Deep Characterization of a Greek Patient with Desmin-Related Myofibrillar Myopathy and Cardiomyopathy.

Desmin is a class III intermediate filament protein highly expressed in cardiac, smooth and striated muscle. Autosomal dominant or recessive mutations in the desmin gene (DES) result in a variety of diseases, including cardiomyopathies and myofibrillar myopathy, collectively called desminopathies. Here we describe the clinical, histological and radiological features of a Greek patient with a myofibrillar myopathy and cardiomyopathy linked to the c.734A>G,p.(Glu245Gly) heterozygous variant in the DES gene. Moreover, through ribonucleic acid sequencing analysis in skeletal muscle we show that this variant provokes a defect in exon 3 splicing and thus should be considered clearly pathogenic.

Genotype-phenotype correlations in valosin-containing protein disease: a retrospective muticentre study.

BACKGROUND: Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations. METHODS: Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene. RESULTS: Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death. CONCLUSION: This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Circulating small RNA signatures differentiate accurately the subtypes of muscular dystrophies: small-RNA next-generation sequencing analytics and functional insights.

Muscular dystrophies are a group of rare and severe inherited disorders mainly affecting the muscle tissue. Duchene Muscular Dystrophy, Myotonic Dystrophy types 1 and 2, Limb Girdle Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy are some of the members of this family of disorders. In addition to the current diagnostic tools, there is an increasing interest for the development of novel non-invasive biomarkers for the diagnosis and monitoring of these diseases. miRNAs are small RNA molecules characterized by high stability in blood thus making them ideal biomarker candidates for various diseases. In this study, we present the first genome-wide next-generation small RNA sequencing in serum samples of five different types of muscular dystrophy patients and healthy individuals. We identified many small RNAs including miRNAs, lncRNAs, tRNAs, snoRNAs and snRNAs, that differentially discriminate the muscular dystrophy patients from the healthy individuals. Further analysis of the identified miRNAs showed that some miRNAs can distinguish the muscular dystrophy patients from controls and other miRNAs are specific to the type of muscular dystrophy. Bioinformatics analysis of the target genes for the most significant miRNAs and the biological role of these genes revealed different pathways that the dysregulated miRNAs are involved in each type of muscular dystrophy investigated. In conclusion, this study shows unique signatures of small RNAs circulating in five types of muscular dystrophy patients and provides a useful resource for future studies for the development of miRNA biomarkers in muscular dystrophies and for their involvement in the pathogenesis of the disorders.

Identification of exosomal muscle-specific miRNAs in serum of myotonic dystrophy patients relating to muscle disease progress.

Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy, which is characterised by progressive muscle wasting and the discovery of reliable blood-based biomarkers could be useful for the disease progress monitoring. There have been some reports showing that the presence of specific miRNAs in blood correlates with DM1. In one of these, our group identified four muscle-specific miRNAs, miR-1, miR-133a, miR-133b and miR-206, which correlated with the progression of muscle wasting observed in DM1 patients. The levels of the four muscle-specific miRNAs were elevated in the serum of DM1 patients compared to healthy participants and were also elevated in the serum of progressive muscle wasting DM1 patients compared to disease-stable DM1 patients. The aim of this work was to characterise the ontology of these four muscle-specific miRNAs in the blood circulation of DM1 patients. Here we show that the four muscle-specific miRNAs are encapsulated within exosomes isolated from DM1 patients. Our results show for the first time, the presence of miRNAs encapsulated within exosomes in blood circulation of DM1 patients. More interestingly, the levels of the four exosomal muscle-specific miRNAs are associated with the progression of muscle wasting in DM1 patients. We propose that exosomal muscle-specific miRNAs may be useful molecular biomarkers for monitoring the progress of muscle wasting in DM1 patients. There has been a growing interest regarding the clinical applications of exosomes and their role in prognosis and therapy of various diseases and the above results contribute towards this way.

Toward understanding tissue-specific symptoms in dolichol-phosphate-mannose synthesis disorders; insight from DPM3-CDG.

The congenital disorders of glycosylation (CDG) are inborn errors of metabolism with a great genetic heterogeneity. Most CDG are caused by defects in the N-glycan biosynthesis, leading to multisystem phenotypes. However, the occurrence of tissue-restricted clinical symptoms in the various defects in dolichol-phosphate-mannose (DPM) synthesis remains unexplained. To deepen our understanding of the tissue-specific characteristics of defects in the DPM synthesis pathway, we investigated N-glycosylation and O-mannosylation in skeletal muscle of three DPM3-CDG patients presenting with muscle dystrophy and hypo-N-glycosylation of serum transferrin in only two of them. In the three patients, O-mannosylation of alpha-dystroglycan (αDG) was strongly reduced and western blot analysis of beta-dystroglycan (ßDG) N-glycosylation revealed a consistent lack of one N-glycan in skeletal muscle. Recently, defective N-glycosylation of ßDG has been reported in patients with mutations in guanosine-diphosphate-mannose pyrophosphorylase B (GMPPB). Thus, we suggest that aberrant O-glycosylation of αDG and N-glycosylation of ßDG in skeletal muscle is indicative of a defect in the DPM synthesis pathway. Further studies should address to what extent hypo-N-glycosylation of ßDG or other skeletal muscle proteins contribute to the phenotype of patients with defects in DPM synthesis. Our findings contribute to our understanding of the tissue-restricted phenotype of DPM3-CDG and other defects in the DPM synthesis pathway.

Polymyositis with mitochondrial pathology or atypical form of sporadic inclusion body myositis: case series and review of the literature.

Polymyositis with mitochondrial pathology (PM-Mito) is a rare form of idiopathic inflammatory myopathy with no definite diagnostic criteria and similarities to both PM and sporadic inclusion body myositis (s-IBM). The aim of this study is to address the dilemma of whether PM-Mito is a subtype of inflammatory myopathy or represents a disease falling into the spectrum of s-IBM. Herein, we report four female patients diagnosed with PM-Mito, highlighting their rather atypical clinical and histopathological characteristics that seem to indicate a diagnosis away from s-IBM. Muscle weakness was rather proximal and symmetrical and lacked the selective pattern observed in s-IBM. Patients had large-scale deletions in mtDNA, reflecting the mitochondrial component in the pathology of the disease. Conclusively, our study adds to the limited data in the literature on whether PM-Mito is a distinct form of myositis or represents a prodromal stage of s-IBM. Although the latter seems to be supported by a substantial body of evidence, there are, however, important differences, such as the different patterns of muscle weakness, and the good response to treatment observed in some patients. Larger-scale studies are certainly needed to clarify pathogenesis and clinical characteristics of PM-Mito patients, especially in therapeutic and prognostic terms.

Stiff-Leg Syndrome Associated with Autoimmune Retinopathy and Its Treatment with IVIg-A Case Report and Review of the Literature.

Antibodies to glutamic acid decarboxylase (GAD) have been predominantly associated with stiff-person syndrome (SPS), which is often accompanied by organ-specific autoimmune diseases, such as late-onset type 1 diabetes. Autoimmune retinal pathology in SPS has recently been suggested to coexist in patients suffering from this disease; however, evidence reporting potential treatment options for the neurological and visual symptoms these patients experience remains scarce. We provide a review of the relevant literature, presenting a rare case of a middle-aged woman with autoimmune retinopathy (AIR) followed by stiff-leg syndrome who responded to intravenous immune globulin treatment (IVIg). Our report adds to previously reported data supporting the efficacy of IVIg in SPS spectrum disorders while also proposing the potential effect of IVIg in treating SPS spectrum patients with coexisting AIR.

Serum miRNAs as biomarkers for the rare types of muscular dystrophy.

Muscular dystrophies are a group of disorders that cause progressive muscle weakness. There is an increasing interest for the development of biomarkers for these disorders and specifically for Duchene Muscular Dystrophy. Limited research however, has been performed on the biomarkers' development for the most rare muscular dystrophies, like the Facioscapulohumeral Muscular Dystrophy, Limb-Girdle Muscular Dystrophy and Myotonic Dystrophy type 2. Here, we aimed to identify novel serum-based miRNA biomarkers for these rare muscular dystrophies, through high-throughput next-generation RNA sequencing. We identified many miRNAs that associate with muscular dystrophy patients compared to controls. Based on a series of selection criteria, the two best candidate miRNAs for each of these disorders were chosen and validated in a larger number of patients. Our results showed that miR-223-3p and miR-206 are promising serum-based biomarkers for Facioscapulohumeral Muscular Dystrophy type 1, miR-143-3p and miR-486-3p for Limb-Girdle Muscular Dystrophy type 2A whereas miR-363-3p and miR-25-3p associate with Myotonic Dystrophy type 2. Some of the identified miRNAs were significantly elevated in the serum of the patients compared to controls, whereas some others were lower. In conclusion, we provide new evidence that certain circulating miRNAs may be used as biomarkers for three types of rare muscular dystrophies.

Effect of aerobic and resistance exercise training on late-onset Pompe disease patients receiving enzyme replacement therapy.

Pompe disease is a rare autosomal recessive disorder characterized by the deficiency of acid α-glycosidase resulting in lysosomal accumulation of glycogen. The late-onset disease form is characterized by progressive skeletal and respiratory muscle dysfunction. In addition to the recently introduced enzyme replacement therapy (ERT), treatments such as protein-enriched diet and exercise training have been proposed, although little is known about their effectiveness on the physical condition of such patients. Aim of the present study was to investigate the effect of exercise training on muscular strength and body composition in five patients with late-onset Pompe disease receiving ERT. All subjects followed a 20 week lasting program of supervised aerobic and progressive resistance exercise training. Before and after the training period, body composition was determined with dual X-ray absorptiometry and isometric muscular strength was measured with a specialized load transducer. Functional capacity was assessed using the 6-min shuttle walk test. A significant increase in muscular strength (15-50% at various body parts, p<0.05) and 6-minute walking distance (203.8 ± 177 m before vs. 248.2 ± 184 m after, p<0.01) was observed after training, whereas total and lower extremities lean body mass did not change significantly. These results suggest that exercise training has a positive effect on muscular strength and functional capacity in patients on ERT with late-onset Pompe disease.

The Relevance of Blepharoptosis in Diagnostic Suspicion of Myopathies.

Blepharoptosis (ptosis) is classified, based on etiology, into mechanical, cerebral, neurogenic, neuromuscular, myogenic, and due to miscellaneous causes. Primary myopathic diseases are rare causes of blepharoptosis and many patients with myogenic ptosis undergo a series of extensive investigations before a myopathy is being considered. In this study, we report four patients with different myopathic disorders who had blepharoptosis as a presenting symptom of their disease. Moreover, we highlight frequent diagnostic errors and difficulties in patients with myopathies who present blepharoptosis. Lack of clear cut aggravation of symptoms by fatigue and response to cholinesterase inhibitors treatment, the association of proximal, distal or extraocular muscle weakness, and positive family history or evidence of a multi systemic disorder should prompt evaluation of an underlying myopathy.

Effect of long term enzyme replacement therapy in late onset Pompe disease: A single-centre experience.

Late onset Pompe disease (LOPD) is a slowly progressive metabolic myopathy with variable clinical severity. The advent of enzyme replacement therapy (ERT) has modified the natural course of the disease, though the treatment effect on adult patients is modest compared to infants with the classic form. This study aims to describe the long-term clinical outcome of the Greek LOPD cohort, as assessed by 6 min walk test, muscle strength using MRC grading scale and spirometry. ERT efficacy was estimated using statistical methodology that is novel in the context of Pompe disease, which at the same time is well-suited to longitudinal studies with small samples and missing data (local non-linear regression analysis). Improvement over baseline was significant at 1 year for motor performance and muscle strength (p < 0.05), and at 2 years for FVC-U and FVC-S (p < 0.05). A subgroup analysis showed that the onset of the disease before adulthood (18 years), a male gender, and a latency of more than 2 years between the onset of symptoms and ERT administration are unfavorable prognostic factors. Conclusively, this study presents longitudinal data from the Greek LOPD cohort supporting previous observations, that therapeutic delay is related to worse prognosis and treatment effects may decline after several years of ERT.

miR-223-3p and miR-24-3p as novel serum-based biomarkers for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients' care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation.

Mild myopathic phenotype in a patient with homozygous c.416C > T mutation in TK2 gene.

The mitochondrial DNA depletion syndrome (MDDS) is characterized by extensive phenotypic variability and is due to nuclear gene mutations resulting in reduced mtDNA copy number. Thymidine kinase 2 (TK2) mutations are well known to be associated with MDDS. Few severely affected cases carrying the c.416C > T mutation in TK2 gene have been described so far. We describe the case of a 14months boy with the aforementioned TK2 gene pathogenic mutation at a homozygous state, presenting with a mild clinical phenotype. In addition to severe mitochondrial pathology on muscle biopsy, there was also histochemical evidence of adenylate deaminase deficiency. Overall, this report serves to further expand the clinical spectrum of TK2 mutations associated with MDDS.

The multifaceted clinical presentation of VCP-proteinopathy in a Greek family.

VCP-proteinopathy is a multisystem neurodegenerative disorder caused by mutations in valosin containing protein. Here, we report the first Greek case of VCP-proteinopathy in a 62 year old patient with a slowly progressing muscular weakness since his mid-40s and a severe deterioration during the last year. He also manifested dementia with prominent neuropsychiatric symptoms, including aggression, apathy, palilalia and obsessions. Brain MRI revealed frontal atrophy, while muscle MRI showed diffuse muscle atrophy. Family history was positive and several members of the family had been diagnosed with motor neuron disease, dementia or behavioral symptoms. Sequencing of the VCP gene revealed a pathogenic heterozygous missense mutation p.R159H. Conclusively, the present report highlights the intrafamilial variability and broadens the phenotypic spectrum of VCP-proteinopathy.