Spinal Muscular Atrophy Phenotype Research Articles

Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network

IntroductionMolecular chaperones and co-chaperones are highly conserved cellular components that perform a variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an assembly chaperone and serves as a paradigm for studying how specific RNAs are identified and paired with their client substrate proteins to form RNPs. SMN is the eponymous component of a large complex, required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs), that localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN protein forms the oligomeric core of this complex, and missense mutations in the human SMN1 gene are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known. However, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood.MethodsGiven the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. We carried out affinity purification mass spectrometry (AP-MS) of Drosophila SMN complexes using fly lines exclusively expressing either wildtype or SMA-causing missense alleles.ResultsBioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially associated with SMA-causing alleles of SMN.DiscussionHsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Complex SMN Hybrids Detected in a Cohort of 31 Patients With Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by the loss or presence of point pathogenic variants in the SMN1 gene. The main positive modifier of the SMA phenotype is the number of copies of the SMN2 gene, a paralog of SMN1, which only produces around 10%-15% of functional SMN protein. The SMN2 copy number is inversely correlated with phenotype severity; however, discrepancies between the SMA type and the SMN2 copy number have been reported. The presence of SMN2-SMN1 hybrids has been proposed as a possible modifier of SMA disease. We studied 31 patients with SMA, followed at a single center and molecularly diagnosed by Multiplex Ligand-Dependent Probe Amplification (MLPA), with a specific next-generation sequencing protocol to investigate their SMN2 genes in depth. Hybrid characterization also included bioinformatics haplotype phasing and specific PCRs to resolve each SMN2-SMN1 hybrid structure. We detected SMN2-SMN1 hybrid genes in 45.2% of the patients (14/31), the highest rate reported to date. This represents a total of 25 hybrid alleles, with 9 different structures, of which only 4 are detectable by MLPA. Of particular interest were 2 patients who presented 4 SMN2-SMN1 hybrid copies each and no pure SMN2 copies, an event reported here for the first time. No clear trend between the presence of hybrids and a milder phenotype was observed, although 5 of the patients with hybrid copies showed a better-than-expected phenotype. The higher hybrid detection rate in our cohort may be due to both the methodology applied, which allows an in-depth characterization of the SMN genes and the ethnicity of the patients, mainly of African origin. Although hybrid genes have been proposed to be beneficial for patients with SMA, our work revealed great complexity and variability between hybrid structures; therefore, each hybrid structure should be studied independently to determine its contribution to the SMA phenotype. Large-scale studies are needed to gain a better understanding of the function and implications of SMN2-SMN1 hybrid copies, improving genotype-phenotype correlations and prediction of the evolution of patients with SMA.

The Spectrum of Childhood-Onset Lower Motor Neuron Disease of Spinal Muscular Atrophy Phenotype Associated with Survival Motor Neuron-2 Gene Deletion

AbstractPediatric lower motor neuron disease is clinically and genetically heterogeneous. We characterized disease progression among children with the spinal muscular atrophy (SMA) phenotype having 2:0 (group A) and 2:1 (group B) of the survival motor neuron 1/2 (SMN1:SMN2) genotype over 1 year. We included children aged 0 to 12 with the SMA phenotype between January 2018 and December 2021. Their demographic, clinical (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders [CHOP-INTEND] scores), electrophysiological, radiological, and genetic data were collected from past medical records. The sequential CHOP-INTEND scores and the compound muscle action potential (CMAP) amplitudes were compared using an analysis of covariance test, controlling for age and sex. A linear regression was run to determine the association between the ages of the patients and the CHOP-INTEND scores. Among nine children in group A and six in group B, the decline of the mean (standard deviation) CHOP-INTEND score from the initial value to the 12th-month follow-up value was significant only in group A. CHOP-INTEND scores did not significantly differ between the two groups at the first admission but were significantly lower in group A at the subsequent visits. Group A patients had significantly lower CMAP amplitudes than patients in group B. There was a moderate, negative association between the age of patients and the CHOP-INTEND scores in group A. Group A patients had a significantly higher age-dependent decline in CHOP-INTEND scores and CMAP values than group B, although their age and the severity of weakness did not significantly differ at presentation.

Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.

Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN protein is the eponymous component of a large complex required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs) and localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially interacted with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Hepatocyte-intrinsic SMN deficiency drives metabolic dysfunction and liver steatosis in spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is typically characterized as a motor neuron disease, but extra-neuronal phenotypes are present in almost every organ in severely affected patients and animal models. Extra-neuronal phenotypes were previously underappreciated as patients with severe SMA phenotypes usually died in infancy; however, with current treatments for motor neurons increasing patient lifespan, impaired function of peripheral organs may develop into significant future comorbidities and lead to new treatment-modified phenotypes. Fatty liver is seen in SMA animal models , but generalizability to patients and whether this is due to hepatocyte-intrinsic Survival Motor Neuron (SMN) protein deficiency and/or subsequent to skeletal muscle denervation is unknown. If liver pathology in SMA is SMN-dependent and hepatocyte-intrinsic, this suggests SMN repleting therapies must target extra-neuronal tissues and motor neurons for optimal patient outcome. Here we showed that fatty liver is present in SMA and that SMA patient-specific iHeps were susceptible to steatosis. Using proteomics, functional studies and CRISPR/Cas9 gene editing, we confirmed that fatty liver in SMA is a primary SMN-dependent hepatocyte-intrinsic liver defect associated with mitochondrial and other hepatic metabolism implications. These pathologies require monitoring and indicate need for systematic clinical surveillance and additional and/or combinatorial therapies to ensure continued SMA patient health.

Risdiplam for the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a devastating disease that is the leading genetic cause of death in infants and young children. It includes a broad spectrum of phenotypes that are classified into clinical groups based on the age of onset and maximum motor function achieved. The most common form of SMA is due to a defect in the survival motor neuron 1 gene (SMN1) localized to 5q11.2-q13.3. The development of clinical symptoms and disease progression is thought to be due to decreased levels of survival motor neuron (SMN) protein. SMA type 1 results in almost inevitable mortality within the first 2 years of life. The first two drugs approved globally for the treatment of SMA were the antisense oligonucleotide nusinersen (Spinraza), and the gene therapy onasemnogene abeparvovec-xioi (Zolgensma). Both interventions have approval and restrictions on use in different countries around the world. Despite these approved therapies, the medical unmet need in SMA (the majority of patients with SMA are not on a disease-modifying therapy) remains high with therapies in the pipeline to address some of the remaining limitations. The third and more recently approved drug for SMA is risdiplam (Evrysdi), an orally administered, centrally and peripherally distributed small molecule that modulates SMN2 pre-mRNA splicing toward the production of full-length SMN2 mRNA to increase functional SMN protein levels. In Russia the drug risdiplam was approved for use on November 26, 2020 with indications for the treatment of SMA in patients aged 2 months and older, and in 2023 the indications were expanded - use is allowed starting from the birth. Risdiplam is widely distributed into the CNS and peripheral tissues including muscles. Following risdiplam administration, SMN protein levels compared with baseline levels increase between 2- and 6-fold depending on the SMA phenotype treated. The risdiplam clinical development program currently has four ongoing clinical trials assessing its safety and efficacy. Clinical trials included more than 450 patients receiving risdiplam to date, has been well tolerated and no treatment-related safety findings leading to study withdrawal have been observed. Data from real clinical practice - more than 11.000 patients worldwide receive therapy with risdiplam, also confirm the safety and good tolerability of the drug.

Mitigating aberrant Cdk5 activation alleviates mitochondrial defects and motor neuron disease symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the top genetic cause of infant mortality, is characterized by motor neuron degeneration. Mechanisms underlying SMA pathogenesis remain largely unknown. Here, we report that the activity of cyclin-dependent kinase 5 (Cdk5) and the conversion of its activating subunit p35 to the more potent activator p25 are significantly up-regulated in mouse models and human induced pluripotent stem cell (iPSC) models of SMA. The increase of Cdk5 activity occurs before the onset of SMA phenotypes, suggesting that it may be an initiator of the disease. Importantly, aberrant Cdk5 activation causes mitochondrial defects and motor neuron degeneration, as the genetic knockout of p35 in an SMA mouse model rescues mitochondrial transport and fragmentation defects, and alleviates SMA phenotypes including motor neuron hyperexcitability, loss of excitatory synapses, neuromuscular junction denervation, and motor neuron degeneration. Inhibition of the Cdk5 signaling pathway reduces the degeneration of motor neurons derived from SMA mice and human SMA iPSCs. Altogether, our studies reveal a critical role for the aberrant activation of Cdk5 in SMA pathogenesis and suggest a potential target for therapeutic intervention.

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

Lessons from Real Life Experience: Importance of In-House Sequencing and Smart Ratio-Based Real-Time PCR Outperform Multiplex Ligation-Dependent Probe Amplification in Prenatal Diagnosis for Spinal Muscular Atrophy: Bench to Bedside Diagnosis.

Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder caused by the presence of pathogenic variants in the SMN gene. As it is the leading inherited cause of infant mortality, identification of SMN gene pathogenic variant carriers is important for diagnostic purposes with effective genetic counseling. Multiple ligation probe analysis (MLPA), a probe-based method, is considered as the gold standard for SMA carrier analysis. However, MLPA might give false-negative results in cases with variations in the probe-binding regions. Here, we present a case born to consanguineous SMA carrier parents. Prenatal diagnosis with MLPA failed to detect the compound heterozygous mutant state of the proband and she was born unfortunately with SMA phenotype. Further analysis with a real-time polymerase chain reaction kit was able to detect the compound heterozygous state of the patient and was confirmed with targeted next-generation sequencing technology.

Efficacy of risdiplam in spinal muscular atrophy: A systematic review and meta-analysis.

This systematic review and meta-analysis aimed to assess the efficacy and safety of risdiplam on motor and respiratory function in spinal muscular atrophy (SMA). We systematically searched Medline, Scopus, Web of Science, and the Cochrane Library from inception to March 2023. We included pre-post studies that determined the effect of risdiplam on the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND), the 32-item Motor Function Measure (MFM32), the Revised Upper Limb Module (RULM), the Hammersmith Functional Motor Scale - Expanded (HFMSE), respiratory function, and the proportion of risdiplam-related adverse events in a population with SMA (phenotypes 1 and 2/3). Meta-analyses were also performed where possible. Eleven studies were included. After 12 months of treatment, 57% of participants with SMA1 achieved a CHOP-INTEND score ≥ 40 points, and more than half were able to feed orally and had head control. In SMA2/3, MFM32, RULM, and HFMSE increased by 2.09 (1.17, 3.01), 1.73 (1.25, 2.20), and 1.00 (0.40, 1.59) points, respectively. Efficacy on respiratory function in SMA2/3 was inconsistent. Finally, 16% of participants experienced adverse events, but serious adverse events could not be quantified due to a lack of cases. The limited available evidence suggests that risdiplam is an effective and safe drug for the treatment of SMA. In addition, long-term clinical benefit may be partly determined by the stage of disease at which treatment is initiated.

Clinical and Functional Characteristics of a New Phenotype of SMA Type I among a National Sample of Spanish Children: A Cross-Sectional Study.

Spinal Muscular Atrophy (SMA) type I has classically presented extremely severe clinical features. New pharmacological treatments have led to a new phenotype of SMA. The aim of this study was to describe the current health and functional status of children with SMA. A cross-sectional study was conducted based on the STROBE guidelines. Patient questionnaires and standardized tools were used. A descriptive analysis was conducted establishing the proportions of subjects for each of the characteristics of interest. In total, 51 genetically confirmed SMA type I subjects were included. Fifty-seven percent received oral feeding, 33% received tube feeding and 10% combined both. Moreover, 21.6% had tracheostomies, and 9.8% needed more than 16 h/d ventilatory support. Regarding orthopedic status, 66.7% had scoliosis, and 68.6% had hip subluxation or dislocation. Up to 67% were able to sit independently, 23.5% walked with support and one child walked independently. Current SMA type I is a different entity from the classic phenotype but also from types II and III. In addition, no differences were found between SMA type I subgroups. These findings may enable the professionals involved in the care of these patients to improve their interventions in terms of prevention and rehabilitation measures for these children.

Base editing rescue of spinal muscular atrophy in cells and in mice.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9-mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA.

Areas of improvement in the medical care of SMA: evidence from a nationwide patient registry in Germany

BackgroundManagement and treatment of spinal muscular atrophy (SMA) has changed in recent years due to the introduction of novel transformative and potentially curative therapies resulting in the emergence of new disease phenotypes. Yet, little is known about the uptake and impact of these therapies in real-world clinical practice. The objective of this study was to describe current motor function, need of assistive devices, and therapeutic and supportive interventions provided by the healthcare system, as well as the socioeconomic situation of children and adults with different SMA phenotypes in Germany. We conducted a cross-sectional, observational study of German patients with genetically confirmed SMA identified and recruited via a nationwide SMA patient registry (www.sma-register.de) within the TREAT-NMD network. Study data was recorded directly from patient-caregiver pairs through a study questionnaire administered online via a dedicated study website.ResultsThe final study cohort consisted of 107 patients with SMA. Of these, 24 were children and 83 adults. In total, about 78% of all participants were taking medication for SMA (predominantly nusinersen and risdiplam). All children with SMA1 were able to sit and 27% of children with SMA2 were able to stand or walk. Impaired upper limb function, scoliosis and bulbar dysfunction were observed more frequently in patients with reduced lower limb performance. Physiotherapy, occupational therapy, and speech therapy, as well as the use of cough assists were less common than indicated by care guidelines. Family planning and educational and employment status appear to be related to motor skill impairment.ConclusionsWe show that the natural history of disease has changed in Germany following improvements in SMA care and the introduction of novel therapies. Yet, a non-trivial proportion of patients remain untreated. We also identified considerable limitations in rehabilitation and respiratory care, as well as low labour-market participation among adults with SMA, calling for action to improve the current situation.

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration.

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

Dysphagia and Lung Disease in Children With Spinal Muscular Atrophy Treated With Disease-Modifying Agents.

Disease-modifying agents (DMAs) for the treatment of spinal muscular atrophy (SMA) have evolved the SMA phenotype with improved survival. Ongoing oropharyngeal dysphagia and respiratory complications are reported. The extent of dysphagia and respiratory morbidity in this population, since DMAs' introduction, has not been well described. A whole-population study involved all children with treated SMA types 1-3 in our facility. Videofluoroscopic swallow studies (type 1 alone), chest CT scans, and clinical data were collected. Thirty-six children were included (n = 9 type 1, n = 14 type 2, and n = 13 type 3; age range 0.3-15.4 years). Abnormal swallowing characteristics were demonstrated in all children with type 1 (n = 8; 100%). Bronchiectasis was found on chest CT: 3 of 9 (33.3%), 2 of 14 (14.3%), and 2 of 13 (15.4%) of type 1, 2, and 3, respectively. Atelectasis, mucus plugging, bronchial wall thickening, and parenchymal changes were common. Swallow impairments were universal in children with type 1. Bronchiectasis was common in all pediatric SMA types, with a prevalence of 1 in 5. Routine monitoring and management of dysphagia/recurrent respiratory infection should be implemented for improvement in lung health.

Risdiplam as an orphan drug treatment of spinal muscular atrophy in adults and children (2 months or older)

ABSTRACT Introduction Spinal Muscular Atrophy (SMA) is caused by autosomal recessive mutations in SMN1 (survival motor neuron1) and results in the loss of motor neurons and progressive muscle weakness. The spectrum of disease severity ranges from early onset with respiratory failure during the first months of life to a milder, slower progressing adult-onset type. The field of SMA treatment has changed significantly over the last years from being a nearly untreatable condition to the marketing of 3 new therapeutic options and the possibility to diagnose the disease very early through newborn screening. Areas covered This article covers and summarizes the published articles of preclinical and clinical data on risdiplam, a new oral centrally and peripherally distributed SMN2 pre-mRNA splicing modifier, together with reviews of abstract of important scientific meetings that have been organized over the past 4 years. Expert opinion The favorable efficacy/safety profile allows risdiplam to address remaining still unmet needs in the recent era of new SMA therapies. In particular, the possibility to administer risdiplam orally at home will make of it an attractive treatment option across all SMA phenotypes. Long-term efficacy and safety are still under evaluation.

Retraction Note: Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.

Retraction Note: Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.

P.41 Adult SMA REACH: development and implementation data collection study in the UK Adult SMA population

Spinal muscular atrophy (SMA) is a form of motor neuron disease caused by a mutation in the survival motor neuron 1 gene (SMN1), which results in a wide disease spectrum affecting children and adults. There are four subtypes of SMA depending on age of onset and maximum acquired motor function. The recent advances in drug development have highlighted an urgent need to establish stronger clinical networks and data collection to gain a better understanding of the impact of new drugs on the natural history of the disease. In collaboration with SMA Reach UK (paediatric and data collection network) and the UK SMA Patient Registry, the Adult SMA Reach study aims to implement a standardised dataset and use systematic data collection to generate knowledge on the natural history and the evolving phenotype of adult SMA, the impact of new treatments and management. Adult SMA REACH is a longitudinal observational study, planning to collect clinical data and outcome measures about SMA patients age ≥16 years with genetically confirmed 5q SMA, with a sample size of approximately 209 treated patients and 140 non-treated patients. A working group of UK neuromuscular clinicians and physiotherapists has designed a specific data collection form and the data collection will be performed using an online database, specifically for this study. The visit schedule will be at baseline and thereafter 4-6 monthly for Nusinersen-treated patients and a minimum of 12 monthly, for non-treated patients or treated with other SMA treatments. Data monitoring will be carried out to ensure data quality. This study will link with the UK SMA Patient Registry and collect vital Patient Reported Outcome Measures. Data from the first year of data collection will be presented in the conference. This study will help us to collect information about the natural history of the disease, the impact of new treatments, monitor the safety of treatments and the implementation of new functional scales. Spinal muscular atrophy (SMA) is a form of motor neuron disease caused by a mutation in the survival motor neuron 1 gene (SMN1), which results in a wide disease spectrum affecting children and adults. There are four subtypes of SMA depending on age of onset and maximum acquired motor function. The recent advances in drug development have highlighted an urgent need to establish stronger clinical networks and data collection to gain a better understanding of the impact of new drugs on the natural history of the disease. In collaboration with SMA Reach UK (paediatric and data collection network) and the UK SMA Patient Registry, the Adult SMA Reach study aims to implement a standardised dataset and use systematic data collection to generate knowledge on the natural history and the evolving phenotype of adult SMA, the impact of new treatments and management. Adult SMA REACH is a longitudinal observational study, planning to collect clinical data and outcome measures about SMA patients age ≥16 years with genetically confirmed 5q SMA, with a sample size of approximately 209 treated patients and 140 non-treated patients. A working group of UK neuromuscular clinicians and physiotherapists has designed a specific data collection form and the data collection will be performed using an online database, specifically for this study. The visit schedule will be at baseline and thereafter 4-6 monthly for Nusinersen-treated patients and a minimum of 12 monthly, for non-treated patients or treated with other SMA treatments. Data monitoring will be carried out to ensure data quality. This study will link with the UK SMA Patient Registry and collect vital Patient Reported Outcome Measures. Data from the first year of data collection will be presented in the conference. This study will help us to collect information about the natural history of the disease, the impact of new treatments, monitor the safety of treatments and the implementation of new functional scales.

P.38 A novel splice site variant in a patient with spinal muscular atrophy and hypoplastic left heart syndrome

Spinal muscular atrophy (SMA) is an autosomal recessive disorder causing degeneration of motor neurones in the anterior horn of the spinal cord. SMA is caused by homozygous in activation of the SMN1 gene but a highly homologous copy (SMN2), which also produces a small amount of functional SMN protein, can mitigate disease severity. We present a patient with a severe SMA phenotype with hypoplastic left heart syndrome (HLHS), characterize a novel spice-site variant in the SMN1 gene and describe the unusual pathological features in muscle biopsy. The patient was born to consanguineous parents in a national heart center due to antenatal diagnosis of HLHS. Postnatal examination (day 4) revealed decreased tone, a lack of anti-gravity movements, absent deep tendon reflexes but no respiratory distress or feeding problems. Due to the combination of suspected muscle disease and HLHS, together with the results below, treatment was discontinued and the patient died on day 10 of life. Whole genome sequencing (WGS) revealed an apparently homozygous variant in SMN1 located in the donor splice-site between exon 7 and intron 7, c.*3+1G>T (NM_000344.3). MLPA identified one copy of SMN1 and SMN2. Analysis of RNA showed a transcript lacking exon 7 and western blotting of SMN protein from muscle showed reduced levels comparable to other SMA1 patients. Muscle biopsy and subsequent immunohistochemical and immunofluorescence showed unusual expression of embryonic myosin heavy chain in the patient compared to controls and other SMA patients. This case highlights the importance of rapid diagnosis of newborns to allow further treatment decisions to be made. Up to 30% of HLHS patients have associated conditions and the combination with SMA has been previously described, whether this is more than the coincidence of two relatively common conditions is unclear. A severe SMA phenotype has been previously seen in other patients with a splice site variant in intron 7.

VP.54 Amifampridine safety and efficacy in spinal muscular atrophy ambulatory patients: a randomized placebo-controlled, crossover, phase 2 trial

Spinal muscular atrophy (SMA) is an autosomal recessive disease where a deficient amount of SMN protein leads to progressive degeneration of bulbar and spinal motor neurons. Therapies for the restoration of SMN production are now available. Yet, fatigue and signs of impaired neuromuscular junction (NMJ) transmission have been documented as possible contributors to SMA phenotype. Amifampridine (3,4-diaminopyridine), a voltage-dependent K+ channel blocker, prolongs depolarization of the presynaptic NMJ terminal, enhancing neuromuscular transmission. Here, we evaluated the safety and efficacy of amifampridine in ambulatory patients with SMA Type 3. SMA-001 was a phase 2, 1:1 randomized, double-blind, placebo-controlled, 2-period, 2-treatment, crossover study. Genetically confirmed SMA Type 3, able to walk unaided for 30 m, not treated with nusinersen in the previous 6 months, entered the run-in phase for at least 21 days during which amifampridine was titrated up to 80 mg/day to reach an optimized stable dose. Then, patients with at least 3-points improvement in Hammersmith Functional Motor Score Extended (HFMSE) were randomized to receive either amifampridine or placebo for 2 weeks, alternatively, for a total of 28 days of double-blind treatment. Safety was evaluated from the first day of Run-In to the end of the study (primary safety outcome). Efficacy was evaluated by changes from randomization of HFMSE (primary efficacy outcome), quality of life, 6-minute walk test, and timed test (secondary outcomes). Descriptive analyses and a mixed effects linear model were used for statistic. Thirteen adult patients were included in the study, no serious adverse events (SAEs) attributed to Amifampridine were reported. Transient paresthesias (33,3%) were the only amifampridine-related AEs reported. Six patients for each sequence of treatment were randomized. Amifampridine treatment led to a statistically significant improvement in HFMSE (LS Mean Difference 0.792; 95% CI from 0.22 to 1.37; p=0.0083), compared to placebo, but not in the secondary endpoints. SMA-001 study provided Class I evidence that AP was safe and effective in treating ambulatory patients affected by SMA type 3.