Neuromuscular disease genetics in under-represented populations: increasing data diversity.

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% 'solved' and ∼13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.

An ultrastructural and genomic study on the SARS-CoV-2 variant B.1.210 circulating during the first wave of COVID-19 pandemic in India.

PURPOSE: The study aims to isolate and understand cytopathogenesis, ultrastructure, genomic characteristics and phylogenetic analysis of SARS-CoV-2 virus of B.1.210 lineage, that circulated in India during first wave of the pandemic. METHODS: Clinical specimen from an interstate traveller from Maharashtra to Karnataka, in May 2020, who was positive by RT PCR for SARS-CoV-2 infection was subjected to virus isolation and Whole Genome Sequencing. Vero cells were used to study cytopathogenesis and ultrastructural features by Transmission Electron Microscopy (TEM). Phylogenetic analysis of the whole genome sequences of several SARS-CoV-2 variants downloaded from GISAID was performed in comparison with the B.1.210 variant identified in this study. RESULTS: The virus was isolated in Vero cells and identified by immunofluorescence assay and RT PCR. The growth kinetics in infected Vero cells revealed a peak viral titre at 24 â€‹h post-infection. Ultrastructural studies revealed distinct morphological changes with accumulation of membrane-bound vesicles containing pleomorphic virions in the cytoplasm, with single or multiple intranuclear filamentous inclusions and dilated rough endoplasmic reticulum with viral particles. Whole genome sequence of the clinical specimen as well as the isolated virus revealed the virus to be of lineage B.1.210 with the D614G mutation in the spike protein. Phylogenetic analysis of the whole genome sequence in comparison with other variants reported globally revealed that the isolated SARS-CoV-2 virus of lineage B.1.210 is closely related to the original Wuhan virus reference sequence. CONCLUSIONS: The SARS-CoV-2 variant B.1.210 virus isolated here showed ultrastructural features and cytopathogenesis similar to that of the virus reported during early phase of pandemic. Phylogenetic analysis showed that the isolated virus is closely related to the original Wuhan virus, thereby suggesting that the SARS-CoV-2 lineage B.1.210 that was circulating in India during the early phase of pandemic is likely to have evolved from the original Wuhan strain.

MLIP-Associated Myopathy: A Case Report and Review of the Literature.

BACKGROUND: Muscular A-type lamin-interacting protein (MLIP) has a regulatory role in myoblast differentiation and organization of myonuclear positioning in skeletal muscle. It is ubiquitously expressed but abundantly in cardiac, skeletal, and smooth muscles. Recently, two studies confirmed the causation of biallelic pathogenic variants in the MLIP gene of a novel myopathy phenotype. OBJECTIVE: Description of the phenotypic spectrum and features of MLIP-related myopathy. METHODS: report a patient with biallelic variants in MLIP gene with the clinical features, and histomorphological findings of MLIP-related myopathy and provide a literature review of the previously reported 12 patients. RESULTS: MLIP-related myopathy is characterized by episodes of rhabdomyolysis, myalgia triggered by mild to moderate exercise, mild muscle weakness, and sometimes cardiac involvement characterized by cardiomyopathy and cardiac rhythm abnormalities. CONCLUSIONS: This report reviews and extends the clinical features of a novel myopathy caused by biallelic pathogenic variants in the MLIP gene.

Basic requirements to establish a neuromuscular laboratory.

Histopathological analysis of muscle biopsy is a prerequisite in the evaluation of neuromuscular disorders, particularly inflammatory myopathies, metabolic myopathies, congenital myopathies, muscular dystrophies and differentiating myopathies and neurogenic disorders with overlapping clinically features. It not only provides useful information that helps in the diagnosis but also treatment and management. Fundamental skills and basic knowledge regarding handling, processing and analyzing a muscle biopsy are required in any specialized or a general pathology lab supporting neuromuscular clinical services. Care during transport of the muscle biopsy, sample receipt in the laboratory and grossing is very important. Standard operating procedure should be followed for the preanalytical steps (freezing and cryomicrotomy), routine and special staining (enzyme and non enzymatic) and immunohistochemistry. A well organized neuromuscular laboratory with good quality management system is necessary for the practice of myopathology. This article gives an overview of establishing such a laboratory.

Approach to the diagnosis of metabolic myopathies.

Metabolic myopathies are a diverse group of genetic disorders that result in impaired energy production. They are individually rare and several have received the 'orphan disorder' status. However, collectively they constitute a relatively common group of disorders that affect not only the skeletal muscle but also the heart, liver, and brain among others. Mitochondrial disorders, with a frequency of 1/8000 population, are the commonest cause of metabolic myopathies. Three main groups that cause metabolic myopathy are glycogen storage disorders (GSD), fatty acid oxidation defects (FAOD), and mitochondrial myopathies. Clinically, patients present with varied ages at onset and neuromuscular features. While newborns and infants typically present with hypotonia and multisystem involvement chiefly affecting the liver, heart, kidney, and brain, patients with onset later in life present with exercise intolerance with or without progressive muscle weakness and myoglobinuria. In general, GSDs result in high-intensity exercise intolerance while, FAODs, and mitochondrial myopathies predominantly manifest during endurance-type activity, fasting, or metabolically stressful conditions. Evaluation of these patients comprises a meticulous clinical examination and a battery of investigations which includes- exercise stress testing, metabolic and biochemical screening, electrophysiological studies, neuro-imaging, muscle biopsy, and molecular genetics. Accurate and early detection of metabolic myopathies allows timely counseling to prevent metabolic crises and helps in therapeutic interventions. This review summarizes the clinical features, diagnostic tests, pathological features, treatment and presents an algorithm to diagnose these three main groups of disorders.

Electron microscopy in the diagnosis of skeletal muscle disorders: Its utility and limitations.

Electron microscopy (EM) has a substantial role in the diagnosis of skeletal muscle disorders. The ultrastructural changes can be observed in muscle fibers and other components of the muscle tissue. EM serves as a confirmatory tool where the diagnosis is already established by enzyme histochemistry staining. Although it is indispensable in the diagnosis of rare forms of congenital myopathies not appreciated by light microscope, such as cylindrical spiral myopathy, zebra body myopathy, fingerprint body myopathy, and intranuclear rod myopathy, in cases not subjected to histochemical staining, it is required for definitive diagnosis in certain groups of muscle disorders, which includes congenital myopathies, metabolic myopathies in particular mitochondrial myopathies and glycogenosis, and in vacuolar myopathies. It does not have diagnostic implications in muscular dystrophies and neurogenic disorders. In the recent past, despite the availability of advanced diagnostic techniques, electron microscopy continues to play a vital role in the diagnosis of skeletal muscle disorders. This review gives an account of ultrastructural features of skeletal muscle disorders, the role of EM in the diagnosis, and its limitations.

Neuronal Ceroid Lipofuscinosis: Clinical and Laboratory Profile in Children from Tertiary Care Centre in South India.

Neuronal ceroid Lipofuscinosis (NCL), inherited disorders of lysosomal storage disorders, constitute the most common progressive encephalopathies with an incidence of 1.3 to 7 in 100,000 live births. We reported clinical, electrophysiological, radiological, ultrastructural, and molecular genetic features of NCL. This is a retrospective review, in a tertiary care center from January 2016 to December 2019. All children with clinical features of NCL and confirmed by pathogenic mutation and/or enzyme assay were included. A total of 60 children (male:female = 3:1) were studied. The commonest type was CLN 2 (41.7%). Neuroregression, seizures, and ataxia were present in all cases. Retinal arterial attenuation was seen in 38.33% cases. Magnetic resonance imaging (MRI) brain was abnormal in all patients, thalamic and caudate nucleus atrophy common in CLN1 (62%). Electroencephalography was abnormal in all children, but photoparoxysmal response at low intermittent photic stimulation frequencies was seen in four children of CLN2. Electron microscopy done in 43 children revealed abnormal inclusions in 20 (46.52%) children. Enzyme study showed low levels in 36 (78%) out of 46 cases. Of these, 21 had low tripeptidyl peptidase and 15 had low palmitoyl protein thioesterase levels. Molecular testing done in 26 cases showed pathogenic variant in 23 (88%) cases. Infantile onset with thalamic atrophy on MRI is common in CLN1 and refractory epilepsy, visual impairment and specific EEG changes are common in CLN2. These features are helpful in selecting enzyme assay for CLN1 versus CLN2. Electron microscopy helped in the diagnosis and genetic testing in subtyping. Thus, a multimode approach played a role in the diagnosis of NCL.

Serum fibroblast growth factor 21 and growth differentiation factor 15: Two sensitive biomarkers in the diagnosis of mitochondrial disorders.

Mitochondrial disorders are often difficult to diagnose because of diverse clinical phenotypes. FGF-21 and GDF-15 are metabolic hormones and promising biomarkers for the diagnosis of these disorders. This study has systematically evaluated serum FGF-21 and GDF-15 levels by ELISA in a well-defined cohort of patients with definite mitochondrial disorders (n = 30), neuromuscular disease controls (n = 36) and healthy controls (n = 36) and aimed to ascertain their utility in the diagnosis of mitochondrial disorders. Both serum FGF-21 and GDF-15 were significantly elevated in patients with mitochondrial disorders, especially in those with muscle involvement. The levels were higher in patients with mitochondrial deletions (both single and multiple) and translation disorders compared to respiratory chain subunit or assembly factor defects.

Utility of Immunohistochemistry and Western Blot in Profiling Clinically Suspected Cases of Congenital Muscular Dystrophy.

OBJECTIVE: Immunocharacterization of congenital muscular dystrophy (CMD) to determine the frequency of various subtypes in a large Indian Cohort. MATERIALS AND METHODS: This retrospective (2014-2017) study was carried on muscle biopsies of clinically suspected cases of CMD with histological evidence of dystrophy/myopathic features. Immunohistochemistry (IHC) to antibodies against laminin (α2, α5,ß1,γ1), Collagen-VI (A1,2,3), and Western blot (WB) for α-dystroglycan and POMT1 was performed. RESULTS: The study included 57 cases, of which 15 cases (26.3%) had mean age at presentation of 3.5 years, M: F = 1.5:1, elevated creatinine kinase (CK) (mean 1657 U/L), global developmental delay, multiple contractures, abnormal facies, white matter hyperintensities and showed laminin-α2 deficiency (Merosin deficient CMD). In addition, secondary reduction in laminin-ß1, over-expression of laminin-α5, and preserved laminin-γ1 was noted. Ullrich CMD constituted 11/57 cases (19.2%) with mean age at presentation of 5.3 years, M: F = 1.2:1 and normal CK. They presented with proximal muscle weakness, soft velvety palms and soles, contractures, and joint hyperextensibility. Collagen-VI (A1,2,3) showed either complete (n = 3) or sarcolemmal specific (n = 8) loss of staining. Out of the remaining 31 cases, WB for α-dystroglycan was performed in 17 cases which showed deficiency in seven (12.3%). Three of these in addition revealed secondary partial loss of laminin-α2. WB for POMT1 showed deficiency in a single case clinically diagnosed Walker-Warburg syndrome, who presented with seizures and classical features of pachygyria, lissencephaly, and cerebellar cyst on MRI. Twenty-four cases (42.2%) remained uncharacterized and need genetic evaluation. CONCLUSION: The study helped in characterizing 57.8% of the proband. Immunotyping helps to direct mutational analysis for targeted genes and offers a potential route for prenatal diagnosis.

A Novel L1 Linker Mutation in DES Resulted in Total Absence of Protein.

Desminopathies (MIM*601419) are clinically heterogeneous, manifesting with myopathy and/or cardiomyopathy and with intra-sarcoplasmic desmin-positive deposits. They have either an autosomal dominant (AD) or recessive (AR) pattern of inheritance. Desmin is a crucial intermediate filament protein regulating various cellular functions in muscle cells. Here, we report a 13-year-old girl, born of second-degree consanguineous parents, with normal developmental milestones, who presented with dilated cardiomyopathy, respiratory insufficiency and predominant distal upper limb weakness. A striking feature on muscle biopsy was the presence of a peripheral chain of nuclei in addition to myopathic features. Immunostaining showed complete lack of desmin expression, further confirmed by western blot analysis. Ultrastructurally, subsarcolemmal granular material, expanded Z-band aggregation, distortion of myofilaments, focal Z-band streaming, lobed and clustered myonuclei were observed. Next-generation sequencing revealed a novel homozygous nonsense mutation c.448C>T, p.R150X in the patient, while the parents were heterozygous carriers. Single mitochondrial DNA deletion and isolated complex IV deficiency were noted. Our findings add to the ever-expanding phenotype and molecular spectrum of desminopathies.

Infective myositis.

Myositis is inflammation especially of the voluntary muscles, characterized by localized or diffuse pain, tenderness on movement or palpation, swelling, and/or weakness. The two main categories of myositis include non-infectious and infectious. Infective myositis may be due to a wide variety of pathogens, including bacteria, fungi, viruses, and parasites. A brief account of the various pathogens causing infective myositis is discussed.

A Dominant C150Y Mutation in FHL1 Induces Structural Alterations in LIM2 Domain Causing Protein Aggregation In Human and Drosophila Indirect Flight Muscles.

FHL1-related myopathies are rare X-linked dominant myopathies. Though clinically classified into several subgroups, spinal and scapuloperoneal muscle involvement are common to all. In this study, we identified c.449G > A, p.C150Y mutation by clinical exome sequencing in two patients from same family (son and mother) of Indian origin who presented with multiple contractures. Muscle biopsy showed numerous intracytoplasmic aggregates intensely stained on HE and MGT. The strong reactions to M-NBT revealed aggregates to be reducing bodies and positively labeled to anti-FHL1 antibody. Ultrastructurally, Z-band streaming and granular and granulofilamentous material were seen. Further, the translational evidence of mutant peptide was confirmed using mass spectrometric analysis. To establish p.C150Y as the cause for protein aggregation, in vivo studies were carried out using transgenic Drosophila model which highlighted Z-band abnormalities and protein aggregates in indirect flight muscles with compromised physiological function. Thus, recapitulating the X-linked human disease phenotype. Additionally, the molecular dynamics simulation analysis unraveled the drastic change in α-helix of LIM2, the region immediately next to site of C150Y mutation that could be the plausible cause for protein aggregation. To the best of our knowledge, this is the first study of p.C150Y mutation in FHL1 identified in Indian patients with in vivo and in silico analysis to establish the cause for protein aggregation in muscle.

Myelopathy in Two Brothers with Respiratory Chain Disorder-Severe Complex 1 Deficiency with Atlantoaxial Dislocation and Long Spinal Arachnoid Cyst: A New Unreported Association.

Billion of years ago bacteria is believed to have entered a eukaryotic cell and converted to mitochondria. The respiratory chain present in it supplies ATP to all cells and therefore the diseases can have any phenotype. Diagnosis may be difficult to confirm by the conventional methods including genetics. Two brothers born to consanguineous parents had global delay, mild visual impairment and drooping of eyelids since birth in the elder child and dysmorphism in the second one. Both had progressive myelopathy due to retroflexed odontoid ,large arachnoid cyst and tethered cord. Muscle biopsy with special stain was normal but respiratory chain assay revealed severe complex 1 deficiency.Elder child underwent surgical decompression of the arachnoid cyst with duroplasty with significant improvement.Atlanto axial dislocation and large arachnoid cyst as cause of myelopathy is not reported in literature in patients with respiratory chain disorders to the best of our knowledge.

Macrophagic Myofasciitis: A Report of Two South Indian Infants.

Macrophagic myofasciitis is a rare inflammatory myopathy characterized by peri-fascicular macrophage infiltration without muscle necrosis. Here we report two children presented in the early infancy. Case 1: a 5-month-old girl presented with lack of neck control and floppiness. On examination, generalized hypotonia, absent deep tendon reflexes, and motor power of 2/5 (Medical Research Council grade) were observed. Case 2: a 17-day-old boy presented with poor feeding, tachypnea, and floppiness. On examination, decreased tone in all limbs and power of <2/5 in all limbs with absent reflexes were observed. Routine investigations including serum Creatine phosphokinase of both babies were normal. Muscle biopsy showed features of macrophagic myofasciitis in both infants. Any floppy infant of lower motor neuron type macrophagic myofasciitis should be considered in addition to inherited causes.

Cerebral small vessel disease with hemorrhagic stroke related to COL4A1 mutation: A case report.

Stroke is a major cause of mortality and morbidity with a wide variety of etiological risk factors. Cerebral small vessel disease (SVD) is an important cause of stroke in the young with several hereditary disorders affecting these small blood vessels. Mutations in the COL4A1 gene (COL4A1) have been shown to be associated with a broad range of disorders including hemorrhagic stroke, myopathy, glaucoma and others. We report a rare case of stroke in an intellectually disabled 18-year-old girl with radiological evidence of basal ganglia microbleeds, periventricular white matter signal changes and porencephalic cyst. Ophthalmic examination revealed bilateral microcornea and Axenfeld-Rieger anomaly. At autopsy there were hemorrhagic lesions at multiple sites within the brain. Histology revealed thickened small-caliber vessels which demonstrated disruption and fragmentation of the basement membrane by collagen type IV alpha 1 immunohistochemistry and by electron microscopy. A missense COL4A1 mutation involving glycine residue was detected in the patient. The present case illustrates the clinicopathological spectrum of COL4A1-related cerebral SVD presenting as hemorrhagic stroke in the young with porencephaly, intellectual disability, and Axenfield-Rieger anomaly and thus adds to the clinical heterogeneity of this genetic disorder.

X-Linked Myopathy with Excessive Autophagy; A Case Report.

X-linked myopathy with excessive autophagy (XMEA) is a rare, slowly progressive muscle disease characterized by membrane-bound sarcoplasmic vacuoles distinct from other forms of myopathies with vacuoles. We report this rare condition in a 5-year-old boy with proximal muscle weakness and morphological evidence of autophagic vacuoles.

Lipid Storage Myopathy with Ketonuria: A Case of Fatty Acid Oxidation-Related Myopathy and Encephalopathy due to Multiple Acyl-CoA Dehydrogenase Deficiency.

Encephalopathy and Myopathy in children of varying ages can be due to variety of causes including Mitochondrial diseases, metabolic diseases like renal tubular acidosis, storage diseases as well as fatty acid oxidation (FAO) disorders. FAO related disorders have variable clinical presentation and manifest in different ages. They may present with hypoglycemia, effort intolerance, multi organ involvement with or without ketonuria. High degree of suspicion and appropriate investigations are mandatory for diagnosis. Here we describe an 11 Year old boy, born to non - consanguineous parents. Presented with exertion induced muscle pain and fatigue of 1year duration, which slowly progressed to severe weakness and vomiting. His reflexes were retained. Therefore metabolic vs inflammatory muscle diseases were considered. Patient had ketonuria with elevated blood levels of medium chain acyl carnitine and long chain acyl carnitine suggestive of MADD. Urine organic acid assessment showed elevated excretion of 2-hydroxyglutarate (2HG), adipate and arabitol. Muscle biopsy showed multiple fine vacuoles on Eosin- hematoxylin stained preparation. Modified Gomori - trichrome stain showed vacuolated fibers with red granular material consistent with ragged red fibers. Oil Red O stains showed vacuolated fibers with 'oil red O' positive material suggesting lipid storage. Above combination of features is consistent of MADD. Genetic evaluation is not done due to financial constraint. Patient was started on high dose riboflavin and carnitine, with which the child became near normal. Our patient is a case of MADD presenting as Reye's syndrome like features and showed excellent response to riboflavin, carnitine, dietary and life style changes. High degree of suspicion is lifesaving.

Sporadic amyotrophic lateral sclerosis (SALS) - skeletal muscle response to cerebrospinal fluid from SALS patients in a rat model.

Skeletal muscle atrophy is the most prominent feature of amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease of motor neurons. However, the contribution of skeletal muscle to disease progression remains elusive. Our previous studies have shown that intrathecal injection of cerebrospinal fluid from sporadic ALS patients (ALS-CSF) induces several degenerative changes in motor neurons and glia of neonatal rats. Here, we describe various pathologic events in the rat extensor digitorum longus muscle following intrathecal injection of ALS-CSF. Adenosine triphosphatase staining and electron microscopic (EM) analysis revealed significant atrophy and grouping of type 2 fibres in ALS-CSF-injected rats. Profound neuromuscular junction (NMJ) damage, such as fragmentation accompanied by denervation, were revealed by α-bungarotoxin immunostaining. Altered expression of key NMJ proteins, rapsyn and calpain, was also observed by immunoblotting. In addition, EM analysis showed sarcolemmal folding, Z-line streaming, structural alterations of mitochondria and dilated sarcoplasmic reticulum. The expression of trophic factors was affected, with significant downregulation of vascular endothelial growth factor (VEGF), marginal reduction in insulin-like growth factor-1 (IGF-1), and upregulation of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF). However, motor neurons might be unable to harness the enhanced levels of BDNF and GDNF, owing to impaired NMJs. We propose that ALS-CSF triggers motor neuronal degeneration, resulting in pathological changes in the skeletal muscle. Muscle damage further aggravates the motor neuronal pathology, because of the interdependency between them. This sets in a vicious cycle, leading to rapid and progressive loss of motor neurons, which could explain the relentless course of ALS.This article has an associated First Person interview with the first author of the paper.

Chikungunya Virus Interacts with Heat Shock Cognate 70 Protein to Facilitate Its Entry into Mosquito Cell Line.

AIM: The study was designed to identify putative Chikungunya virus (CHIKV) receptor/s on C6/36 cells that facilitate viral entry. METHODS: The virus overlay protein binding assay (VOPBA) was adopted to identify CHIKV-interacting bands present in C6/36 cell membrane and identity of the protein was established by mass spectrometry. The role of this protein as a putative CHIKV receptor on C6/36 cells was confirmed by infection inhibition assay. Cell surface localization of the identified protein was studied by indirect immunofluorescence assay (IFA) on nonpermeabilized cells and by flow cytometry. Interaction between this protein and CHIKV was confirmed by co-immunoprecipation (Co-IP) and Western blotting. The effect of depletion of the identified protein by quercetin was demonstrated by infection inhibition assay. RESULTS: A 70-kDa protein was identified as a CHIKV-interacting protein by VOPBA. MALDI-TOF analysis followed by homology search revealed that this protein could be heat shock cognate 70 (HSC 70). Anti-HSC 70 antibodies blocked CHIKV entry into C6/36 cells in a dose-dependent manner. IFA and flow cytometry analysis demonstrated HSC 70 localization on C6/36 cell surface. Co-IP experiments confirmed the interaction between HSC 70 and CHIKV envelope. Quercetin- and YM-01-treated C6/36 cells exhibited dose-dependent infection inhibition. CONCLUSION: HSC 70 serves as a putative CHIKV receptor on C6/36 cells.