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.

Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores.

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing.

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.

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.

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.

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.

Cardiomyopathy with lethal arrhythmias associated with inactivation of KLHL24.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, yet the genetic cause of up to 50% of cases remains unknown. Here, we show that mutations in KLHL24 cause HCM in humans. Using genome-wide linkage analysis and exome sequencing, we identified homozygous mutations in KLHL24 in two consanguineous families with HCM. Of the 11 young affected adults identified, 3 died suddenly and 1 had a cardiac transplant due to heart failure. KLHL24 is a member of the Kelch-like protein family, which acts as substrate-specific adaptors to Cullin E3 ubiquitin ligases. Endomyocardial and skeletal muscle biopsies from affected individuals of both families demonstrated characteristic alterations, including accumulation of desmin intermediate filaments. Knock-down of the zebrafish homologue klhl24a results in heart defects similar to that described for other HCM-linked genes providing additional support for KLHL24 as a HCM-associated gene. Our findings reveal a crucial role for KLHL24 in cardiac development and function.

Progressive external ophthalmoplegia associated with novel MT-TN mutations.

OBJECTIVES: To describe two patients with progressive external ophthalmoplegia (PEO) and mitochondrial myopathy associated with mutations in mitochondrial DNA, encoding the tRNAAsn gene (MT-TN), which have not previously been published with clinical descriptions. MATERIALS & METHODS: Two unrelated patients with PEO were clinically examined. Muscle biopsy was performed and investigated by exome sequencing, enzyme histochemistry, and immunohistochemistry. The level of heteroplasmy was investigated in single muscle fibers and in other tissues. RESULTS: Patient 1 was a 52-year-old man with ptosis, PEO, and exercise intolerance since childhood. Muscle biopsy demonstrated mitochondrial myopathy with frequent cytochrome c oxidase (COX)-deficient fibers and a heteroplasmic mutation, m.5669G>A in the MT-TN gene, resulting in a substitution of a highly conserved C to T in the T stem of tRNAAsn . Patient 2 was a 66-year-old woman with ptosis, PEO, and exercise intolerance since many years. Muscle biopsy demonstrated mitochondrial myopathy with frequent COX-deficient fibers. She had a novel m.5702delA mutation in MT-TN, resulting in loss of a highly conserved U in the anticodon stem of tRNAAsn . Single fiber analysis in both cases showed highly significant differences in mutation load between COX-deficient and COX-normal fibers and a high threshold level for COX deficiency. The mutations were not found in blood, urine sediment or buccal cells. CONCLUSION: We describe two MT-TN mutations associated with PEO and mitochondrial myopathy, and their pathogenicity was demonstrated. Together with previous reports, the results indicate that MT-TN is a hot spot for mutations causing sporadic PEO.

Loss of supervillin causes myopathy with myofibrillar disorganization and autophagic vacuoles.

The muscle specific isoform of the supervillin protein (SV2), encoded by the SVIL gene, is a large sarcolemmal myosin II- and F-actin-binding protein. Supervillin (SV2) binds and co-localizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibrillar Z-lines. Despite its important role in muscle cell physiology suggested by various in vitro studies, there are so far no reports of any human disease caused by SVIL mutations. We here report four patients from two unrelated, consanguineous families with a childhood/adolescence onset of a myopathy associated with homozygous loss-of-function mutations in SVIL. Wide neck, anteverted shoulders and prominent trapezius muscles together with variable contractures were characteristic features. All patients showed increased levels of serum creatine kinase but no or minor muscle weakness. Mild cardiac manifestations were observed. Muscle biopsies showed complete loss of large supervillin isoforms in muscle fibres by western blot and immunohistochemical analyses. Light and electron microscopic investigations revealed a structural myopathy with numerous lobulated muscle fibres and considerable myofibrillar alterations with a coarse and irregular intermyofibrillar network. Autophagic vacuoles, as well as frequent and extensive deposits of lipoproteins, including immature lipofuscin, were observed. Several sarcolemma-associated proteins, including dystrophin and sarcoglycans, were partially mis-localized. The results demonstrate the importance of the supervillin (SV2) protein for the structural integrity of muscle fibres in humans and show that recessive loss-of-function mutations in SVIL cause a distinctive and novel myopathy.

RBCK1-related disease: A rare multisystem disorder with polyglucosan storage, auto-inflammation, recurrent infections, skeletal, and cardiac myopathy-Four additional patients and a review of the current literature.

In this article, we report four new patients, from three kindreds, with pathogenic variants in RBCK1 and a multisystem disorder characterised by widespread polyglucosan storage. We describe the clinical presentation of progressive skeletal and cardiac myopathy, combined immunodeficiencies and auto-inflammation, illustrate in detail the histopathological findings in multiple tissue types, and report muscle MRI findings.

TANGO2 deficiency as a cause of neurodevelopmental delay with indirect effects on mitochondrial energy metabolism.

Exome sequencing has recently identified mutations in the gene TANGO2 (transport and Golgi organization 2) as a cause of developmental delay associated with recurrent crises involving rhabdomyolysis, cardiac arrhythmias, and metabolic derangements. The disease is not well understood, in part as the cellular function and subcellular localization of the TANGO2 protein remain unknown. Furthermore, the clinical syndrome with its heterogeneity of symptoms, signs, and laboratory findings is still being defined. Here, we describe 11 new cases of TANGO2-related disease, confirming and further expanding the previously described clinical phenotype. Patients were homozygous or compound heterozygous for previously described exonic deletions or new frameshift, splice site, and missense mutations. All patients showed developmental delay with ataxia, dysarthria, intellectual disability, or signs of spastic diplegia. Of importance, we identify two subjects (aged 12 and 17 years) who have never experienced any overt episode of the catabolism-induced metabolic crises typical for the disease. Mitochondrial complex II activity was mildly reduced in patients investigated in association with crises but normal in other patients. In one deceased patient, post-mortem autopsy revealed heterotopic neurons in the cerebral white matter, indicating a possible role for TANGO2 in neuronal migration. Furthermore, we have addressed the subcellular localization of several alternative isoforms of TANGO2, none of which were mitochondrial but instead appeared to have a primarily cytoplasmic localization. Previously described aberrations in Golgi morphology were not observed in cultured skin fibroblasts.

Cardiomyopathy as presenting sign of glycogenin-1 deficiency-report of three cases and review of the literature.

We describe a new type of cardiomyopathy caused by a mutation in the glycogenin-1 gene (GYG1). Three unrelated male patients aged 34 to 52 years with cardiomyopathy and abnormal glycogen storage on endomyocardial biopsy were homozygous for the missense mutation p.Asp102His in GYG1. The mutated glycogenin-1 protein was expressed in cardiac tissue but had lost its ability to autoglucosylate as demonstrated by an in vitro assay and western blot analysis. It was therefore unable to form the primer for normal glycogen synthesis. Two of the patients showed similar patterns of heart dilatation, reduced ejection fraction and extensive late gadolinium enhancement on cardiac magnetic resonance imaging. These two patients were severely affected, necessitating cardiac transplantation. The cardiomyocyte storage material was characterized by large inclusions of periodic acid and Schiff positive material that was partly resistant to alpha-amylase treatment consistent with polyglucosan. The storage material had, unlike normal glycogen, a partly fibrillar structure by electron microscopy. None of the patients showed signs or symptoms of muscle weakness but a skeletal muscle biopsy in one case revealed muscle fibres with abnormal glycogen storage. Glycogenin-1 deficiency is known as a rare cause of skeletal muscle glycogen storage disease, usually without cardiomyopathy. We demonstrate that it may also be the cause of severe cardiomyopathy and cardiac failure without skeletal muscle weakness. GYG1 should be included in cardiomyopathy gene panels.

Novel myopathy in a newborn with Shwachman-Diamond syndrome and review of neonatal presentation.

Shwachman-Diamond-Bodian syndrome (SDS) is a pleiotropic disorder in which the main features are bone marrow dysfunction and pancreatic insufficiency. Skeletal changes can occur, and in rare cases manifest as severe congenital thoracic dystrophy. We report a newborn boy with asphyxia, narrow thorax, and severe hypotonia initially suggesting a neuromuscular disease. The muscle biopsy showed myopathic changes with prominent variability in muscle fiber size and abnormal expression of developmental isoforms of myosin. The myofibrils showed focal loss and disorganization of myofilaments, and thickening of the Z-discs including some abortive nemaline rods. The boy became permanently dependent on assisted ventilation. Pancreatic insufficiency was subsequently diagnosed, explaining the malabsorption and failure to thrive. Except transitory thrombocytopenia and leukopenia, no major hematological abnormalities were noted. He had bilateral nephrocalcinosis with preserved renal function. Transitory liver dysfunction with elevated transaminase levels and parenchymal changes on ultrasound were registered. The clinical diagnosis was confirmed by detection of compound heterozygous mutations in SBDS using whole-exome sequencing: a recurrent intronic mutation causing aberrant splicing (c.258+2T>C) and a novel missense variant in a highly conserved codon (c.41A>G, p.Asn14Ser), considered to be damaging for the protein structure by in silico prediction programs. The carrier status of the parents has been confirmed. This case illustrates the challenges in differential diagnosis of pronounced neonatal hypotonia with asphyxia and highlights the muscular involvement in SDS. To our knowledge, this is the first report of myopathy evidenced in a patient with clinically and molecularly confirmed SDS.

A new muscle glycogen storage disease associated with glycogenin-1 deficiency.

We describe a slowly progressive myopathy in 7 unrelated adult patients with storage of polyglucosan in muscle fibers. Genetic investigation revealed homozygous or compound heterozygous deleterious variants in the glycogenin-1 gene (GYG1). Most patients showed depletion of glycogenin-1 in skeletal muscle, whereas 1 showed presence of glycogenin-1 lacking the C-terminal that normally binds glycogen synthase. Our results indicate that either depletion of glycogenin-1 or impaired interaction with glycogen synthase underlies this new form of glycogen storage disease that differs from a previously reported patient with GYG1 mutations who showed profound glycogen depletion in skeletal muscle and accumulation of glycogenin-1.

Analysis of an independent tumor suppressor locus telomeric to Tp53 suggested Inpp5k and Myo1c as novel tumor suppressor gene candidates in this region.

BACKGROUND: Several reports indicate a commonly deleted chromosomal region independent from, and distal to the TP53 locus in a variety of human tumors. In a previous study, we reported a similar finding in a rat tumor model for endometrial carcinoma (EC) and through developing a deletion map, narrowed the candidate region to 700 kb, harboring 19 genes. In the present work real-time qPCR analysis, Western blot, semi-quantitative qPCR, sequencing, promoter methylation analysis, and epigenetic gene expression restoration analyses (5-aza-2'-deoxycytidine and/or trichostatin A treatments) were used to analyze the 19 genes located within the candidate region in a panel of experimental tumors compared to control samples. RESULTS: Real-time qPCR analysis suggested Hic1 (hypermethylated in cancer 1), Inpp5k (inositol polyphosphate-5-phosphatase K; a.k.a. Skip, skeletal muscle and kidney enriched inositol phosphatase) and Myo1c (myosin 1c) as the best targets for the observed deletions. No mutation in coding sequences of these genes was detected, hence the observed low expression levels suggest a haploinsufficient mode of function for these potential tumor suppressor genes. Both Inpp5k and Myo1c were down regulated at mRNA and/or protein levels, which could be rescued in gene expression restoration assays. This could not be shown for Hic1. CONCLUSION: Innp5k and Myo1c were identified as the best targets for the deletions in the region. INPP5K and MYO1C are located adjacent to each other within the reported independent region of tumor suppressor activity located at chromosome arm 17p distal to TP53 in human tumors. There is no earlier report on the potential tumor suppressor activity of INPP5K and MYO1C, however, overlapping roles in phosphoinositide (PI) 3-kinase/Akt signaling, known to be vital for the cell growth and survival, are reported for both. Moreover, there are reports on tumor suppressor activity of other members of the gene families that INPP5K and MYO1C belong to. Functional significance of these two candidate tumor suppressor genes in cancerogenesis pathways remains to be investigated.

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.

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.