1134Tyr) in trans with the previously identified p.Thr251Ile/Pro587Leu double mutant. Biochemical characterization of the purified recombinant POLγA variants showed that the p.His1134Tyr mutation caused severe polymerase dysfunction. The p.Thr251Ile/Pro587Leu mutation caused reduced polymerase function in conditions of low dNTP concentration that mimic postmitotic tissues. Critically, when p.His1134Tyr and p.Thr251Ile/Pro587Leu were combined under these conditions, mtDNA replication was severely diminished and featured prominent stalling. Our data provide a molecular explanation for the patient´s mtDNA depletion and clinical features, particularly in tissues such as brain and muscle that have low dNTP concentration.