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.

Proteomic Investigation of Differential Interactomes of Glypican 1 and a Putative Disease-Modifying Variant of Ataxia.

In a currently 13-year-old girl of consanguineous Turkish parents, who developed unsteady gait and polyneuropathy at the ages of 3 and 6 years, respectively, we performed whole genome sequencing and identified a biallelic missense variant c.424C>T, p.R142W in glypican 1 (GPC1) as a putative disease-associated variant. Up to date, GPC1 has not been associated with a neuromuscular disorder, and we hypothesized that this variant, predicted as deleterious, may be causative for the disease. Using mass spectrometry-based proteomics, we investigated the interactome of GPC1 WT and the missense variant. We identified 198 proteins interacting with GPC1, of which 16 were altered for the missense variant. This included CANX as well as vacuolar ATPase (V-ATPase) and the mammalian target of rapamycin complex 1 (mTORC1) complex members, whose dysregulation could have a potential impact on disease severity in the patient. Importantly, these proteins are novel interaction partners of GPC1. At 10.5 years, the patient developed dilated cardiomyopathy and kyphoscoliosis, and Friedreich's ataxia (FRDA) was suspected. Given the unusually severe phenotype in a patient with FRDA carrying only 104 biallelic GAA repeat expansions in FXN, we currently speculate that disturbed GPC1 function may have exacerbated the disease phenotype. LC-MS/MS data are accessible in the ProteomeXchange Consortium (PXD040023).

Ecology of fear in highly invasive fish revealed by robots.

Invasive species threaten biodiversity and ecosystem functioning. We develop an innovative experimental approach, integrating biologically inspired robotics, time-series analysis, and computer vision, to build a detailed profile of the effects of non-lethal stress on the ecology and evolution of mosquitofish (Gambusia holbrooki)-a global pest. We reveal that brief exposures to a robotic predator alter mosquitofish behavior, increasing fear and stress responses, and mitigate the impact of mosquitofish on native tadpoles (Litoria moorei) in a cause-and-effect fashion. Effects of predation risk from the robot carry over to routine activity and feeding rate of mosquitofish weeks after exposure, resulting in weight loss, variation in body shape, and reduction in the fertility of both sexes-impairing survival, reproduction, and ecological success. We capitalize on evolved responses of mosquitofish to reduce predation risk-neglected in biological control practices-and provide scientific foundations for widespread use of state-of-the-art robotics in ecology and evolution research.

Genomic variants causing mitochondrial dysfunction are common in hereditary lower motor neuron disease.

Hereditary lower motor neuron diseases (LMND) other than 5q-spinal muscular atrophy (5q-SMA) can be classified according to affected muscle groups. Proximal and distal forms of non-5q-SMA represent a clinically and genetically heterogeneous spectrum characterized by significant overlaps with axonal forms of Charcot-Marie-Tooth (CMT) disease. A consensus for the best approach to molecular diagnosis needs to be reached, especially in light of continuous novel gene discovery and falling costs of next-generation sequencing (NGS). We performed exome sequencing (ES) in 41 families presenting with non-5q-SMA or axonal CMT, 25 of which had undergone a previous negative neuromuscular disease (NMD) gene panel analysis. The total diagnostic yield of ES was 41%. Diagnostic success in the cohort with a previous NMD-panel analysis was significantly extended by ES, primarily due to novel gene associated-phenotypes and uncharacteristic phenotypic presentations. We recommend early ES for individuals with hereditary LMND presenting uncharacteristic or significantly overlapping features. As mitochondrial dysfunction was the underlying pathomechanism in 47% of the solved individuals, we highlight the sensitivity of the anterior horn cell and peripheral nerve to mitochondrial imbalance as well as the necessity to screen for mitochondrial disorders in individuals presenting predominant lower motor neuron symptoms.

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy.

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.

De novo DNM1L variant presenting with severe muscular atrophy, dystonia and sensory neuropathy.

DNM1L encodes dynamin-related protein 1 (DRP1), a multi-domain GTPase essential for mitochondrial and peroxisomal division. Autosomal dominant and recessive variants in DNM1L cause encephalopathy due to defective mitochondrial and peroxisomal fission 1 (EMPF1), which presents as a complex and clinically heterogeneous neurological disorder of variable severity, often accompanied by seizures. Clinical features are diverse, and no clear phenotype-genotype correlations were drawn to date. DNM1L-related sensory neuropathy has recently been reported as a predominant feature in one case with a de novo variant in the GTPase domain. Herein we present a second case with DNM1L-related sensory neuropathy as the predominant underlying feature without motor neuron involvement, which resulted in severe muscular atrophy and generalized dystonia.

Acute Citalopram administration alters zebrafish social dynamics in a behavioral teleporting experiment.

In the Star Trek universe, Scotty can "beam you up" and teleport you to a location. Even though science fiction is yet to be reality, is teleporting still possible? Recently, we have developed a robotic platform that is capable of teleporting the behavior of fish from one tank to another through biologically-inspired robotic replicas which map the behavior of a fish from a tank to the other one. Our results indicate that behavioral teleporting is a promising tool to study the biological determinants of behavior. Here we demonstrate its use in pharmacological research, by investigating the effects of selective serotonin reuptake inhibitors of social interactions.

Acute Citalopram administration modulates anxiety in response to the context associated with a robotic stimulus in zebrafish.

BACKGROUND: Anxiety represents one of the most urgent health challenges in Western Countries, where it is associated with major medical and societal costs. A common therapeutic approach is the use of selective serotonin reuptake inhibitors, such as Citalopram. However, this treatment of choice is characterized by incomplete efficacy and potential side effects. Preclinical research is needed to detail the mechanisms underlying therapeutic efficacy of available treatments. METHODS: Zebrafish, a rapidly emerging model species, constitutes an excellent candidate for high-throughput studies in behavioral pharmacology. Here, we present a robotics-based experimental paradigm to investigate the effects of acute Citalopram administration on conditioned place aversion. We trained adult subjects in a three-partitioned tank, consisting of one central and two lateral compartments: the latter were associated either with a fear eliciting robotic stimulus or with an empty environment. Following training, we implemented an automated three-dimensional tracking system to assess the spatial association and detail individual phenotype in a stimulus-free test session. RESULTS: We observed a linear dose-response profile with respect to geotaxis, with increasing Citalopram concentrations reducing the tendency to swim near the bottom of the tank. Although control subjects failed to exhibit the predicted conditioned aversion, we found preliminary evidence that Citalopram may affect sexes differentially, with male subjects showing increased conditioned aversion at low Citalopram concentration. CONCLUSIONS: Experimental paradigms based on robotics and three-dimensional tracking can contribute methodological advancements in zebrafish behavioral psychopharmacology.

Novel variants broaden the phenotypic spectrum of PLEKHG5-associated neuropathies.

BACKGROUND AND PURPOSE: Pathogenic variants in PLEKHG5 have been reported to date to be causative in three unrelated families with autosomal recessive intermediate Charcot-Marie-Tooth disease (CMT) and in one consanguineous family with spinal muscular atrophy (SMA). PLEKHG5 is known to be expressed in the human peripheral nervous system, and previous studies have shown its function in axon terminal autophagy of synaptic vesicles, lending support to its underlying pathogenetic mechanism. Despite this, there is limited knowledge of the clinical and genetic spectrum of disease. METHODS: We leverage the diagnostic utility of exome and genome sequencing and describe novel biallelic variants in PLEKHG5 in 13 individuals from nine unrelated families originating from four different countries. We compare our phenotypic and genotypic findings with a comprehensive review of cases previously described in the literature. RESULTS: We found that patients presented with variable disease severity at different ages of onset (8-25 years). In our cases, weakness usually started proximally, progressing distally, and can be associated with intermediate slow conduction velocities and minor clinical sensory involvement. We report three novel nonsense and four novel missense pathogenic variants associated with these PLEKHG5-associated neuropathies, which are phenotypically spinal muscular atrophy (SMA) or intermediate Charcot-Marie-Tooth disease. CONCLUSIONS: PLEKHG5-associated neuropathies should be considered as an important differential in non-5q SMAs even in the presence of mild sensory impairment and a candidate causative gene for a wide range of hereditary neuropathies. We present this series of cases to further the understanding of the phenotypic and molecular spectrum of PLEKHG5-associated diseases.

De Novo and Inherited Variants in GBF1 are Associated with Axonal Neuropathy Caused by Golgi Fragmentation.

Distal hereditary motor neuropathies (HMNs) and axonal Charcot-Marie-Tooth neuropathy (CMT2) are clinically and genetically heterogeneous diseases characterized primarily by motor neuron degeneration and distal weakness. The genetic cause for about half of the individuals affected by HMN/CMT2 remains unknown. Here, we report the identification of pathogenic variants in GBF1 (Golgi brefeldin A-resistant guanine nucleotide exchange factor 1) in four unrelated families with individuals affected by sporadic or dominant HMN/CMT2. Genomic sequencing analyses in seven affected individuals uncovered four distinct heterozygous GBF1 variants, two of which occurred de novo. Other known HMN/CMT2-implicated genes were excluded. Affected individuals show HMN/CMT2 with slowly progressive distal muscle weakness and musculoskeletal deformities. Electrophysiological studies confirmed axonal damage with chronic neurogenic changes. Three individuals had additional distal sensory loss. GBF1 encodes a guanine-nucleotide exchange factor that facilitates the activation of members of the ARF (ADP-ribosylation factor) family of small GTPases. GBF1 is mainly involved in the formation of coatomer protein complex (COPI) vesicles, maintenance and function of the Golgi apparatus, and mitochondria migration and positioning. We demonstrate that GBF1 is present in mouse spinal cord and muscle tissues and is particularly abundant in neuropathologically relevant sites, such as the motor neuron and the growth cone. Consistent with the described role of GBF1 in Golgi function and maintenance, we observed marked increase in Golgi fragmentation in primary fibroblasts derived from all affected individuals in this study. Our results not only reinforce the existing link between Golgi fragmentation and neurodegeneration but also demonstrate that pathogenic variants in GBF1 are associated with HMN/CMT2.

Zebrafish exhibit associative learning for an aversive robotic stimulus.

Zebrafish have quickly emerged as a species of choice in preclinical research, holding promise to advance the field of behavioral pharmacology through high-throughput experiments. Besides biological and heuristic considerations, zebrafish also constitute a fundamental tool that fosters the replacement of mammals with less sentient experimental subjects. Notwithstanding these features, experimental paradigms to investigate emotional and cognitive domains in zebrafish are still limited. Studies on emotional memories have provided sound methodologies to investigate fear conditioning in zebrafish, but these protocols may still benefit from a reconsideration of the independent variables adopted to elicit aversion. Here, we designed a fear-conditioning paradigm in which wild-type zebrafish were familiarized over six training sessions with an empty compartment and a fear-eliciting one. The fearful stimulus was represented by three zebrafish replicas exhibiting a fully synchronized and polarized motion as they were maneuvered along 3D trajectories by a robotic platform. When allowed to freely swim between the two compartments in the absence of the robotic stimulus (test session), zebrafish displayed a marked avoidance of the stimulus-paired one. To investigate whether fear conditioning was modulated by psychoactive compounds, two groups of zebrafish were administered ethanol (0.25% and 1.00%, ethanol/water, by volume) a few minutes before the test session. We observed that ethanol administration abolished the conditioned avoidance of the stimulus-paired compartment. Ultimately, this study confirms that robotic stimuli may be used in the design of fear-conditioning paradigms, which are sensitive to pharmacological manipulations.

Behavioral Teleporting of Individual Ethograms onto Inanimate Robots: Experiments on Social Interactions in Live Zebrafish.

Social behavior is widespread in the animal kingdom, and it remarkably influences human personal and professional lives. However, a thorough understanding of the mechanisms underlying social behavior is elusive. Integrating the seemingly different fields of robotics and preclinical research could bring new insight on social behavior. Toward this aim, we established "behavioral teleporting" as an experimental solution to independently manipulate multiple factors underpinning social interactions. Behavioral teleporting consists of real-time transfer of the complete ethogram of a live zebrafish onto a remotely-located robotic replica. Through parallel and simultaneous behavioral teleporting, we studied the interaction between two live fish swimming in remotely-located tanks: each live fish interacted with an inanimate robot that mirrored the behavior of the other fish, and the morphology of each robot was independently tailored. Our results indicate that behavioral teleporting can preserve natural interaction between two live animals, while allowing fine control over morphological features that modulate social behavior.

Hereditary polyneuropathy with optic atrophy due to PDXK variant leading to impaired Vitamin B6 metabolism.

PDXK encodes for a pyridoxal kinase, which converts inactive B6 vitamers to the active cofactor pyridoxal 5'-phosphate (PLP). Recently, biallelic pathogenic variants in PDXK were shown to cause axonal Charcot-Marie-Tooth disease with optic atrophy that responds to PLP supplementation. We present two affected siblings carrying a novel biallelic missense PDXK variant with a similar phenotype with earlier onset. After detection of a novel PDXK variant using Whole Exome Sequencing, we confirmed pathogenicity through in silico protein structure analysis, determination of pyridoxal kinase activity using liquid chromatography-tandem mass spectrometry, and measurement of plasma PLP concentrations using high performance liquid chromatography. Our in silico analysis shows a potential effect on PDXK dimer stability, as well as a putative effect on posttranslational ubiquitination that is predicted to lead to increased protein degradation. We demonstrate that the variant leads to almost complete loss of PDXK enzymatic activity and low PLP levels. Our patients' early diagnosis and prompt PLP replacement restored the PLP plasma levels, enabling long-term monitoring of clinical outcomes. We recommend that patients presenting with similar phenotype should be screened for PDXK mutations, as this is a rare opportunity for treatment.

Correction: The genomic and clinical landscape of fetal akinesia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Twenty-Five Years of Spinal Muscular Atrophy Research: From Phenotype to Genotype to Therapy, and What Comes Next.

Twenty-five years ago, the underlying genetic cause for one of the most common and devastating inherited diseases in humans, spinal muscular atrophy (SMA), was identified. Homozygous deletions or, rarely, subtle mutations of SMN1 cause SMA, and the copy number of the nearly identical copy gene SMN2 inversely correlates with disease severity. SMA has become a paradigm and a prime example of a monogenic neurological disorder that can be efficiently ameliorated or nearly cured by novel therapeutic strategies, such as antisense oligonucleotide or gene replacement therapy. These therapies enable infants to survive who might otherwise have died before the age of two and allow individuals who have never been able to sit or walk to do both. The major milestones on the road to these therapies were to understand the genetic cause and splice regulation of SMN genes, the disease's phenotype-genotype variability, the function of the protein and the main affected cellular pathways and tissues, the disease's pathophysiology through research on animal models, the windows of opportunity for efficient treatment, and how and when to treat patients most effectively.This review aims to bridge our knowledge from phenotype to genotype to therapy, not only highlighting the significant advances so far but also speculating about the future of SMA screening and treatment.

The genomic and clinical landscape of fetal akinesia.

PURPOSE: Fetal akinesia has multiple clinical subtypes with over 160 gene associations, but the genetic etiology is not yet completely understood. METHODS: In this study, 51 patients from 47 unrelated families were analyzed using next-generation sequencing (NGS) techniques aiming to decipher the genomic landscape of fetal akinesia (FA). RESULTS: We have identified likely pathogenic gene variants in 37 cases and report 41 novel variants. Additionally, we report putative pathogenic variants in eight cases including nine novel variants. Our work identified 14 novel disease-gene associations for fetal akinesia: ADSSL1, ASAH1, ASPM, ATP2B3, EARS2, FBLN1, PRG4, PRICKLE1, ROR2, SETBP1, SCN5A, SCN8A, and ZEB2. Furthermore, a sibling pair harbored a homozygous copy-number variant in TNNT1, an ultrarare congenital myopathy gene that has been linked to arthrogryposis via Gene Ontology analysis. CONCLUSION: Our analysis indicates that genetic defects leading to primary skeletal muscle diseases might have been underdiagnosed, especially pathogenic variants in RYR1. We discuss three novel putative fetal akinesia genes: GCN1, IQSEC3 and RYR3. Of those, IQSEC3, and RYR3 had been proposed as neuromuscular disease-associated genes recently, and our findings endorse them as FA candidate genes. By combining NGS with deep clinical phenotyping, we achieved a 73% success rate of solved cases.

Application of symbolic recurrence to experimental data, from firearm prevalence to fish swimming.

Recurrence plots and recurrence quantification analysis are powerful tools to study the behavior of dynamical systems. What we learn through these tools is typically determined by the choice of a distance threshold in the phase space, which introduces arbitrariness in the definition of recurrence. Not only does symbolic recurrence overcome this difficulty, but also it offers a richer representation that book-keeps the recurrent portions of the phase space. Using symbolic recurrences, we can construct recurrence plots, perform quantification analysis, and examine causal links between dynamical systems from their time-series. Although previous efforts have demonstrated the feasibility of such a symbolic framework on synthetic data, the study of real time-series remains elusive. Here, we seek to bridge this gap by systematically examining a wide range of experimental datasets, from firearm prevalence and media coverage in the United States to the effect of sex on the interaction of swimming fish. This work offers a compelling demonstration of the potential of symbolic recurrence in the study of real-world applications across different research fields while providing a computer code for researchers to perform their own time-series explorations.

Novel mutations in SLC6A5 with benign course in hyperekplexia.

Infants suffering from life-threatening apnea, stridor, cyanosis, and increased muscle tone may often be misdiagnosed with infantile seizures and inappropriately treated because of lack and delay in genetic diagnosis. Here, we report a patient with increased muscle tone after birth and hypertonic attacks with life-threatening apnea but no epileptiform patterns in EEG recordings. We identified novel compound heterozygous variants in SLC6A5 (NM_004211.4:c.[1429T > C];[1430delC]) by trio whole-exome sequencing, containing a base deletion inherited by the asymptomatic mother leading to a frameshift (c.1430delC, p.Ser477PhefsTer9) and a de novo base exchange leading to an amino acid change (c.1429T > C, p.Ser477Pro). To date, there are four known disease-associated genes for primary hyperekplexia, all of which are involved in the functioning of glycinergic synapses. SLC6A5 encodes the sodium- and chloride-dependent glycine transporter 2 (GlyT2), which recaptures glycine, a major inhibitory transmitter in the brainstem and spinal cord. The diagnosis altered the patient's medical care to his benefit because SLC6A5 mutations with rather benign courses of hyperekplexia may be spared of needless pharmacotherapy. Symptoms eventually decreased in frequency until about once in 2 mo at 2 yr age. We present the first report of halting hyperekplexia episodes by maternal soothing in multiple instances. We highlight the importance of clarifying the genetic diagnosis by rapid next-generation sequencing techniques in this group of infantile apneic attacks with hyperekplexia due to the broad differential diagnoses.