RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions.

The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating ß-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

A form of muscular dystrophy associated with pathogenic variants in JAG2.

JAG2 encodes the Notch ligand Jagged2. The conserved Notch signaling pathway contributes to the development and homeostasis of multiple tissues, including skeletal muscle. We studied an international cohort of 23 individuals with genetically unsolved muscular dystrophy from 13 unrelated families. Whole-exome sequencing identified rare homozygous or compound heterozygous JAG2 variants in all 13 families. The identified bi-allelic variants include 10 missense variants that disrupt highly conserved amino acids, a nonsense variant, two frameshift variants, an in-frame deletion, and a microdeletion encompassing JAG2. Onset of muscle weakness occurred from infancy to young adulthood. Serum creatine kinase (CK) levels were normal or mildly elevated. Muscle histology was primarily dystrophic. MRI of the lower extremities revealed a distinct, slightly asymmetric pattern of muscle involvement with cores of preserved and affected muscles in quadriceps and tibialis anterior, in some cases resembling patterns seen in POGLUT1-associated muscular dystrophy. Transcriptome analysis of muscle tissue from two participants suggested misregulation of genes involved in myogenesis, including PAX7. In complementary studies, Jag2 downregulation in murine myoblasts led to downregulation of multiple components of the Notch pathway, including Megf10. Investigations in Drosophila suggested an interaction between Serrate and Drpr, the fly orthologs of JAG1/JAG2 and MEGF10, respectively. In silico analysis predicted that many Jagged2 missense variants are associated with structural changes and protein misfolding. In summary, we describe a muscular dystrophy associated with pathogenic variants in JAG2 and evidence suggests a disease mechanism related to Notch pathway dysfunction.

Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Measurement properties of the Inclusion Body Myositis Functional Rating Scale.

OBJECTIVES: To evaluate the validity, reliability, responsiveness and meaningful change threshold of the Inclusion Body Myositis (IBM) Functional Rating Scale (FRS). METHODS: Data from a large 20-month multicentre, randomised, double-blind, placebo-controlled trial in IBM were used. Convergent validity was tested using Spearman correlation with other health outcomes. Discriminant (known groups) validity was assessed using standardised effect sizes (SES). Internal consistency was tested using Cronbach's alpha. Intrarater reliability in stable patients and equivalence of face-to-face and telephone administration were tested using intraclass correlation coefficients (ICCs) and Bland-Altman plots. Responsiveness was assessed using a standardised response mean (SRM). An receiver operator characteristic (ROC) curve anchor-based approach was used to determine clinically meaningful IBMFRS change. RESULTS: Among the 150 patients, mean (SD) IBMFRS total score was 27.4 (4.6). Convergent validity was supported by medium to large correlations (rs modulus: 0.42-0.79) and discriminant validity by moderate to large group differences (SES=0.51-1.59). Internal consistency was adequate (overall Cronbach's alpha: 0.79). Test-retest reliability (ICCs=0.84-0.87) and reliability of telephone versus face-to-face administration (ICCs=0.93-0.95) were excellent, with Bland-Altman plots showing good agreement. Responsiveness in the worsened group defined by various external constructs was large at both 12 (SRM=-0.76 to -1.49) and 20 months (SRM=-1.12 to -1.57). In ROC curve analysis, a drop in two IBMFRS total score points was shown to represent a meaningful decline. CONCLUSIONS: When administered by trained raters, the IBMFRS is a reliable, valid and responsive tool that can be used to evaluate the impact of IBM and its treatment on physical function, with a 2-point reduction representing meaningful decline. TRIAL REGISTRATION NUMBER: NCT02753530.

A diagnostic framework to identify vestibular involvement in multi-sensory neurological disease.

BACKGROUND AND PURPOSE: Identifying vestibular causes of dizziness and unsteadiness in multi-sensory neurological disease can be challenging, with problems typically attributed to central or peripheral nerve involvement. Acknowledging vestibular dysfunction as part of the presentation provides an opportunity to access targeted vestibular rehabilitation, for which extensive evidence exists. A diagnostic framework was developed and validated to detect vestibular dysfunction, benign paroxysmal positional vertigo or vestibular migraine. The specificity and sensitivity of the diagnostic framework was tested in patients with primary mitochondrial disease. METHODS: Adults with a confirmed diagnosis of primary mitochondrial disease were consented, between September 2020 and February 2022. Participants with and without dizziness or unsteadiness underwent remote physiotherapy assessment and had in-person detailed neuro-otological assessment. The six framework question responses were compared against objective neuro-otological assessment or medical notes. The output was binary, with sensitivity and specificity calculated. RESULTS: Seventy-four adults completed the study: age range 20-81 years (mean 48 years, ±SD 15.05 years); ratio 2:1 female to male. The framework identified a vestibular diagnosis in 35 participants, with seven having two diagnoses. The framework was able to identify vestibular diagnoses in adults with primary mitochondrial disease, with a moderate (40-59) to very high (90-100) sensitivity and positive predictive value, and moderate to high (60-74) to very high (90-100) specificity and negative predictive value. CONCLUSIONS: Overall, the clinical framework identified common vestibular diagnoses with a moderate to very high specificity and sensitivity. This presents an opportunity for patients to access effective treatment in a timely manner, to reduce falls and improve quality of life.

Imaging swallowing function and the mechanisms driving dysphagia in inclusion body myositis.

Sporadic inclusion body myositis (IBM) is a progressive condition which commonly affects patients aged above 40. IBM does not respond to immunosuppression and no proven treatments are available. Up to 80% of patients develop some degree of swallowing impairment during the disease course. Dysphagia is a source of marked morbidity in IBM and predisposes patients to life-threatening complications such as aspiration pneumonia. The pathophysiology behind dysphagia in IBM is not fully understood. Evidence from imaging demonstrates that impaired swallowing is predominantly underpinned by oropharyngeal deficits. Changes in cricopharyngeal physiology is thought to be an important factor influencing dysphagia in IBM. However, it is unclear whether this is secondary to structural changes within the cricopharyngeus itself or driven by impairment of the muscles promoting pharyngeal clearance. The approach to dysphagia in IBM patients is limited by a lack of validated instruments to reliably assess swallowing function and an absence of effective therapeutic interventions derived from controlled trials targeting dysphagia. Imaging modalities such as the video fluoroscopic swallowing study (VFSS) are commonly used to evaluate dysphagia in IBM. Whilst VFSS is a commonly used technique in clinical practice; cumulative radiation exposure with repeated testing can be a limitation. Alternative imaging techniques could be developed further as outcome measures for assessing swallowing.In this review, we provide an overview of imaging techniques used to assess swallowing and the insight provided from such investigations into the mechanisms behind dysphagia in IBM. We suggest future directions for evaluation and outcome measurement of dysphagia in this population.

In silico versus functional characterization of genetic variants: lessons from muscle channelopathies.

Accurate determination of the pathogenicity of missense genetic variants of uncertain significance is a huge challenge for implementing genetic data in clinical practice. In silico predictive tools are used to score variants' pathogenicity. However, their value in clinical settings is often unclear, as they have not usually been validated against robust functional assays. We compared nine widely used in silico predictive tools, including more recently developed tools (EVE and REVEL) with detailed cell-based electrophysiology, for 126 CLCN1 variants discovered in patients with the skeletal muscle channelopathy myotonia congenita. We found poor accuracy for most tools. The highest accuracy was obtained with MutationTaster (84.58%) and REVEL (82.54%). Both of these scores showed poor specificity, although specificity was better using EVE. Combining methods based on concordance improved performance overall but still lacked specificity. Our calculated statistics for the predictive tools were different to reported values for other genes in the literature, suggesting that the utility of the tools varies between genes. Overall, current predictive tools for this chloride channel are not reliable for clinical use, and tools with better specificity are urgently required. Improving the accuracy of predictive tools is a wider issue and a huge challenge for effective clinical implementation of genetic data.

Unexpected frequency of the pathogenic AR CAG repeat expansion in the general population.

CAG repeat expansions in exon 1 of the AR gene on the X chromosome cause spinal and bulbar muscular atrophy, a male-specific progressive neuromuscular disorder associated with a variety of extra-neurological symptoms. The disease has a reported male prevalence of approximately 1:30 000 or less, but the AR repeat expansion frequency is unknown. We established a pipeline, which combines the use of the ExpansionHunter tool and visual validation, to detect AR CAG expansion on whole-genome sequencing data, benchmarked it to fragment PCR sizing, and applied it to 74 277 unrelated individuals from four large cohorts. Our pipeline showed sensitivity of 100% [95% confidence interval (CI) 90.8-100%], specificity of 99% (95% CI 94.2-99.7%), and a positive predictive value of 97.4% (95% CI 84.4-99.6%). We found the mutation frequency to be 1:3182 (95% CI 1:2309-1:4386, n = 117 734) X chromosomes-10 times more frequent than the reported disease prevalence. Modelling using the novel mutation frequency led to estimate disease prevalence of 1:6887 males, more than four times more frequent than the reported disease prevalence. This discrepancy is possibly due to underdiagnosis of this neuromuscular condition, reduced penetrance, and/or pleomorphic clinical manifestations.

Kennedy's disease.

A 57-year-old man developed worsening early morning headaches, muscle cramps and falls over 12 months. He had widespread fasciculation and was diagnosed with motor neurone disease, and treated with nocturnal hypoventilation. Based on this diagnosis, he made significant personal and financial decisions including retiring and selling his house. He subsequently developed a lump in his right breast and was found to have gynaecomastia. This triggered genetic testing for Kennedy's disease leading to the correct diagnosis. This case highlights an unusual presentation of a rare disease leading to misdiagnosis and major repercussions for the patient. Recent genetic analysis from the 100 000 genome project suggests Kennedy's disease may be four times more prevalent in the population than previously thought, highlighting the need to consider genetic testing, especially if there is a suggestion of multisystem disease.

Expanding SPTAN1 monoallelic variant associated disorders: From epileptic encephalopathy to pure spastic paraplegia and ataxia.

PURPOSE: Nonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants. METHODS: We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients. RESULTS: Statistically significant enrichment of rare (minor allele frequency < 1 × 10-5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10-5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants. CONCLUSION: We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.

Episodic Ataxia Type 1: Natural History and Effect on Quality of Life.

Episodic ataxia type 1 (EA1) is a rare autosomal potassium channelopathy, due to mutations in KCNA1. Patients have childhood onset of intermittent attacks of ataxia, dizziness or imbalance. In order to quantify the natural history of EA1, its effect on quality of life and in preparation for future clinical trials, we set up an international multi-centre study of EA1. We recruited thirty-three participants with EA1: twenty-three completed 1-year follow-up and eighteen completed 2-year follow-up. There was very little accumulation of disability or impairment over the course of the 2 years of the study. The outcome measures of ataxia (SARA and functional rating of ataxia) and the activities of daily living scale were largely stable over time. Self-reported health-related quality of life (SF-36) scores were lower across all domains than controls, in keeping with a chronic condition. Physical subdomain scores appeared to deteriorate over time, which seems to be driven by the female participants in the study. This is an interesting finding and warrants further study. Attacks of EA1 reported by participants in real time via an interactive voice response system showed that symptoms were not stereotyped; however, attack duration and frequency was stable between individuals. This large prospective study is the first ever completed in subjects with EA1. We document the natural history of the disorder over 2 years. These data will enable the development of outcome measures for clinical trials of treatment.

De novo KCNA6 variants with attenuated KV 1.6 channel deactivation in patients with epilepsy.

OBJECTIVE: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. METHODS: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two-electrode voltage clamp. RESULTS: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-lining S6 α-helix of KV 1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-expressed with wild-type KV 1.6 or KV 1.1 subunits. SIGNIFICANCE: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain-of-function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

Muscle MRI in periodic paralysis shows myopathy is common and correlates with intramuscular fat accumulation.

INTRODUCTION/AIMS: The periodic paralyses are muscle channelopathies: hypokalemic periodic paralysis (CACNA1S and SCN4A variants), hyperkalemic periodic paralysis (SCN4A variants), and Andersen-Tawil syndrome (KCNJ2). Both episodic weakness and disabling fixed weakness can occur. Little literature exists on magnetic resonance imaging (MRI) in muscle channelopathies. We undertake muscle MRI across all subsets of periodic paralysis and correlate with clinical features. METHODS: A total of 45 participants and eight healthy controls were enrolled and underwent T1-weighted and short-tau-inversion-recovery (STIR) MRI imaging of leg muscles. Muscles were scored using the modified Mercuri Scale. RESULTS: A total of 17 patients had CACNA1S variants, 16 SCN4A, and 12 KCNJ2. Thirty-one (69%) had weakness, and 9 (20%) required a gait-aid/wheelchair. A total of 78% of patients had intramuscular fat accumulation on MRI. Patients with SCN4A variants were most severely affected. In SCN4A, the anterior thigh and posterior calf were more affected, in contrast to the posterior thigh and posterior calf in KCNJ2. We identified a pattern of peri-tendinous STIR hyperintensity in nine patients. There were moderate correlations between Mercuri, STIR scores, and age. Intramuscular fat accumulation was seen in seven patients with no fixed weakness. DISCUSSION: We demonstrate a significant burden of disease in patients with periodic paralyses. MRI intramuscular fat accumulation may be helpful in detecting early muscle involvement, particularly in those without fixed weakness. Longitudinal studies are needed to assess the role of muscle MRI in quantifying disease progression over time and as a potential biomarker in clinical trials.

Factors associated with the severity of COVID-19 outcomes in people with neuromuscular diseases: Data from the International Neuromuscular COVID-19 Registry.

BACKGROUND AND PURPOSE: Clinical outcome information on patients with neuromuscular diseases (NMDs) who have been infected with SARS-CoV-2 is limited. The aim of this study was to determine factors associated with the severity of COVID-19 outcomes in people with NMDs. METHODS: Cases of NMD, of any age, and confirmed/presumptive COVID-19, submitted to the International Neuromuscular COVID-19 Registry up to 31 December 2021, were included. A mutually exclusive ordinal COVID-19 severity scale was defined as follows: (1) no hospitalization; (2) hospitalization without oxygenation; (3) hospitalization with ventilation/oxygenation; and (4) death. Multivariable ordinal logistic regression analyses were used to estimate odds ratios (ORs) for severe outcome, adjusting for age, sex, race/ethnicity, NMD, comorbidities, baseline functional status (modified Rankin scale [mRS]), use of immunosuppressive/immunomodulatory medication, and pandemic calendar period. RESULTS: Of 315 patients from 13 countries (mean age 50.3 [±17.7] years, 154 [48.9%] female), 175 (55.5%) were not hospitalized, 27 (8.6%) were hospitalized without supplemental oxygen, 91 (28.9%) were hospitalized with ventilation/supplemental oxygen, and 22 (7%) died. Higher odds of severe COVID-19 outcomes were observed for: age ≥50 years (50-64 years: OR 2.4, 95% confidence interval [CI] 1.33-4.31; >64 years: OR 4.16, 95% CI 2.12-8.15; both vs. <50 years); non-White race/ethnicity (OR 1.81, 95% CI 1.07-3.06; vs. White); mRS moderately severe/severe disability (OR 3.02, 95% CI 1.6-5.69; vs. no/slight/moderate disability); history of respiratory dysfunction (OR 3.16, 95% CI 1.79-5.58); obesity (OR 2.24, 95% CI 1.18-4.25); ≥3 comorbidities (OR 3.2, 95% CI 1.76-5.83; vs. ≤2; if comorbidity count used instead of specific comorbidities); glucocorticoid treatment (OR 2.33, 95% CI 1.14-4.78); and Guillain-Barré syndrome (OR 3.1, 95% CI 1.35-7.13; vs. mitochondrial disease). CONCLUSIONS: Among people with NMDs, there is a differential risk of COVID-19 outcomes according to demographic and clinical characteristics. These findings could be used to develop tailored management strategies and evidence-based recommendations for NMD patients.

Translating genetic and functional data into clinical practice: a series of 223 families with myotonia.

High-throughput DNA sequencing is increasingly employed to diagnose single gene neurological and neuromuscular disorders. Large volumes of data present new challenges in data interpretation and its useful translation into clinical and genetic counselling for families. Even when a plausible gene is identified with confidence, interpretation of the clinical significance and inheritance pattern of variants can be challenging. We report our approach to evaluating variants in the skeletal muscle chloride channel ClC-1 identified in 223 probands with myotonia congenita as an example of these challenges. Sequencing of CLCN1, the gene that encodes CLC-1, is central to the diagnosis of myotonia congenita. However, interpreting the pathogenicity and inheritance pattern of novel variants is notoriously difficult as both dominant and recessive mutations are reported throughout the channel sequence, ClC-1 structure-function is poorly understood and significant intra- and interfamilial variability in phenotype is reported. Heterologous expression systems to study functional consequences of CIC-1 variants are widely reported to aid the assessment of pathogenicity and inheritance pattern. However, heterogeneity of reported analyses does not allow for the systematic correlation of available functional and genetic data. We report the systematic evaluation of 95 CIC-1 variants in 223 probands, the largest reported patient cohort, in which we apply standardized functional analyses and correlate this with clinical assessment and inheritance pattern. Such correlation is important to determine whether functional data improves the accuracy of variant interpretation and likely mode of inheritance. Our data provide an evidence-based approach that functional characterization of ClC-1 variants improves clinical interpretation of their pathogenicity and inheritance pattern, and serve as reference for 34 previously unreported and 28 previously uncharacterized CLCN1 variants. In addition, we identify novel pathogenic mechanisms and find that variants that alter voltage dependence of activation cluster in the first half of the transmembrane domains and variants that yield no currents cluster in the second half of the transmembrane domain. None of the variants in the intracellular domains were associated with dominant functional features or dominant inheritance pattern of myotonia congenita. Our data help provide an initial estimate of the anticipated inheritance pattern based on the location of a novel variant and shows that systematic functional characterization can significantly refine the assessment of risk of an associated inheritance pattern and consequently the clinical and genetic counselling.

Andersen-Tawil syndrome: deep phenotyping reveals significant cardiac and neuromuscular morbidity.

Andersen-Tawil syndrome is a neurological channelopathy caused by mutations in the KCNJ2 gene that encodes the ubiquitously expressed Kir2.1 potassium channel. The syndrome is characterized by episodic weakness, cardiac arrythmias and dysmorphic features. However, the full extent of the multisystem phenotype is not well described. In-depth, multisystem phenotyping is required to inform diagnosis and guide management. We report our findings following deep multimodal phenotyping across all systems in a large case series of 69 total patients, with comprehensive data for 52. As a national referral centre, we assessed point prevalence and showed it is higher than previously reported, at 0.105 per 100 000 population in England. While the classical phenotype of episodic weakness is recognized, we found that a quarter of our cohort have fixed myopathy and 13.5% required a wheelchair or gait aid. We identified frequent fat accumulation on MRI and tubular aggregates on muscle biopsy, emphasizing the active myopathic process underpinning the potential for severe neuromuscular disability. Long exercise testing was not reliable in predicting neuromuscular symptoms. A normal long exercise test was seen in five patients, of whom four had episodic weakness. Sixty-seven per cent of patients treated with acetazolamide reported a good neuromuscular response. Thirteen per cent of the cohort required cardiac defibrillator or pacemaker insertion. An additional 23% reported syncope. Baseline electrocardiograms were not helpful in stratifying cardiac risk, but Holter monitoring was. A subset of patients had no cardiac symptoms, but had abnormal Holter monitor recordings which prompted medication treatment. We describe the utility of loop recorders to guide management in two such asymptomatic patients. Micrognathia was the most commonly reported skeletal feature; however, 8% of patients did not have dysmorphic features and one-third of patients had only mild dysmorphic features. We describe novel phenotypic features including abnormal echocardiogram in nine patients, prominent pain, fatigue and fasciculations. Five patients exhibited executive dysfunction and slowed processing which may be linked to central expression of KCNJ2. We report eight new KCNJ2 variants with in vitro functional data. Our series illustrates that Andersen-Tawil syndrome is not benign. We report marked neuromuscular morbidity and cardiac risk with multisystem involvement. Our key recommendations include proactive genetic screening of all family members of a proband. This is required, given the risk of cardiac arrhythmias among asymptomatic individuals, and a significant subset of Andersen-Tawil syndrome patients have no (or few) dysmorphic features or negative long exercise test. We discuss recommendations for increased cardiac surveillance and neuropsychometry testing.

Forecasting stroke-like episodes and outcomes in mitochondrial disease.

In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.

A Method for Evaluating Brain Deformation Under Sagittal Blunt Impacts Using a Half-Skull Human-Scale Surrogate.

Trauma to the brain is a biomechanical problem where the initiating event is a dynamic loading (blunt, inertial, blast) to the head. To understand the relationship between the mechanical parameters of the injury and the spatial and temporal deformation patterns in the brain, there is a need to develop a reusable and adaptable experimental traumatic brain injury (TBI) model that can measure brain motion under varying parameters. In this effort, we aim to directly measure brain deformation (strain and strain rates) in different brain regions in a human head model using a drop tower. METHODS: Physical head models consisting of a half, sagittal plane skull, brain, and neck were constructed and subjected to crown and frontal impacts at two impact speeds. All tests were recorded with a high-speed camera at 1000 frames per second. Motion of visual markers within brain surrogates were used to track deformations and calculate spatial strain histories in 6 brain regions of interest. Principal strains, strain rates and strain impulses were calculated and reported. RESULTS: Higher impact velocities corresponded to higher strain values across all impact scenarios. Crown impacts were characterized by high, long duration strains distributed across the parietal, frontal and hippocampal regions whereas frontal impacts were characterized by sharply rising and falling strains primarily found in the parietal, frontal, hippocampal and occipital regions. High strain rates were associated with short durations and impulses indicating fast but short-lived strains. 2.23 m/s (5 mph) crown impacts resulted in 53% of the brain with shear strains higher than 0.15 verses 32% for frontal impacts. CONCLUSIONS: The results reveal large differences in the spatial and temporal strain responses between crown and forehead impacts. Overall, the results suggest that for the same speed, crown impact leads to higher magnitude strain patterns than a frontal impact. The data provided by this model provides unique insight into the spatial and temporal deformation patterns that have not been provided by alternate surrogate models. The model can be used to investigate how anatomical, material and loading features and parameters can affect deformation patterns in specific regions of interest in the brain.