Investigating the effects of dopamine on short- and long-latency afferent inhibition.

Short- and long-latency afferent inhibition (SAI and LAI respectively) are phenomenon whereby the motor evoked potential induced by transcranial magnetic stimulation (TMS) is inhibited by a sensory afferent volley consequent to nerve stimulation. It remains unclear whether dopamine participates in the genesis or modulation of SAI and LAI. The present study aimed to determine if SAI and LAI are modulated by levodopa (l-DOPA). In this placebo-controlled, double-anonymized study Apo-Levocarb (100 mg l-DOPA in combination with 25 mg carbidopa) and a placebo were administered to 32 adult males (mean age 24 ± 3 years) in two separate sessions. SAI and LAI were evoked by stimulating the median nerve and delivering single-pulse TMS over the motor hotspot corresponding to the first dorsal interosseous muscle of the right hand. SAI and LAI were quantified before and 1 h following ingestion of drug or placebo corresponding to the peak plasma concentration of Apo-Levocarb. The results indicate that Apo-Levocarb increases SAI and does not significantly alter LAI. These findings support literature demonstrating increased SAI following exogenous dopamine administration in neurodegenerative disorders. KEY POINTS: Short- and long-latency afferent inhibition (SAI and LAI respectively) are measures of corticospinal excitability evoked using transcranial magnetic stimulation. SAI and LAI are reduced in conditions such as Parkinson's disease which suggests dopamine may be involved in the mechanism of afferent inhibition. 125 mg of Apo-Levocarb (100 mg dopamine) increases SAI but not LAI. This study increases our understanding of the pharmacological mechanism of SAI and LAI.

Skeletal muscle mitochondrial morphology negatively affected in mice lacking Xin.

Altered mitochondrial structure and function are implicated in the functional decline of skeletal muscle. Numerous cytoskeletal proteins are known to affect mitochondrial homeostasis, but this complex network is still being unraveled. Here, we investigated mitochondrial alterations in mice lacking the cytoskeletal adapter protein, XIN (XIN-/-). XIN-/- and wild-type littermate male and female mice were fed a chow or high-fat diet (HFD; 60% kcal fat) for 8 weeks before analyses of their skeletal muscles were conducted. Immuno-electron microscopy (EM) and immunofluorescence staining revealed XIN in the mitochondria and peri-mitochondrial areas, as well as the myoplasm. Intermyofibrillar mitochondria in chow-fed XIN-/- mice were notably different from wild-type (large, and/or swollen in appearance). Succinate dehydrogenase and Cytochrome Oxidase IV staining indicated greater evidence of mitochondrial enzyme activity in XIN-/- mice. No difference in body mass gains or glucose handling was observed between cohorts with HFD. However, EM revealed significantly greater mitochondrial density with evident structural abnormalities (swelling, reduced cristae density) in XIN-/- mice. Absolute Complex I and II-supported respiration was not different between groups, but relative to mitochondrial density, was significantly lower in XIN-/-. These results provide the first evidence for a role of XIN in maintaining mitochondrial morphology and function.

ABHD16A deficiency causes a complicated form of hereditary spastic paraplegia associated with intellectual disability and cerebral anomalies.

ABHD16A (abhydrolase domain-containing protein 16A, phospholipase) encodes the major phosphatidylserine (PS) lipase in the brain. PS lipase synthesizes lysophosphatidylserine, an important signaling lipid that functions in the mammalian central nervous system. ABHD16A has not yet been associated with a human disease. In this report, we present a cohort of 11 affected individuals from six unrelated families with a complicated form of hereditary spastic paraplegia (HSP) who carry bi-allelic deleterious variants in ABHD16A. Affected individuals present with a similar phenotype consisting of global developmental delay/intellectual disability, progressive spasticity affecting the upper and lower limbs, and corpus callosum and white matter anomalies. Immunoblot analysis on extracts from fibroblasts from four affected individuals demonstrated little to no ABHD16A protein levels compared to controls. Our findings add ABHD16A to the growing list of lipid genes in which dysregulation can cause complicated forms of HSP and begin to describe the molecular etiology of this condition.

Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Nav) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Nav channels. Functional characterization of mutant FHF2A co-expressed with wild-type Nav1.6 (SCN8A) revealed that mutant FHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Nav channel function.

Evaluation of the diagnostic accuracy of exome sequencing and its impact on diagnostic thinking for patients with rare disease in a publicly funded health care system: A prospective cohort study.

PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases. METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests. RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses. CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test.

Missense variant in PIGM associated with severe cystic encephalomalacia and portal vein thrombosis: Phenotypic and genotypic expansion of the glycosylphosphatidylinositol biosynthesis defect-1 (GPIBD1).

Glycosylphosphatidylinositols (GPIs) are a type of glycolipid responsible for anchoring many important proteins to the cell membrane surface. Defects in the synthesis of GPIs can lead to a group of multisystem disorders known as the inherited GPI deficiencies (IGDs). Homozygosity for the c.-270C > G variant in the promoter of PIGM has been associated with a IGD subtype known as glycosylphosphatidylinositol biosynthesis defect-1 (GPIBD1). The several cases reported in the literature have been described to have a milder neurologic phenotype in comparison to the other IGDs and have been treated with sodium phenylbutyrate with some degree of success. These patients typically present with portal and hepatic vein thrombosis and mostly develop absence seizures. Here we describe a patient homozygous for a nonsynonymous variant in PIGM who deceased at 9 weeks of life and had multiple physical dysmorphisms (rocker bottom feet, midline cleft palate, thickened and lichenified skin), portal vein thrombosis, CNS structural anomalies (progressive multicystic encephalomalacia and ventriculomegaly), and a neurological phenotype of a diffuse encephalopathy. This is the first known case report of a PIGM-related IGD/CDG due to a coding variant.

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated with torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with AMC5-TOR1A have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with foetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71%, with higher mortality in males. Death occurred at a median age of 1.2 months (1 week-9 years), due to respiratory failure, cardiac arrest or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival.

Variants in CLDN5 cause a syndrome characterized by seizures, microcephaly and brain calcifications.

The blood-brain barrier ensures CNS homeostasis and protection from injury. Claudin-5 (CLDN5), an important component of tight junctions, is critical for the integrity of the blood-brain barrier. We have identified de novo heterozygous missense variants in CLDN5 in 15 unrelated patients who presented with a shared constellation of features including developmental delay, seizures (primarily infantile onset focal epilepsy), microcephaly and a recognizable pattern of pontine atrophy and brain calcifications. All variants clustered in one subregion/domain of the CLDN5 gene and the recurrent variants demonstrate genotype-phenotype correlations. We modelled both patient variants and loss of function alleles in the zebrafish to show that the variants analogous to those in patients probably result in a novel aberrant function in CLDN5. In total, human patient and zebrafish data provide parallel evidence that pathogenic sequence variants in CLDN5 cause a novel neurodevelopmental disorder involving disruption of the blood-brain barrier and impaired neuronal function.

Circulating exosome-like vesicle and skeletal muscle microRNAs are altered with age and resistance training.

The age-related loss of skeletal muscle mass and functionality, known as sarcopenia, is a critical risk factor for morbidity and all-cause mortality. Resistance exercise training (RET) is the primary countermeasure to fight sarcopenia and ageing. Altered intercellular communication is a hallmark of ageing, which is not well elucidated. Circulating extracellular vesicles (EVs), including exosomes, contribute to intercellular communication by delivering microRNAs (miRNAs), which modulate post-translational modifications, and have been shown to be released following exercise. There is little evidence regarding how EVs or EV-miRNAs are altered with age or RET. Therefore, we sought to characterize circulating EVs in young and older individuals, prior to and following a 12-week resistance exercise programme. Plasma EVs were isolated using size exclusion chromatography and ultracentrifugation. We found that ageing reduced circulating expression markers of CD9, and CD81. Using late-passage human myotubes as a model for ageing in vitro, we show significantly lower secreted exosome-like vesicles (ELVs). Further, levels of circulating ELV-miRNAs associated with muscle health were lower in older individuals at baseline but increased following RET to levels comparable to young. Muscle biopsies show similar age-related reductions in miRNA expressions, with largely no effect of training. This is reflected in vitro, where aged myotubes show significantly reduced expression of endogenous and secreted muscle-specific miRNAs (myomiRs). Lastly, proteins associated with ELV and miRNA biogenesis were significantly higher in both older skeletal muscle tissues and aged human myotubes. Together we show that ageing significantly affects ELV and miRNA cargo biogenesis, and release. RET can partially normalize this altered intercellular communication. KEY POINTS: We show that ageing reduces circulating expression of exosome-like vesicle (ELV) markers, CD9 and CD81. Using late-passage human skeletal myotubes as a model of ageing, we show that secreted ELV markers are significantly reduced in vitro. We find circulating ELV miRNAs associated with skeletal muscle health are lower in older individuals but can increase following resistance exercise training (RET). In skeletal muscle, we find altered expression of miRNAs in older individuals, with no effect of RET. Late-passage myotubes also appear to have aberrant production of endogenous myomiRs with lower abundance than youthful counterparts In older skeletal muscle and late-passage myotubes, proteins involved with ELV- and miRNA biogenesis are upregulated.

Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.

COVID-19-Associated Critical Illness Myopathy with Direct Viral Effects.

Coronavirus disease 2019 (COVID-19) severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2 infection) can lead to intensive care unit (ICU) admission and critical illness myopathy (CIM). We examined 3 ICU patients with COVID-19 who required mechanical ventilation for pneumonia and developed CIM. Pathological examination of the skeletal muscle biopsies revealed myopathic changes consistent with CIM, variable inflammation with autophagic vacuoles, SARS-CoV immunostaining + fibers/granules, and electron microscopy findings of mitochondrial abnormalities and coronavirus-like particles. Although mitochondrial dysfunction with compromised energy production is a critical pathogenic mechanism of non-COVID-19-associated CIM, in our series of COVID-19-associated CIM, myopathic changes including prominent mitochondrial damage suggest a similar mechanism and association with direct SARS-CoV-2 muscle infection. ANN NEUROL 2022;91:568-574.

Cerebrospinal fluid and plasma metabolomics of acute endurance exercise.

Metabolomics has emerged as a powerful new tool in precision medicine. No studies have yet been published on the metabolomic changes in cerebrospinal fluid (CSF) produced by acute endurance exercise. CSF and plasma were collected from 19 young active adults (13 males and 6 females) before and 60 min after a 90-min monitored outdoor run. The median age, BMI, and VO2 max of subjects was 25 years (IQR 22-31), 23.2 kg/m2 (IQR 21.7-24.5), and 47 ml/kg/min (IQR 38-51), respectively. Targeted, broad-spectrum metabolomics was performed by liquid chromatography, tandem mass spectrometry (LC-MS/MS). In the CSF, purines and pyrimidines accounted for 32% of the metabolic impact after acute endurance exercise. Branch chain amino acids, amino acid neurotransmitters, fatty acid oxidation, phospholipids, and Krebs cycle metabolites traceable to mitochondrial function accounted for another 52% of the changes. A narrow but important channel of metabolic communication was identified between the brain and body by correlation network analysis. By comparing these results to previous work in experimental animal models, we found that over 80% of the changes in the CSF correlated with a cascade of mitochondrial and metabolic changes produced by ATP signaling. ATP is released as a co-neurotransmitter and neuromodulator at every synapse studied to date. By regulating brain mitochondrial function, ATP release was identified as an early step in the kinetic cascade of layered benefits produced by endurance exercise.

Human skeletal muscle mitochondrial responses to single-leg intermittent or continuous cycle exercise training matched for absolute intensity and total work.

There is renewed interest in the potential for interval (INT) training to increase skeletal muscle mitochondrial content including whether the response differs from continuous (CONT) training. Comparisons of INT and CONT exercise are impacted by the manner in which protocols are "matched", particularly with respect to exercise intensity, as well as inter-individual differences in training responses. We employed single-leg cycling to facilitate a within-participant design and test the hypothesis that short-term INT training would elicit a greater increase in mitochondrial content than work- and intensity-matched CONT training. Ten young healthy adults (five males and five females) completed 12 training sessions over 4 weeks with each leg. Legs were randomly assigned to complete either 30 min of CONT exercise at a challenging sustainable workload (~50% single-leg peak power output; Wpeak) or INT exercise that involved 10 × 3-min bouts at the same absolute workload. INT bouts were interspersed with 1 min of recovery at 10% Wpeak and each CONT session ended with 10 min at 10% Wpeak. Absolute and mean intensity, total training time, and volume were thus matched between legs but the pattern of exercise differed. Contrary to our hypothesis, biomarkers of mitochondrial content including citrate synthase maximal activity, mitochondrial protein content and subsarcolemmal mitochondrial volume increased after CONT (p < 0.05) but not INT training. Both training modes increased single-leg Wpeak (p < 0.01) and time to exhaustion at 70% of single-leg Wpeak (p < 0.01). In a work- and intensity-matched comparison, short-term CONT training increased skeletal muscle mitochondrial content whereas INT training did not.

Expanding the Boundaries of RNA Sequencing as a Diagnostic Tool for Rare Mendelian Disease.

Gene-panel and whole-exome analyses are now standard methodologies for mutation detection in Mendelian disease. However, the diagnostic yield achieved is at best 50%, leaving the genetic basis for disease unsolved in many individuals. New approaches are thus needed to narrow the diagnostic gap. Whole-genome sequencing is one potential strategy, but it currently has variant-interpretation challenges, particularly for non-coding changes. In this study we focus on transcriptome analysis, specifically total RNA sequencing (RNA-seq), by using monogenetic neuromuscular disorders as proof of principle. We examined a cohort of 25 exome and/or panel "negative" cases and provided genetic resolution in 36% (9/25). Causative mutations were identified in coding and non-coding exons, as well as in intronic regions, and the mutational pathomechanisms included transcriptional repression, exon skipping, and intron inclusion. We address a key barrier of transcriptome-based diagnostics: the need for source material with disease-representative expression patterns. We establish that blood-based RNA-seq is not adequate for neuromuscular diagnostics, whereas myotubes generated by transdifferentiation from an individual's fibroblasts accurately reflect the muscle transcriptome and faithfully reveal disease-causing mutations. Our work confirms that RNA-seq can greatly improve diagnostic yield in genetically unresolved cases of Mendelian disease, defines strengths and challenges of the technology, and demonstrates the suitability of cell models for RNA-based diagnostics. Our data set the stage for development of RNA-seq as a powerful clinical diagnostic tool that can be applied to the large population of individuals with undiagnosed, rare diseases and provide a framework for establishing minimally invasive strategies for doing so.

Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions.

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.

Truncating Mutations in UBAP1 Cause Hereditary Spastic Paraplegia.

The diagnostic gap for rare neurodegenerative diseases is still considerable, despite continuous advances in gene identification. Many novel Mendelian genes have only been identified in a few families worldwide. Here we report the identification of an autosomal-dominant gene for hereditary spastic paraplegia (HSP) in 10 families that are of diverse geographic origin and whose affected members all carry unique truncating changes in a circumscript region of UBAP1 (ubiquitin-associated protein 1). HSP is a neurodegenerative disease characterized by progressive lower-limb spasticity and weakness, as well as frequent bladder dysfunction. At least 40% of affected persons are currently undiagnosed after exome sequencing. We identified pathological truncating variants in UBAP1 in affected persons from Iran, USA, Germany, Canada, Spain, and Bulgarian Roma. The genetic support ranges from linkage in the largest family (LOD = 8.3) to three confirmed de novo mutations. We show that mRNA in the fibroblasts of affected individuals escapes nonsense-mediated decay and thus leads to the expression of truncated proteins; in addition, concentrations of the full-length protein are reduced in comparison to those in controls. This suggests either a dominant-negative effect or haploinsufficiency. UBAP1 links endosomal trafficking to the ubiquitination machinery pathways that have been previously implicated in HSPs, and UBAP1 provides a bridge toward a more unified pathophysiology.

Routine lung volume recruitment in boys with Duchenne muscular dystrophy: a randomised clinical trial.

BACKGROUND: Impaired cough results in airway secretion retention, atelectasis and pneumonia in individuals with Duchenne muscular dystrophy (DMD). Lung volume recruitment (LVR) stacks breaths to inflate the lungs to greater volumes than spontaneous effort. LVR is recommended in DMD clinical care guidelines but is not well studied. We aimed to determine whether twice-daily LVR, compared with standard of care alone, attenuates the decline in FVC at 2 years in boys with DMD. METHODS: In this multicentre, assessor-blinded, randomised controlled trial, boys with DMD, aged 6-16 years with FVC >30% predicted, were randomised to receive conventional treatment or conventional treatment plus manual LVR twice daily for 2 years. The primary outcome was FVC % predicted at 2 years, adjusted for baseline FVC % predicted, age and ambulatory status. Secondary outcomes included change in chest wall distensibility (maximal insufflation capacity minus FVC) and peak cough flow. RESULTS: Sixty-six boys (36 in LVR group, 30 in control) were evaluated (median age (IQR): 11.5 years (9.5-13.5), median baseline FVC (IQR): 85% predicted (73-96)). Adjusted mean difference in FVC between groups at 2 years was 1.9% predicted (95% CI -6.9% to 10.7%; p=0.68) in the direction of treatment benefit. We found no differences in secondary outcomes. CONCLUSION: There was no difference in decline in FVC % predicted with use of twice-daily LVR for boys with DMD and relatively normal lung function. The burden associated with routine LVR may outweigh the benefit. Benefits of LVR to maintain lung health in boys with worse baseline lung function still need to be clarified. TRIAL REGISTRATION NUMBER: NCT01999075.

Nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants enhances autophagic clearance in Pompe disease.

Alglucosidase alpha is an orphan drug approved for enzyme replacement therapy (ERT) in Pompe disease (PD); however, its efficacy is limited in skeletal muscle because of a partial blockage of autophagic flux that hinders intracellular trafficking and enzyme delivery. Adjunctive therapies that enhance autophagic flux and protect mitochondrial integrity may alleviate autophagic blockage and oxidative stress and thereby improve ERT efficacy in PD. In this study, we compared the benefits of ERT combined with a ketogenic diet (ERT-KETO), daily administration of an oral ketone precursor (1,3-butanediol; ERT-BD), a multi-ingredient antioxidant diet (ERT-MITO; CoQ10, α-lipoic acid, vitamin E, beetroot extract, HMB, creatine, and citrulline), or co-therapy with the ketone precursor and multi-ingredient antioxidants (ERT-BD-MITO) on skeletal muscle pathology in GAA-KO mice. We found that two months of 1,3-BD administration raised circulatory ketone levels to ≥1.2 mM, attenuated autophagic buildup in type 2 muscle fibers, and preserved muscle strength and function in ERT-treated GAA-KO mice. Collectively, ERT-BD was more effective vs. standard ERT and ERT-KETO in terms of autophagic clearance, dampening of oxidative stress, and muscle maintenance. However, the addition of multi-ingredient antioxidants (ERT-BD-MITO) provided the most consistent benefits across all outcome measures and normalized mitochondrial protein expression in GAA-KO mice. We therefore conclude that nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants may provide an alternative to ketogenic diets for inducing ketosis and enhancing autophagic flux in PD patients.

Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects.

The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Comprehensive genetic sequence and copy number analysis for Charcot-Marie-Tooth disease in a Canadian cohort of 2517 patients.

Charcot-Marie-Tooth disease (CMT) is one of the most common Mendelian disorders characterised by genetic heterogeneity, progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. In this report, we describe genetic testing data including comprehensive sequencing and copy number analysis of 34 CMT-related genes in a Canadian cohort of patients with suspected CMT. We have demonstrated a notable gender testing bias, with an overall diagnostic yield of 15% in males and 21% in females. We have identified a large number of novel pathogenic variants as well as variants of unknown clinical significance in CMT-related genes. In this largest to date analysis of gene CNVs in CMT, in addition to the common PMP22 deletion/duplication, we have described a significant contribution of pathogenic CNVs in several CMT-related genes. This study significantly expand the mutational spectrum of CMT genes, while demonstrating the clinical utility of a comprehensive sequence and copy number next-generation sequencing-based clinical genetic testing in patients with suspected diagnosis of CMT.