Identification of a mosaic MTOR variant in purified neuronal DNA in a patient with focal cortical dysplasia using a novel depth electrode harvesting technique.

OBJECTIVE: Recent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery, which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by nonbrain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD). METHODS: Depth electrode tips were collected, pooled by brain region and seizure onset zone, and nuclei were isolated and sorted using fluorescence-activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital polymerase chain reaction (PCR). RESULTS: An 11-year-old male with drug-resistant genetic-structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (variant allele frequency = .78% in affected neuronal sample; variant was absent in all other samples). SIGNIFICANCE: Our findings confirm that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high-quality cells of the cell type of interest.

Valosin-Containing Protein (VCP): A Review of Its Diverse Molecular Functions and Clinical Phenotypes.

In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

Therapeutic developments for valosin-containing protein mediated multisystem proteinopathy.

PURPOSE OF REVIEW: Missense mutations in valosin-containing protein (VCP) can lead to a multisystem proteinopathy 1 (MSP1) with any combination of limb-girdle distribution inclusion body myopathy (IBM) (present in about 90% of cases), Paget's disease of bone, and frontotemporal dementia (IBMPFD). VCP mutations lead to gain of function activity with widespread disarray in cellular function, with enhanced ATPase activity, increased binding with its cofactors, and reduced mitofusin levels. RECENT FINDINGS: This review highlights novel therapeutic approaches in VCP-MSP in in-vitro and in-vivo models. Furthermore, we also discuss therapies targeting mitochondrial dysfunction, autophagy, TDP-43 pathways, and gene therapies in other diseases with similar pathway involvement which can also be applicable in VCP-MSP. SUMMARY: Being a rare disease, it is challenging to perform large-scale randomized control trials (RCTs) in VCP-MSP. However, it is important to recognize potential therapeutic targets, and assess their safety and efficacy in preclinical models, to initiate RCTs for potential therapies in this debilitating disease.

A National Spinal Muscular Atrophy Registry for Real-World Evidence.

BACKGROUND: Spinal muscular atrophy (SMA) is a devastating rare disease that affects individuals regardless of ethnicity, gender, and age. The first-approved disease-modifying therapy for SMA, nusinursen, was approved by Health Canada, as well as by American and European regulatory agencies following positive clinical trial outcomes. The trials were conducted in a narrow pediatric population defined by age, severity, and genotype. Broad approval of therapy necessitates close follow-up of potential rare adverse events and effectiveness in the larger real-world population. METHODS: The Canadian Neuromuscular Disease Registry (CNDR) undertook an iterative multi-stakeholder process to expand the existing SMA dataset to capture items relevant to patient outcomes in a post-marketing environment. The CNDR SMA expanded registry is a longitudinal, prospective, observational study of patients with SMA in Canada designed to evaluate the safety and effectiveness of novel therapies and provide practical information unattainable in trials. RESULTS: The consensus expanded dataset includes items that address therapy effectiveness and safety and is collected in a multicenter, prospective, observational study, including SMA patients regardless of therapeutic status. The expanded dataset is aligned with global datasets to facilitate collaboration. Additionally, consensus dataset development aimed to standardize appropriate outcome measures across the network and broader Canadian community. Prospective outcome studies, data use, and analyses are independent of the funding partner. CONCLUSION: Prospective outcome data collected will provide results on safety and effectiveness in a post-therapy approval era. These data are essential to inform improvements in care and access to therapy for all SMA patients.

Skeletal Phenotypes Due to Abnormalities in Mitochondrial Protein Homeostasis and Import.

Mitochondrial disease represents a collection of rare genetic disorders caused by mitochondrial dysfunction. These disorders can be quite complex and heterogeneous, and it is recognized that mitochondrial disease can affect any tissue at any age. The reasons for this variability are not well understood. In this review, we develop and expand a subset of mitochondrial diseases including predominantly skeletal phenotypes. Understanding how impairment ofdiverse mitochondrial functions leads to a skeletal phenotype will help diagnose and treat patients with mitochondrial disease and provide additional insight into the growing list of human pathologies associated with mitochondrial dysfunction. The underlying disease genes encode factors involved in various aspects of mitochondrial protein homeostasis, including proteases and chaperones, mitochondrial protein import machinery, mediators of inner mitochondrial membrane lipid homeostasis, and aminoacylation of mitochondrial tRNAs required for translation. We further discuss a complex of frequently associated phenotypes (short stature, cataracts, and cardiomyopathy) potentially explained by alterations to steroidogenesis, a process regulated by mitochondria. Together, these observations provide novel insight into the consequences of impaired mitochondrial protein homeostasis.

High diagnostic yield and novel variants in very late-onset spasticity.

Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotype-phenotype correlation in these conditions that should be the subject of future study.

Emerging therapies for mitochondrial disorders.

Mitochondrial disorders are a diverse group of debilitating conditions resulting from nuclear and mitochondrial DNA mutations that affect multiple organs, often including the central and peripheral nervous system. Despite major advances in our understanding of the molecular mechanisms, effective treatments have not been forthcoming. For over five decades patients have been treated with different vitamins, co-factors and nutritional supplements, but with no proven benefit. There is therefore a clear need for a new approach. Several new strategies have been proposed acting at the molecular or cellular level. Whilst many show promise in vitro, the clinical potential of some is questionable. Here we critically appraise the most promising preclinical developments, placing the greatest emphasis on diseases caused by mitochondrial DNA mutations. With new animal and cellular models, longitudinal deep phenotyping in large patient cohorts, and growing interest from the pharmaceutical industry, the field is poised to make a breakthrough.

Exome sequencing in undiagnosed inherited and sporadic ataxias.

Inherited ataxias are clinically and genetically heterogeneous, and a molecular diagnosis is not possible in most patients. Having excluded common sporadic, inherited and metabolic causes, we used an unbiased whole exome sequencing approach in 35 affected individuals, from 22 randomly selected families of white European descent. We defined the likely molecular diagnosis in 14 of 22 families (64%). This revealed de novo dominant mutations, validated disease genes previously described in isolated families, and broadened the clinical phenotype of known disease genes. The diagnostic yield was the same in both young and older-onset patients, including sporadic cases. We have demonstrated the impact of exome sequencing in a group of patients notoriously difficult to diagnose genetically. This has important implications for genetic counselling and diagnostic service provision.

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis.

Titin founder mutation is a common cause of myofibrillar myopathy with early respiratory failure.

OBJECTIVE: Titin gene (TTN) mutations have been described in eight families with hereditary myopathy with early respiratory failure (HMERF). Some of the original patients had features resembling myofibrillar myopathy (MFM), arguing that TTN mutations could be a much more common cause of inherited muscle disease, especially in presence of early respiratory involvement. METHODS: We studied 127 undiagnosed patients with clinical presentation compatible with MFM. Sanger sequencing for the two previously described TTN mutations in HMERF (p.C30071R in the 119th fibronectin-3 (FN3) domain, and p.R32450W in the kinase domain) was performed in all patients. Patients with mutations had detailed review of their clinical records, muscle MRI findings and muscle pathology. RESULTS: We identified five new families with the p.C30071R mutation who were clinically similar to previously reported cases, and muscle pathology demonstrated diagnostic features of MFM. Two further families had novel variants in the 119th FN3 domain (p.P30091L and p.N30145K). No patients were identified with mutations at position p.32450. CONCLUSIONS: Mutations in TTN are a cause of MFM, and titinopathy is more common than previously thought. The finding of the p.C30071R mutation in 3.9% of our study population is likely due to a British founder effect. The occurrence of novel FN3 domain variants, although still of uncertain pathogenicity, suggests that other mutations in this domain may cause MFM, and that the disease is likely to be globally distributed. We suggest that HMERF due to mutations in the TTN gene be nosologically classified as MFM-titinopathy.

Brain-body mechanisms contribute to sexual dimorphism in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common form of human motor neuron disease. It is characterized by the progressive degeneration of upper and lower motor neurons, leading to generalized motor weakness and, ultimately, respiratory paralysis and death within 3-5 years. The disease is shaped by genetics, age, sex and environmental stressors, but no cure or routine biomarkers exist for the disease. Male individuals have a higher propensity to develop ALS, and a different manifestation of the disease phenotype, than female individuals. However, the mechanisms underlying these sex differences remain a mystery. In this Review, we summarize the epidemiology of ALS, examine the sexually dimorphic presentation of the disease and highlight the genetic variants and molecular pathways that might contribute to sex differences in humans and animal models of ALS. We advance the idea that sexual dimorphism in ALS arises from the interactions between the CNS and peripheral organs, involving vascular, metabolic, endocrine, musculoskeletal and immune systems, which are strikingly different between male and female individuals. Finally, we review the response to treatments in ALS and discuss the potential to implement future personalized therapeutic strategies for the disease.

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Life-Saving Treatments for Spinal Muscular Atrophy: Global Access and Availability.

Background and Objectives: Spinal muscular atrophy (SMA) is a neurodegenerative disorder manifesting with progressive muscle weakness and atrophy. SMA type 1 used to be fatal within the first 2 years of life, but is now treatable with therapies targeting splicing modification and gene replacement. Nusinersen, risdiplam, and onasemnogene abeparvovec-xioi improve survival, motor strength, endurance, and ability to thrive, allowing many patients to potentially attain a normal life; all have been recently approved by major regulatory agencies. Although these therapies have revolutionized the world of SMA, they are associated with a high economic burden, and access to these therapies is limited in some countries. The primary objective of this study was to compare the availability and implementation of treatment of SMA from different regions of the world. Methods: In this qualitative study, we surveyed health care providers from 21 countries regarding their experiences caring for patients with SMA. The main outcome measures were provider survey responses on newborn screening, drug availability/access, barriers to treatment, and related questions. Results: Twenty-four providers from 21 countries with decades of experience (mean 26 years) in treating patients with SMA responded to the survey. Nusinersen was the most available therapy for SMA. Our survey showed that while genetic testing is usually available, newborn screening is still unavailable in many countries. The provider-reported treatment cost also varied between countries, and economic burden was a major barrier in treating patients with SMA. Discussion: Overall, this survey highlights the global inequality in managing patients with SMA. The spread of newborn screening is essential in ensuring improved access to care for patients with SMA. With the advancement of neurotherapeutics, more genetic diseases will soon be treatable, and addressing the global inequality in clinical care will require novel approaches to mitigate such inequality in the future.

Titin mutation segregates with hereditary myopathy with early respiratory failure.

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.

Primary lateral sclerosis.

Like motor neuron diseases (MNDs) refer to a constellation of primarily sporadic neurodegenerative diseases characterized by a progressive loss of upper and/or lower motor neurons. Primary lateral sclerosis (PLS) is considered a neurodegenerative disorder that is characterized by a gradually progressive course affecting the central motor systems, designated by the phrase "upper motor neurons." Despite significant development in neuroimaging, neurophysiology, and molecular biology, there is a growing consensus that PLS is of unknown etiology. Currently there is no disease-modifying treatment for PLS, or prospective randomized trials being carried out, partly due to the rarity of the disease and lack of significant understanding of the underlying pathophysiology. Consequently, the approach to treatment remains largely symptomatic. In this chapter we provide an overview of primary lateral sclerosis including clinical and electrodiagnostic considerations, differential diagnosis, updates in genetics and pathophysiology, and future directions for research.