Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

Whole-exome analyses of congenital muscular dystrophy and congenital myopathy patients from India reveal a wide spectrum of known and novel mutations.

BACKGROUND AND PURPOSE: Congenital muscular dystrophies (CMDs) and congenital myopathies (CMs) are a group of genetically and clinically heterogeneous degenerative primary muscle disorders with onset at birth or during infancy. Due to vast heterogeneity, clinical examination and protein-based analyses often fail to identify the genetic causes of these diseases. The aim of this study was to genetically diagnose a cohort of 36 difficult-to-diagnose CMD and CM cases of Indian origin using next-generation sequencing methods. METHODS: Whole-exome sequencing (WES) was performed to identify pathogenic mutations in previously reported CMD and CM-related genes using variant calling and stringent variant filtration process. Subsequently, in silico homology modelling and molecular dynamics simulations (MDS) studies were undertaken for a number of novel and missense variants. RESULTS: A total of 33 and 21 rare and deleterious mutations were identified in 28 genes previously reported in CMD and CM based on OMIM, ClinVar and Orphanet, respectively. We could accurately diagnose 54% patients (n = 12/22) in the CMD group and 35% patients (n = 5/14) in the CM group. Furthermore, MDS studies for mutations located in LMNA, LAMA2 and RYR1 suggest that the wild-type proteins are more stable than their mutant counterparts, implying a potential mechanism of pathogenesis. CONCLUSION: The WES findings led us to identify reported as well as novel variants for the first time in Indian patients with CMD and CM. This allowed us to achieve an accurate genetic diagnosis, which was difficult using conventional diagnostic tools. Transferring these WES findings to clinical practice will help guide clinical care of the affected patients and inform genetic counselling.

Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder.

Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSDs) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSDs (n = 7), compared to controls (n = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n = 5) (vs. control, n = 5) by high-throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSDs. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSDs. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases. Cover Image for this Issue: doi: 10.1111/jnc.14177.

Natural history of a cohort of Duchenne muscular dystrophy children seen between 1998 and 2014: An observational study from South India.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. There are no large studies describing its natural course from India. MATERIALS AND METHODS: Immunohistochemically/genetically confirmed DMD patients diagnosed between 1998 and 2014 were ambispectively included. The main aim was to study the natural course of motor milestones, i.e., age at onset of wheelchair status, bedbound state, and age at death, which were considered as primary outcome measures. We also correlated the DMD genotype with the motor milestones and other phenotypic features. RESULTS: A total of 500 DMD patients were included and 275 participated in the study. The mean age at symptom onset was 3.7 ± 1.9 years, mean age at presentation was 8.1 ± 2.5 years, and mean duration of illness was 4.4 ± 2.6 years. On following them over 15 years, 155 (56.4%) had attained at least one of the primary outcome measures. Wheelchair status was attained in 124 (45.1%) [mean age: 10.4 ± 1.6 years] and bedbound state in 24 (8.7%; mean age: 11.8 ± 2.2 years) patients. Seven patients (2.6%) died during the follow-up period (mean age: 15.2 ± 2.4 years). There was no significant impact of the genotypic or phenotypic features on the primary outcome. CONCLUSION: The pattern of major motor milestones (primary outcome measures) in this large cohort is comparable with that of the Western population despite variability in medical care. The genotypic pattern was also similar to other large studies, which suggests that DMD is a more homogeneous disorder with limited ethnic variability in its geno-phenotypic expression.

Manganese- and 1-methyl-4-phenylpyridinium-induced neurotoxicity display differences in morphological, electrophysiological and genome-wide alterations: implications for idiopathic Parkinson's disease.

Idiopathic Parkinson's disease and manganese-induced atypical parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to levodopa distinguish both, differences at the neuronal level are largely unknown. We studied the morphological, neurophysiological and molecular differences in dopaminergic neurons exposed to the Parkinson's disease toxin 1-methyl-4-phenylpyridinium ion (MPP+ ) and manganese (Mn), followed by validation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Mn mouse models. Morphological analysis highlighted loss of neuronal processes in the MPP+ and not the Mn model. Cellular network dynamics of dopaminergic neurons characterized by spike frequency and inter-spike intervals indicated major neuronal population (~ 93%) with slow discharge rates (0-5 Hz). While MPP+ exposure suppressed the firing of these neurons, Mn neither suppressed nor elevated the neuronal activity. High-throughput transcriptomic analysis revealed up-regulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP+ and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic/mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeleton. Analysis of the Janus kinase-signal transducer and activator of transcription pathway with implications for neuritogenesis and neuronal proliferation revealed contrasting profile in both models. Genome-wide DNA methylomics revealed differences between both models and substantiated the epigenetic basis of the difference in the Janus kinase-signal transducer and activator of transcription pathway. We conclude that idiopathic Parkinson's disease and atypical parkinsonism have divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and evolution of novel therapeutics. Cover Image for this issue: doi. 10.1111/jnc.13821.

MLPA identification of dystrophin mutations and in silico evaluation of the predicted protein in dystrophinopathy cases from India.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations in the DMD gene. The aim of this study was to predict the effect of gene mutations on the dystrophin protein and study its impact on clinical phenotype. METHODS: In this study, 415 clinically diagnosed patients were tested for mutations by Multiplex ligation dependent probe amplification (MLPA). Muscle biopsy was performed in 34 patients with negative MLPA. Phenotype-genotype correlation was done using PROVEAN, hydrophobicity and eDystrophin analysis. We have utilized bioinformatics tools in order to evaluate the observed mutations both at the level of primary as well as secondary structure. RESULTS: Mutations were identified in 75.42% cases, of which there were deletions in 91.6% and duplications in 8.30%. As per the reading frame rule, 84.6% out-of frame and 15.3% in-frame mutations were noted. Exon 50 was the most frequently deleted exon and the exon 45-52 region was the hot-spot for deletions in this cohort. There was no correlation noted between age of onset or creatine kinase (CK) values with extent of gene mutation. The PROVEAN analysis showed a deleterious effect in 94.5% cases and a neutral effect in 5.09% cases. Mutations in exon 45-54 (out of frame) and exon 46-54 (in-frame) regions in the central rod domain of dystrophin showed more negative scores compared to other domains in the present study. Hydrophobicity profile analysis showed that the hydrophobic regions I & III were equally affected. Analysis of deletions in hinge III hydrophobic region by the eDystrophin programme also predicted a hybrid repeat seen to be associated with a BMD like disease progression, thus making the hinge III region relatively tolerant to mutations. CONCLUSIONS: We found that, while the predictions made by the software utilized might have overall significance, the results were not convincing on a case by case basis. This reflects the inadequacy of the currently available tools and also underlines the possible inadequacy of MLPA to detect other minor mutations that might enhance or suppress the effect of the primary mutation in this large gene. Next Generation Sequencing or targeted Sanger sequencing on a case by case basis might improve phenotype- genotype correlation.

Novel magnetic resonance imaging findings in a patient with short chain acyl CoA dehydrogenase deficiency.

Reports on magnetic resonance imaging findings in patients with short chain acyl -Coenzyme A dehydrogenase (SCAD) deficiency, an autosomal recessive disorder caused by mutations in the acyl-Coenzyme A dehydrogenase (ACADS), are limited. Many asymptomatic carriers of ACAD variants have also been described necessitating careful evaluation of clinical and biochemical findings for an accurate diagnosis. Here we report a an infant with short chain acyl -Coenzyme A dehydrogenase (SCAD) deficiency diagnosed based on the characteristic biochemical findings and confirmed by genetic testing. He presented with refractory seizures and neuro regression at 4 months of age. His metabolic work up revealed elevated butyryl carnitine in plasma and ethyl malonic acid in urine. Magnetic resonance imaging of the brain showed cortical and basal ganglia signal changes with cortical swelling. Serial scans showed progression of the lesions resulting in cystic leukomalacia with brain atrophy. Exome sequencing revealed a novel homozygous nonsense variation, c.1146C > G (p.Y382Ter) in exon ten of ACADS which was further validated by Sanger sequencing. Both parents were heterozygous carriers. Follow up at 15 months showed gross psychomotor retardation and refractory seizures despite being on optimal doses of anti-epileptic medications, carnitine and multivitamin supplementation. This report expands the phenotypic and genotypic spectrum of SCAD deficiency.

Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function.

Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from dysferlinopathy (Dysfy), polymyositis (PM), and distal myopathy with rimmed vacuoles (DMRV) displayed morphological and biochemical evidences of mitochondrial dysfunction. Proteomic analysis revealed down-regulation of electron transport chain (ETC) subunits, assembly factors, and tricarboxylic acid (TCA) cycle enzymes, with 80 proteins common among the three pathologies. Mitochondrial proteins from muscle pathologies also displayed higher Trp oxidation that could alter the local structure. Cover image for this issue: doi: 10.1111/jnc.13324.

Huppke-Brendel syndrome in a seven months old boy with a novel 2-bp deletion in SLC33A1.

Huppke -Brendel syndrome is a new addition to the evolving spectrum of copper metabolism defects. It is an autosomal recessive disorder characterized by congenital cataract, impaired hearing, and developmental delay with low copper and ceruloplasmin. It is caused by defects in SLC33A1 that codes for acetyl CoA transporter protein. Reports on variation in this gene causing human disease is extremely scarce and the metabolic link between this gene and copper metabolism is yet to be identified. Here we report a seven months old infant with Huppke-Brendel Syndrome. In addition to the already reported features, he also had hypo pigmented hair and hypogonadism. His magnetic resonance imaging revealed hypo myelination and cerebellar hypoplasia. Clinical exome sequencing revealed a homozygous two base pair deletion, c.542_543delTG (p.Val181GlyfsTer6) in exon 1 of the SLC33A1. This report expands the phenotypic and genotypic spectrum of Huppke Brendel syndrome.

Mitochondrial alterations and oxidative stress in an acute transient mouse model of muscle degeneration: implications for muscular dystrophy and related muscle pathologies.

Muscular dystrophies (MDs) and inflammatory myopathies (IMs) are debilitating skeletal muscle disorders characterized by common pathological events including myodegeneration and inflammation. However, an experimental model representing both muscle pathologies and displaying most of the distinctive markers has not been characterized. We investigated the cardiotoxin (CTX)-mediated transient acute mouse model of muscle degeneration and compared the cardinal features with human MDs and IMs. The CTX model displayed degeneration, apoptosis, inflammation, loss of sarcolemmal complexes, sarcolemmal disruption, and ultrastructural changes characteristic of human MDs and IMs. Cell death caused by CTX involved calcium influx and mitochondrial damage both in murine C2C12 muscle cells and in mice. Mitochondrial proteomic analysis at the initial phase of degeneration in the model detected lowered expression of 80 mitochondrial proteins including subunits of respiratory complexes, ATP machinery, fatty acid metabolism, and Krebs cycle, which further decreased in expression during the peak degenerative phase. The mass spectrometry (MS) data were supported by enzyme assays, Western blot, and histochemistry. The CTX model also displayed markers of oxidative stress and a lowered glutathione reduced/oxidized ratio (GSH/GSSG) similar to MDs, human myopathies, and neurogenic atrophies. MS analysis identified 6 unique oxidized proteins from Duchenne muscular dystrophy samples (n = 6) (versus controls; n = 6), including two mitochondrial proteins. Interestingly, these mitochondrial proteins were down-regulated in the CTX model thereby linking oxidative stress and mitochondrial dysfunction. We conclude that mitochondrial alterations and oxidative damage significantly contribute to CTX-mediated muscle pathology with implications for human muscle diseases.

Raman spectroscopic studies on screening of myopathies.

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld(1) (up(1)), heldup(2) (hdp(2)), myosin heavy chain(7) (Mhc(7)), actin88F(KM88) (Act88F(KM88)), upheld(101) (up(101)), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and ß-sheet and/or random coil protein content along with a decrease in α-helix protein. Interestingly, the 12th day samples of up(1) and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up(1) and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up(101) and hdp(2) (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up(1)). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.

Clinical and Neuroimaging Features in Two Children with Mutations in the Mitochondrial ND5 Gene.

Mutations in the mitochondrial-encoded nicotinamide adenine dinucleotide dehydrogenase 5 gene (MT-ND5) has been implicated as an important genetic cause of childhood mitochondrial encephalomyopathies. This study reports the clinical and magnetic resonance imaging findings in two pediatric patients with mutations in the ND5 gene of mitochondrial DNA. The 8-month-old boy with m.13513 G>A mutation presented with infantile basal ganglia stroke syndrome secondary to mineralizing angiopathy. The 7-year-old girl with the m.13514A>G mutation had episodic regression, progressive ataxia, optic atrophy, and hyperactivity. Magnetic resonance imaging of the brain showed bilateral symmetrical signal intensity changes in the thalamus, tectal plate, and inferior olivary nucleus, which subsided on follow-up image. Both the patients had a stable course. Familiarity with the various phenotypic and magnetic resonance imaging findings and the clinical course in childhood mitochondrial encephalomyopathies may help the physician in targeted metabolic-genetic testing and prognostication.

A prospective study on the immunophenotypic characterization of limb girdle muscular dystrophies 2 in India.

OBJECTIVE: In this prospective study conducted over 2 years, 300 nonconsecutive cases of autosomal recessive limb girdle muscular dystrophies (AR-LGMD) were characterized, based on phenotypic features, biochemical findings, electrophysiological studies, muscle immunohistochemistry (IHC), and western blot (WB) analysis. METHODS: Muscle biopsy was performed in 280 index cases. 226 biopsies were subjected for IHC, and, 176 of these for WB analysis. RESULTS: A total of 246 patients were finally analyzed. This figure included 20 affected siblings. LGMD2B was the most common form and comprised of 33.3% (n = 82) of the entire cohort. This was followed by alpha-dystroglycanopathies with 61 (24.79%) patients (LGMD2I in 15, 2K in 10 and combined deficiency of both in the remaining). LGMD 2C-F was present in 35 (14.23%) cases and LGMD2A in 22 (10.2%) cases, and were identified by routine WB, densitometry method and autocatalytic assay. LGMD2G was present in 8 patients (3.25%), and LGMD2H and 2J in 2 cases each, respectively. CONCLUSIONS: For the first time, we have identified patients with LGMD2G, 2H, 2I, and 2K by the WB technique. These may be the common forms of autosomal recessive (AR)-LGMD's among Indian patients and need identification for prognostication and appropriate counseling. Although not a nationwide study, our data is sufficient to provide information about the relative proportions of various LGMD2 subtypes in India. Diagnosing LGMD2 based on classical clinical features, IHC and WB is fairly sensitive and specific; however, further genetic studies are required to confirm the diagnosis.

GNE myopathy in India.

BACKGROUND: GNE myopathy is a clinicopathologically distinct distal myopathy with autosomal-recessive inheritance. The GNE gene mutations are known to cause this form of distal myopathy MATERIALS AND METHODS: Over the last 6 years, a total of 54 patients from 48 families were diagnosed to have GNE myopathy based on the clinical and histopathological findings. We have reported on 23 cases earlier and from this cohort 12 patients from 11 families underwent genetic testing for GNE mutation. RESULTS: Nine patients belonging to eight families were confirmed as GNE myopathy by genetic analysis. There were six women and three men. Mean age of onset was 26.7 ± 5.47 years (20-36 years) and mean age at clinical examination was 32.3 ± 4.2 years (28-39 years). Mean duration of the illness was 5.7 ± 4.7 years (1-14 years). All had characteristic clinical features of progressive weakness and wasting of the anterior part of leg muscles, adductors of thighs and hamstrings with relative sparing of the quadriceps muscles. Biopsy from the tibialis anterior muscles revealed the presence of rimmed vacuoles. Mutation analysis of the GNE gene revealed that c. 2086G > A (p.Val696Met) change was common in our series like Thailand and six of eight families carried this mutation, heterozygously. CONCLUSION: These results show the presence of a common mutation in GNE gene in Southeast Asia.

New mutation of the desmin gene identified in an extended Indian pedigree presenting with distal myopathy and cardiac disease.

In this report, we describe a new mutation located in the coiled 1B domain of desmin and associated with a predominant cardiac involvement and a high degree of cardiac sudden death in a large Indian pedigree with 12 affected members. The index cases was 38-year-old man who presented with progressive difficulty in gripping footwear of 5 years duration with the onset in the left lower limb followed by right lower limb in 6 months. 3 years from onset, he developed lower limb proximal and truncal muscle weakness. There was mild atrophy of the shoulder girdle muscles with grade 3 weakness, moderate wasting of thigh and anterior leg muscles with proximal muscle weakness and foot drop. At 40 years, he had a pacemaker implanted. The 9 exons and intronic boundaries of the desmin gene were sequenced and a heterozygous nucleotide change c. 734A > G in exon 3 was identified.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy.

PURPOSE: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. METHODS: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. RESULTS: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. CONCLUSIONS: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Diagnosis of primary mitochondrial disorders -Emphasis on myopathological aspects.

Mitochondrial disorders are one of the most common neurometabolic disorders affecting all age groups. The phenotype-genotype heterogeneity in these disorders can be attributed to the dual genetic control on mitochondrial functions, posing a challenge for diagnosis. Though the advancement in the high-throughput sequencing and other omics platforms resulted in a "genetics-first" approach, the muscle biopsy remains the benchmark in most of the mitochondrial disorders. This review focuses on the myopathological aspects of primary mitochondrial disorders. The utility of muscle biopsy is not limited to analyse the structural abnormalities; rather it also proves to be a potential tool to understand the deranged sub-cellular functions.

Inhibition of mitochondrial complex II in neuronal cells triggers unique pathways culminating in autophagy with implications for neurodegeneration.

Mitochondrial dysfunction and neurodegeneration underlie movement disorders such as Parkinson's disease, Huntington's disease and Manganism among others. As a corollary, inhibition of mitochondrial complex I (CI) and complex II (CII) by toxins 1-methyl-4-phenylpyridinium (MPP+) and 3-nitropropionic acid (3-NPA) respectively, induced degenerative changes noted in such neurodegenerative diseases. We aimed to unravel the down-stream pathways associated with CII inhibition and compared with CI inhibition and the Manganese (Mn) neurotoxicity. Genome-wide transcriptomics of N27 neuronal cells exposed to 3-NPA, compared with MPP+ and Mn revealed varied transcriptomic profile. Along with mitochondrial and synaptic pathways, Autophagy was the predominant pathway differentially regulated in the 3-NPA model with implications for neuronal survival. This pathway was unique to 3-NPA, as substantiated by in silico modelling of the three toxins. Morphological and biochemical validation of autophagy markers in the cell model of 3-NPA revealed incomplete autophagy mediated by mechanistic Target of Rapamycin Complex 2 (mTORC2) pathway. Interestingly, Brain Derived Neurotrophic Factor (BDNF), which was elevated in the 3-NPA model could confer neuroprotection against 3-NPA. We propose that, different downstream events are activated upon neurotoxin-dependent CII inhibition compared to other neurotoxins, with implications for movement disorders and regulation of autophagy could potentially offer neuroprotection.

Ethylmalonic encephalopathy ETHE1 p. D165H mutation alters the mitochondrial function in human skeletal muscle proteome.

Ethylmalonic encephalopathy (EE) is a rare autosomal recessive inborn error of metabolism. To study the molecular effects of ETHE1 p. D165H mutation, we employed mass spectrometry-based mitochondrial proteome and phosphoproteome profiling in the human skeletal muscle. Eighty-six differentially altered proteins were identified, of which thirty-seven mitochondrial proteins were differentially expressed, and most of the proteins (37%) were down-regulated in the OXPHOS complex-IV. Also, nine phosphopeptides that correspond to eight mitochondrial proteins were significantly affected in EE patient. These altered proteins recognized are involved in several pathways and molecular functions, predominantly in oxidoreductase activity. This is the first study that has integrated proteome and phosphoproteome of skeletal muscle and identified multiple proteins associated in the pathogenesis of EE.