Myosin post-translational modifications and function in the presence of myopathy-linked truncating MYH2 mutations.

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1) MyHCs presence/post-translational modifications using LC/MS; 2) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3) myosin activation with an unloaded in vitro motility assay; and 4) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2) faster actomyosin kinetics; and 3) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.

COVID-19-associated Guillain-Barré syndrome in the early pandemic experience in Lombardia (Italy).

OBJECTIVE: To estimate the incidence and describe clinical characteristics and outcome of GBS in COVID-19 patients (COVID19-GBS) in one of the most hit regions during the first pandemic wave, Lombardia. METHODS: Adult patients admitted to 20 Neurological Units between 1/3-30/4/2020 with COVID19-GBS were included as part of a multi-center study organized by the Italian society of Hospital Neuroscience (SNO). RESULTS: Thirty-eight COVID19-GBS patients had a mean age of 60.7 years and male frequency of 86.8%. CSF albuminocytological dissociation was detected in 71.4%, and PCR for SARS-CoV-2 was negative in 19 tested patients. Based on neurophysiology, 81.8% of patients had a diagnosis of AIDP, 12.1% of AMSAN, and 6.1% of AMAN. The course was favorable in 76.3% of patients, stable in 10.5%, while 13.2% worsened, of which 3 died. The estimated occurrence rate in Lombardia ranges from 0.5 to 0.05 GBS cases per 1000 COVID-19 infections depending on whether you consider positive cases or estimated seropositive cases. When we compared GBS cases with the pre-pandemic period, we found a reduction of cases from 165 to 135 cases in the 2-month study period in Lombardia. CONCLUSIONS: We detected an increased incidence of GBS in COVID-19 patients which can reflect a higher risk of GBS in COVID-19 patients and a reduction of GBS events during the pandemic period possibly due to a lower spread of more common respiratory infectious diseases determined by an increased use of preventive measures.

ATPase Domain AFG3L2 Mutations Alter OPA1 Processing and Cause Optic Neuropathy.

OBJECTIVE: Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed. METHODS: We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts. RESULTS: Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells. INTERPRETATION: This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.

p.Asn1180Ile mutation of SCN4A gene in an Italian family with myopathy and myotonic syndrome.

INTRODUCTION: Mutations of the skeletal muscle sodium channel gene SCN4A are associated with several neuromuscular disorders including hyper/hypokaliemic periodic paralysis, paramyotonia congenita and sodium channel myotonia. These disorders are distinguished from dystrophic myotonias by the absence of progressive weakness and extramuscular systemic involvement. METHODS: We present an Italian family with 2 subjects carrying a p.Asn1180Ile mutation in SCN4A gene showing a peculiar clinical picture characterized by the association of myopathic features and myotonia. RESULTS: The clinical, electromyographic and histological findings of these patients are reported. The possible pathogenicity of the mutation was tested by three different software, all giving positive results. DISCUSSION: This is the first report of a dominant, heterozygous mutation in SCN4A causing a complex phenotype of non-congenital myopathy and myotonic syndrome. We suggest that, in patients with myotonia and myopathy not related to dystrophic myotonias, the sequence analysis of SCN4A gene should be performed.

Mitochondrial epilepsy: a cross-sectional nationwide Italian survey.

Many aspects of epilepsy in mitochondrial disorders (MDs) need to be further clarified. To this aim, we explored retrospectively a cohort of individuals with MDs querying the "Nationwide Italian Collaborative Network of Mitochondrial Diseases" (NICNMD) database (1467 patients included since 2010 to December 2016). We collected information on age at epilepsy onset, seizure type and frequency, genetic findings, and antiepileptic drugs (AEDs). At the time of our survey, 147/1467 (10%) patients in the NICNMD database had epilepsy. Complete information was available only for 98 patients, 52 males and 46 females, aged 5-92 years (mean age 40.4 ± 18.4; 14/98 children/teenagers and 84 adults). Epilepsy was the presenting feature of MD in 46/98 (47%) individuals, with onset at a median age of 19 years (range, 0.2-68; < 3 years in 14/97 (14%), 3-19 years in 36/97 (37%), > 19 years in 47/97 (49%)). Moreover, 91/98 patients (93%) displayed multiple seizures, with daily or weekly frequency in 25/91 (28%). Interictal EEG was abnormal in 70/78 (90%) patients, displaying abnormal background (47/70; 67%) and/or interictal paroxysms (53/70; 76%). Eighty of 90 patients (89%) displayed a 50-100% reduction of seizures on AEDs; levetiracetam was the most commonly used. Forty-one patients (42%) carried the m.3243A>G mutation, 16 (16%) the m.8344A>G, and 9 (9%) nuclear DNA (nDNA) mutations. Individuals with early-onset seizures mainly carried nDNA mutations and had a more severe epilepsy phenotype, higher seizure frequency, and disorganized background EEG activity. A better definition of epilepsy in MDs may foster the diagnostic workup, management, and treatment of affected patients, and allow more homogeneous patient stratification.

Homozygous variant in OTX2 and possible genetic modifiers identified in a patient with combined pituitary hormone deficiency, ocular involvement, myopathy, ataxia, and mitochondrial impairment.

Here we report on a singleton patient affected by a complicated congenital syndrome characterized by growth delay, retinal dystrophy, sensorineural deafness, myopathy, ataxia, combined pituitary hormone deficiency, associated with mitochondrial impairment. Targeted clinical exome sequencing led to the identification of a homozygous missense variant in OTX2. Since only dominant mutations within OTX2 have been associated with cases of syndromic microphthalmia, retinal dystrophy with or without pituitary dysfunctions, this represents the first report of an OTX2 recessive mutation. Part of the phenotype, including ataxia, myopathy and multiple mitochondrial respiratory chain defects, seemed not related to OTX2. Further analysis of next generation sequencing (NGS) data revealed additional candidate variants: a homozygous variant in LETM1, and heterozygous rare variants in AFG3L2 and POLG. All three genes encode mitochondrial proteins and the last two are known to be associated with ataxia, a neurological sign present also in the father of the proband. With our study, we aim to encourage the integration of NGS data with a detailed analysis of clinical description and family history in order to unravel composite genotypes sometimes associated with complicated phenotypes.

Value of structured reporting in neuromuscular disorders.

OBJECTIVE: To assess whether structured reports (SRs) of MRI in patients with inherited neuromuscular disorders (IND) provide more clinically relevant information than non-structured reports (NSRs) and whether neuroradiologists' expertise affects completeness of reports. MATERIAL AND METHODS: Lower limbs' MRI reports of patients with IND produced by neuroradiologists with different level of expertise (> 15 years vs. < 15 years of experience in reading IND-MRI) before and after implementation of a SR template were included. Reports were assessed for the presence of 9 key features relevant for IND management. Reports and images were evaluated by neurologists who assessed: disease-specific muscular involvement pattern; presence of sufficient information to order the appropriate genetic/diagnostic tests; presence of sufficient information to make therapeutic decision/perform biopsy and necessity to review MRI images. Mann-Whitney and Fisher's exact tests were used to compare the number of key features for NSR and SR and neurologists' answers for reports produced by neuroradiologists with different experience. RESULTS: Thirty-one SRs and 101 NSRs were reviewed. A median of 8 and 6 key features was present in SR and NSR, respectively (p value < 0.0001). When reports were produced by less expert neuroradiologists, neurologists recognized muscular involvement pattern, had sufficient information for clinical decision-making/perform biopsy more often with SR than NSR (p values: < 0.0001), and needed to evaluate images less often with SR (p value: 0.0001). When reports produced by expert neuroradiologists were evaluated, no significant difference in neurologists' answers was observed. CONCLUSION: SR of IND-MRI contained more often clinically relevant information considered important for disease management than NSR. Radiologist's expertise affects completeness of NSR reports.

Mutations in DNA2 link progressive myopathy to mitochondrial DNA instability.

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.

Mitochondrial myopathy with dystrophic features due to a novel mutation in the MTTM gene.

INTRODUCTION: A 61-year-old woman with a 5-year history of progressive muscle weakness and atrophy had a muscle biopsy characterized by a combination of dystrophic features (necrotic fibers and endomysial fibrosis) and mitochondrial alterations [ragged-red, cytochrome c oxidase (COX)-negative fibers]. METHODS: Sequencing of the whole mtDNA, assessment of the mutation load in muscle and accessible nonmuscle tissues, and single fiber polymerase chain reaction. RESULTS: Muscle mitochondrial DNA (mtDNA) sequencing revealed a novel heteroplasmic mutation (m.4403G>A) in the gene (MTTM) that encodes tRNA(Met). The mutation was not present in accessible nonmuscle tissues from the patient or 2 asymptomatic sisters. CONCLUSIONS: The clinical features and muscle morphology in this patient are very similar to those described in a previous patient with a different mutation, also in MTTM, which suggests that mutations in this gene confer a distinctive "dystrophic" quality. This may be a diagnostic clue in patients with isolated mitochondrial myopathy.

Mitochondrial disease heterogeneity: a prognostic challenge.

Mitochondrial diseases are a heterogeneous group of progressive, genetically transmitted, multisystem disorders caused by impaired mitochondrial function. The disease course for individuals with mitochondrial myopathies varies greatly from patient to patient because disease progression largely depends on the type of disease and on the degree of involvement of various organs which makes the prognosis unpredictable both within the same family and among families with the same mutation. This is particularly, but not exclusively, true for mitochondrial disorders caused by mtDNA point mutations, which are maternally inherited and subject to the randomness of the heteroplasmy. For this reason, the prognosis cannot be given by single mitochondrial disease, but should be formulated by any single mitochondrial disease-related event or complication keeping in mind that early recognition and treatment of symptoms are crucial for the prognosis. The following approach can help prevent severe organ dysfunctions or at least allow early diagnosis and treatment of disease-related complications.

NGS-Based Genetic Analysis in a Cohort of Italian Patients with Suspected Inherited Myopathies and/or HyperCKemia.

Introduction/Aims HyperCKemia is considered a hallmark of neuromuscular diseases. It can be either isolated or associated with cramps, myalgia, weakness, myoglobinuria, or rhabdomyolysis, suggesting a metabolic myopathy. The aim of this work was to investigate possible genetic causes in order to help diagnose patients with recurrent hyperCKemia or clinical suspicion of inherited metabolic myopathy. Methods A cohort of 139 patients (90 adults and 49 children) was analyzed using a custom panel containing 54 genes associated with hyperCKemia. Results A definite genetic diagnosis was obtained in 15.1% of cases, while candidate variants or variants of uncertain significance were found in a further 39.5%. Similar percentages were obtained in patients with infantile or adult onset, with some different causative genes. RYR1 was the gene most frequently identified, either with single or compound heterozygous variants, while ETFDH variants were the most common cause for recessive cases. In one patient, mRNA analysis allowed identifying a large LPIN1 deletion missed by DNA sequencing, leading to a certain diagnosis. Conclusion These data confirm the high genetic heterogeneity of hyperCKemia and metabolic myopathies. The reduced diagnostic yield suggests the existence of additional genes associated with this condition but also allows speculation that a significant number of cases presenting with hyperCKemia or muscle symptoms are due to extrinsic, not genetic, factors.

Sex differences in ischemic stroke during COVID-19 first outbreak in northern Italy.

INTRODUCTION: COVID-19 pandemic had a great impact on outcome in SARS-CoV-2 positive patients with ischemic stroke during the first wave in Italy. Few data are available on outcome stratified by sex. METHODS: The Italian Society of Hospital Neuroscience conducted a multi-center, retrospective, observational study on neurological complications in COVID-19 patients with ischemic stroke. All the patients admitted from March 1st to April 30th, 2020 in 20 Neurology Units in Northern Italy were recruited. Demographical and clinical features, treatment and outcome data were compared focusing on sex differences. RESULTS: 812 patients with ischemic stroke were enrolled, of whom 129 with COVID-19; males were 53.8%. In-hospital mortality in COVID-19 patients was 35.3% in males and 27.9% in females while 8.5% in male and 5.8% in female patients without COVID-19. SARS-CoV-2 positive patients had a higher frequency of stroke of undetermined etiology, than negative ones (32.8% vs 22.5%; p = 0.02), especially in females compared to males (36.1% vs 27.9%), albeit without statistical significance. Male patients with SARS-CoV-2 were more likely to require cPAP (30.9% vs 14.8%; p = 0.03), endotracheal tube (14.9% vs 3.3%; p = 0.02) and reperfusion strategies (29.4% vs 11.5%; p = 0.01) than females, as well as to have a higher CRP and D-dimer. These elements together with older age, a total anterior circulation stroke and lymphopenia were predictors of a worse outcome. DISCUSSION: Our study detected some differences due to sex in ischemic stroke with and without COVID-19, supporting the possibility to perform sex analyses for SARS-CoV-2 positive patients for a better clinical management.

Steroid-responsive Hashimoto encephalopathy mimicking Creutzfeldt-Jakob disease.

Hashimoto's encephalopathy (HE) is a rare neurological disorder with a heterogeneous group of neurological symptoms associated with high titres of anti-thyroid antibodies. Clinical manifestations may include encephalopathic features such as seizures, behavioural and psychiatric manifestations, movement disorders and coma. The objective of this presentation is to describe a patient with this rare and controversial clinical syndrome mimicking Creutzfeldt-Jakob disease, associated with a Hashimoto euthyroid thyroiditis and with a significant response to high dose intravenous prednisone. The responsiveness of this syndrome to steroids suggests that this disorder involves immune pathogenic mechanisms, as previous reviews reported.

Leber's Hereditary Optic Neuropathy: A Report on Novel mtDNA Pathogenic Variants.

Leber's hereditary optic neuropathy (LHON) is due to missense point mutations affecting mitochondrial DNA (mtDNA); 90% of cases harbor the m.3460G>A, m.11778G>A, and m.14484T>C primary mutations. Here, we report and discuss five families with patients affected by symptomatic LHON, in which we found five novel mtDNA variants. Remarkably, these mtDNA variants are located in complex I genes, though without strong deleterious effect on respiration in cellular models: this finding is likely linked to the tissue specificity of LHON. This study observes that in the case of a strong clinical suspicion of LHON, it is recommended to analyze the whole mtDNA sequence, since new rare mtDNA pathogenic variants causing LHON are increasingly identified.

Impact of SARS-CoV-2 infection on acute intracerebral haemorrhage in northern Italy.

INTRODUCTION: Growing evidence has been published as to the impact of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) on cerebrovascular events over the last few months, with considerable attention paid to ischemic strokes. Conversely, little is known about the clinical course of intracerebral haemorrhage (ICH) and simultaneous SARS-CoV-2 infection. METHOD: The Italian Society of Hospital Neurosciences (SNO) promoted a multicentre, retrospective, observational study (SNO-COVID-19), involving 20 Neurological Departments in Northern Italy. Clinical data on patients with acute cerebrovascular diseases, admitted from March 1st to April 30th, 2020, were collected. A comparison was made of the demographical and clinical features of both SARS-CoV-2 positive and negative patients with ICH. RESULTS: 949 patients were enrolled (average age 73.4 years; 52.7% males); 135 patients had haemorrhagic stroke and 127 (13.4%) had a primary ICH. Only 16 patients with ICH (12.6%) had laboratory confirmed SARS-CoV-2 infection, both symptomatic and asymptomatic. SARS-CoV-2 related pneumonia or respiratory distress (OR 5.4), lobar location (OR 5.0) and previous antiplatelet or anticoagulant treatment (OR 2.9) were the only factors significantly associated with increased mortality in ICH. SARS-CoV-2 infection, regardless of respiratory involvement, led to a non-significantly increased risk of in-hospital death (37.5% vs 23.4%, p = 0.2). DISCUSSION: ICH patients with COVID-19 did not experience an increase in mortality as striking as ischemic stroke. The inflammatory response and respiratory complications could justify the slight increase of death in ICH. Bleeding sites and previous antiplatelet or anticoagulant treatment were the only other predictors of a worse outcome.

MYH2 myopathy, a new case expands the clinical and pathological spectrum of the recessive form.

BACKGROUND: Hereditary myosin myopathies are a group of rare muscle disorders, caused by mutations in genes encoding for skeletal myosin heavy chains (MyHCs). MyHCIIa is encoded by MYH2 and is expressed in fast type 2A and 2B muscle fibers. MYH2 mutations are responsible for an autosomal dominant (AD) progressive myopathy, characterized by the presence of rimmed vacuoles and by a reduction in the number and size of type 2A fibers, and a recessive early onset myopathy characterized by complete loss of type 2A fibers. Recently, a patient with a homozygous mutation but presenting a dominant phenotype has been reported. METHODS: The patient was examined thoroughly and two muscle biopsies were performed through the years. NGS followed by confirmation in Sanger sequencing was used to identify the genetic cause. RESULTS: We describe the second case presenting with late-onset ophthalmoparesis, ptosis, diffuse muscle weakness, and histopathological features typical for AD forms but with a recessive MYH2 genotype. CONCLUSION: This report contributes to expand the clinical and genetic spectrum of MYH2 myopathies and to increase the awareness of these very rare diseases.