Survival Motor Neuron Research Articles

Analysis of a Chinese pedigree affected with Spinal muscular atrophy due to compound heterozygous variants of SMN gene

To analyze variants of SMN gene in a Chinese pedigree affected with Spinal muscular atrophy (SMA). A Chinese pedigree diagnosed at the Nanchang First Hospital in January 2020 was selected as the study subject. Peripheral blood samples were collected for the extraction of DNA. All exons of the SMN gene were detected by multiple ligation-dependent probe amplification (MLPA). Potential variants of the SMN gene were also detected by Whole exome sequencing (WES), and the result was verified by Sanger sequencing. cDNA extracted from fresh blood sample was used as a template to verify the location of variant on the SMN genes. The proband was found to harbor a heterozygous deletion of the SMN1 Exon7+Exon8, and a heterozygous c.81G>A variant. The SMN1 Exon7+Exon8 deletion was inherited from her father and grandmother, whilst the c.81G>A variant was inherited from her mother and maternal grandfather. Her aunt was also a carrier of the heterozygous deletion, while her paternal aunt, her husband, and their daughter were not. cDNA amplification and Sanger sequencing confirmed that the c.81G>A variant was located in the SMN1 gene. MLPA combined with NGS and Sanger sequencing can identify compound heterozygous variants of the SMN gene in the SMA patients.

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.

An Effective and Universal Long-Read Sequencing-Based Approach for SMN1 2 + 0 Carrier Screening through Family Trio Analysis

Abstract Background Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by professional organizations to facilitate informed reproductive options. However, genetic screening for SMN1 2 + 0 carriers, accounting for 3%–8% of all SMA carriers, has been challenging due to the large gene size and long distance between the 2 SMN genes. Methods Here we repurposed a previously developed long-read sequencing-based approach, termed comprehensive analysis of SMA (CASMA), to identify SMN1 2 + 0 carriers through haplotype analysis in family trios (CASMA-trio). Bioinformatics pipelines were developed for accurate haplotype analysis and SMN1 2 + 0 deduction. Seventy-nine subjects from 24 families composed of, at the minimum, 3 were enrolled, and CASMA-trio was employed to determine whether an index subject with 2 SMN1 copies was a 2 + 0 carrier in these families. For the proof-of-principle, SMN2 2 + 0 was also analyzed. Results Among the 16 subjects with 2 SMN1 copies, CASMA-trio identified 5 subjects from 4 families as SMN1 2 + 0 carriers, which was consistent with pedigree analysis involving an affected proband. CASMA-trio also identified SMN2 2 + 0 in six out of 43 subjects with 2 SMN2 copies. Additionally, CASMA-trio successfully determined the distribution pattern of SMN1 and SMN2 genes on 2 alleles in all 79 subjects. Conclusions CASMA-trio represents an effective and universal approach for SMN1 2 + 0 carriers screening, as it does not reply on the presence of an affected proband, certain single-nucleotide polymorphisms, ethnicity-specific haplotypes, or complicated single-nucleotide polymorphism analysis across 3 generations. Incorporating CASMA-trio into existing SMA carrier screening programs will greatly reduce residual risk ratio.

Gene therapy in spinal muscular atrophy

Infantile SMA is a neuromuscular disease caused by the motor neuron degeneration, depending on the age of appearance of clinical signs and the evolution of the disease, three types of decreasing severity have been defined. SMA is caused by mutations or deletions of the SMN1 gene and disease. Various therapies aimed at increasing SMN protein levels have been developed. Gene therapy is part of the therapeutic arsenal now available for the treatment of SMA under certain conditions. It uses the scAAV9 vector carrying a functional copy of SMN1 to restore SMN protein expression at the cellular level. Because the adeno-associated virus genome is maintained as it is an episome, a single intravenous administration is sufficient to producing a long-lasting therapeutic effect. The effectiveness of gene replacement therapy in patients with SMA has been demonstrated in various studies. It is now clear that treatment as early as possible provides better clinical results. However, this treatment must be carried out in a suitable medical environment, with close monitoring initially due to potentially serious side effects. In France, this treatment has been available since 2019. A national committee of experts involved in the treatment of pediatric SMA patients has established that pediatric patients with SMA decide on the indications for disease-modifying therapies (DMT) in children. The French Spinal Muscular Atrophy Registry (SMA France Registry) was established in January 2020. The registry includes all patients with genetically confirmed SMN1-related SMA. All patients treated with GT are systematically included in the registry. As of July 21, 2023: 72 patients with SMA have been treated with GT in France since June 2019. The arrival of new treatments reveals new clinical phenotypes of SMA which constitute a new management challenge. Treatment as early as possible is also a very important factor for a favorable outcome and calls for presymptomatic screening. However, the arrival of these new treatments, extremely expensive raises other socio-economic questions. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

SMN haploinsufficiency promotes ischemia/ reperfusion-induced AKI-to-CKD transition by endoplasmic reticulum stress activation.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are interconnected syndromes that represent a global public health challenge. Here, we identified a specific role of survival of motor neuron (SMN) in ischemia/reperfusion (I/R)-induced kidney injury and progression of CKD. SMN was an essential protein in all cell type and was reported to play important roles in multiple fundamental cellular homeostatic pathways. However, the function of SMN in experimental models of I/R-induced kidney fibrosis has not extensively studied. Genetic ablation of SMN or small interfering RNA-base knockdown of SMN expression aggravated the tubular injury and interstitial fibrosis. Administration of scAAV9-CB-SMN or epithelial cell overexpression of SMN reduced I/R-induced kidney dysfunction and attenuated AKI-to-CKD transition, indicating that SMN is vital for the preservation and recovery of tubular phenotype. Our data showed that the endoplasmic reticulum stress (ERS) induced by I/R was persistent and became progressively more severe in the kidney without SMN. On the contrary, overexpression of SMN prevented against I/R-induced ERS and tubular cell damage. In summary, our data collectively substantiate a critical role of SMN in regulating the ERS activation and phenotype of AKI-to-CKD transition that may contribute to renal pathology during injury and repair.

The Impact of p70S6 Kinase-Dependent Phosphorylation of Gemin2 in UsnRNP Biogenesis.

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.

DDX20: A Multifunctional Complex Protein.

DEAD-box decapping enzyme 20 (DDX20) is a putative RNA-decapping enzyme that can be identified by the conserved motif Asp-Glu-Ala-Asp (DEAD). Cellular processes involve numerous RNA secondary structure alterations, including translation initiation, nuclear and mitochondrial splicing, and assembly of ribosomes and spliceosomes. DDX20 reportedly plays an important role in cellular transcription and post-transcriptional modifications. On the one hand, DDX20 can interact with various transcription factors and repress the transcriptional process. On the other hand, DDX20 forms the survival motor neuron complex and participates in the assembly of snRNP, ultimately affecting the RNA splicing process. Finally, DDX20 can potentially rely on its RNA-unwinding enzyme function to participate in microRNA (miRNA) maturation and act as a component of the RNA-induced silencing complex. In addition, although DDX20 is not a key component in the innate immune system signaling pathway, it can affect the nuclear factor kappa B (NF-κB) and p53 signaling pathways. In particular, DDX20 plays different roles in tumorigenesis development through the NF-κB signaling pathway. This process is regulated by various factors such as miRNA. DDX20 can influence processes such as viral replication in cells by interacting with two proteins in Epstein-Barr virus and can regulate the replication process of several viruses through the innate immune system, indicating that DDX20 plays an important role in the innate immune system. Herein, we review the effects of DDX20 on the innate immune system and its role in transcriptional and post-transcriptional modification processes, based on which we provide an outlook on the future of DDX20 research in innate immunity and viral infections.

Clinical SMN1 and SMN2 Gene-Specific Sequencing to Enhance the Clinical Sensitivity of Spinal Muscular Atrophy Diagnostic Testing

Purpose. Therapeutic advances in the treatment of spinal muscular atrophy (SMA) prompt the need for robust and efficient molecular diagnosis of this disease. Approximately five percent of SMA cases are attributable to one copy of SMN1 with a hypomorphic or inactivating variant in trans with a deleted or converted allele. These intragenic variants are challenging to definitively localize to SMN1 due to its sequence homology with the SMN2 gene. To enhance the clinical sensitivity of SMA diagnostic testing, we present an optimized gene-specific sequencing assay to localize variants to either SMN1 or SMN2. Methods. SMN1 and SMN2 genes are independently amplified by long-range allele-specific PCR. Long-range products are used in subsequent nested PCR reactions to amplify the coding exons of SMN1 and SMN2. The resulting products are sequenced using standard Sanger-based methodologies and analyzed for disease-associated alterations. Results. 83 probands suspicious for a clinical diagnosis of SMA with a nondiagnostic SMN dosage result were sequenced for intragenic variants in the SMN1 gene. Gene-specific sequencing revealed likely disease-associated variants in SMN1 in 42 cases (50.6%). Of the 42 variants, 27 are unique including 16 loss-of-function variants, 9 missense variants, 1 in-frame deletion variant, and 1 splice site variant. Conclusions. Herein, we describe an optimized assay for clinical sequencing of the full coding region of SMN1 and SMN2. This assay uses standard techniques and equipment readily available to most molecular diagnostic laboratories.

Researches of calcium-activated chloride channel ANO1 intervening amyotrophic lateral sclerosis progression by activating EGFR and CaMKII signaling

BackgroundANO1 is closely correlated with the activation of EGFR and CaMKII, while EGFR and CaMKII show low activation in amyotrophic lateral sclerosis (ALS) models. Therefore, we designed experiments to verify that ANO1 may play a protective role on motor neurons in ALS by activating EGFR and CaMKII. MethodsThe expression changes of ANO1, EGFR, CaMKII, pEGFR, and pCaMKII, cell survival status, and apoptosis were studied by western blot, real-time quantitative PCR, immunofluorescence, immunohistochemistry, CCK-8, and flow cytometry. The role of ANO1 in the ALS model by activating EGFR and CaMKII was studied by applying corresponding activators, inhibitors, gene silencing, and overexpression. ResultsIn hSOD1G93A transgenic animals and cell lines, low expression of ANO1 and low activation of EGFR and CaMKII were identified. ANO1 expression decreased gradually with the progression of ALS. Overexpression of ANO1 in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice increased cell viability and decreased cell apoptosis. After the application of ANO1 inhibitor CaCC-inhA01 in hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR activator EGF and CaMKII activator Carbachol, increased cell viability and reduced cell apoptosis. After ANO1 was overexpressed in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR inhibitor AEE788 and CaMKII inhibitor KN93 decreased cell viability and increased cell apoptosis. ConclusionsOur results suggest that ANO1 plays an important role in the survival of ALS motor neurons. ANO1 can increase cell activity and reduce apoptosis by activating EGFR and CaMKII signals.

The Neuroprotective Role of TERT Influences the Expression of SOD1 in Motor Neurons and Mouse Brain: Implications for fALS

Amyotrophic lateral sclerosis (ALS) disease is characterized by degeneration of motor neurons and elevation of brain oxidative stress. Previous studies demonstrated the neuroprotective effects of Telomerase reverse transcriptase (TERT) from oxidative stress. We showed that increasing TERT expression in the brain of the Tg hSOD1G93A mouse ALS model attenuated the disease pathology and increased the survival of motor neurons exposed to oxidative stress. How TERT increased the survival of motor neurons exposed to oxidative stress is not yet clear. Here we investigated the consequence of TERT depletion in motor neuron cells under normal and oxidative stress conditions and in mouse brains of TERT knockout mice, on the expression and activity of SOD1 and catalase enzymes. Depletion of mouse TERT caused mitochondrial dysfunction and impaired catalase and SOD1 activity. Compensation with hTERT restored the activity of SOD1. SOD1 expression increased in the brain of TERT KO and in ALS mice and decreased in the brain of WT mice treated with telomerase-increasing compounds. We suggest that the ability of TERT to protect neurons from oxidative stress affects the expression and activity of SOD1, in a TERT-dependent manner, and supports the notion of TERT as a therapeutic target for neurodegenerative diseases like ALS.

Neurofilaments as a biomarker of spinal muscular atrophy: review

Spinal muscular atrophy (SMA) is an autosomal recessive, disabling neuromuscular disease characterized by the death of motor neurons in the spinal cord, giving rise in the development both of muscle weakness and, subsequently, flaccid tetraparesis, swallowing and breathing disorders. There are 4 types of SMA, depending on the age of manifestation, the most severe is type I (not counting type 0 — prenatal type).
 Modern diagnosis of SMA includes a molecular genetic study looking for mutations in the SMN1 gene and determining the copy number of the SMN2 gene. Instrumental and biochemical methods for evaluating the effectiveness of therapy for spinal muscular atrophy are currently under study. Neurofilament proteins have been investigated as potential biomarkers for several diseases characterized by axonal damage and degeneration. In clinical studies, there are isolated data on the use of blood neurofilaments as markers of SMA. This review considers the literature data of foreign authors and clinical studies of neurofilaments as perspective biomarkers of SMA, both heavy and light chains.

Exploring factors impacting haplotype-based noninvasive prenatal diagnosis for single-gene recessive disorders.

Haplotype-based noninvasive prenatal diagnosis (NIPD) is applicable for various recessive single-gene disorders in proband families. However, a comprehensive exploration of critical factors influencing the assay performance, such as fetal fraction, informative single nucleotide polymorphism (SNP) count, and recombination events, has yet to be performed. It is critical to identify key factors affecting NIPD performance, including its accuracy and success rate, and their impact on clinical diagnostics to guide clinical practice. We conducted a prospective study, recruiting 219 proband families with singleton pregnancies at risk for eight recessive single-gene disorders (Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, methylmalonic acidemia, hemophilia A, hemophilia B, non-syndromic hearing loss, and congenital adrenal hyperplasia) at 7-14 weeks of gestation. Haplotype-based NIPD was performed by evaluating the relative haplotype dosage (RHDO) in maternal circulation, and the results were validated via invasive prenatal diagnosis or newborn follow-ups. Among the 219 families, the median gestational age at first blood draw was 8+5 weeks. Initial testing succeeded for 190 families and failed for 29 due to low fetal fraction (16), insufficient informative SNPs (9), and homologous recombination near pathogenic variation (4). Among low fetal fraction families, successful testing was achieved for 11 cases after a redraw, while 5 remained inconclusive. Test failures linked to insufficient informative SNPs correlated with linkage disequilibrium near the genes, with F8 and MMUT exhibiting the highest associated failure rates (14.3% and 25%, respectively). Homologous recombination was relatively frequent around the DMD and SMN1 genes (8.8% and 4.8%, respectively) but led to detection failure in only 44.4% (4/9) of such cases. All NIPD results from the 201 successful families were consistent with invasive diagnostic findings or newborn follow-up. Fetal fraction, informative SNPs count, and homologous recombination are pivotal to NIPD performance. Redrawing blood effectively improves the success rate for low fetal fraction samples. However, informative SNPs count and homologous recombination rates vary significantly across genes, necessitating careful consideration in clinical practice. We have designed an in silico method based on linkage disequilibrium data to predict the number of informative SNPs. This can identify genomic regions where there might be an insufficient number of SNPs, thereby guiding panel design. With these factors properly accounted for, NIPD is highly accurate and reliable.

FUS-dependent microRNA deregulations identify TRIB2 as a druggable target for ALS motor neurons

MicroRNAs (miRNAs) modulate mRNA expression, and their deregulation contributes to various diseases including amyotrophic lateral sclerosis (ALS). As fused in sarcoma (FUS) is a causal gene for ALS and regulates biogenesis of miRNAs, we systematically analyzed the miRNA repertoires in spinal cords and hippocampi from ALS-FUS mice to understand how FUS-dependent miRNA deregulation contributes to ALS. miRNA profiling identified differentially expressed miRNAs between different central nervous system (CNS) regions as well as disease states. Among the up-regulated miRNAs, miR-1197 targets the pro-survival pseudokinase Trib2. A reduced TRIB2 expression was observed in iPSC-derived motor neurons from ALS patients. Pharmacological stabilization of TRIB2 protein with a clinically approved cancer drug rescues the survival of iPSC-derived human motor neurons, including those from a sporadic ALS patient. Collectively, our data indicate that miRNA profiling can be used to probe the molecular mechanisms underlying selective vulnerability, and TRIB2 is a potential therapeutic target for ALS.

Identifying Clinical and Genetic Characteristics of Spinal Muscular Atrophy Patients and Families in Saudi Arabia.

Spinal muscular atrophy (SMA) is an inherited, neuromuscular disease characterized by the deterioration of spinal motor neurons, causing progressive muscular atrophyand weakening. It is an autosomal recessive disease with the mutation of the survival motor neuron 1 (SMN1) gene as a hallmark. Evidence suggests that the SMN2 gene modulates the severity of the disease. SMA is classified based on the maximum motor function achieved. This study aims to describe the genetic makeup and characteristics of an SMA cohort in the Kingdom of Saudi Arabia (KSA). Data from families presenting with SMA children was collected between January 2018 and December 2020. Blood samples were collected from patients and family members. Genetic testing for SMA and mutations was performed at a European central lab. Seventeen families were enrolled in the study, including 52 children. Among 34 parents, 28 were carriers with heterozygous deletion (82.3%), one (2.9%) had no deletion detected by multiplex ligation-dependent probe amplification (MLPA) but had point mutation by sequencing, one (2.9%) had homozygous deletion and was symptomatic, three (8.8%) had no deletion or point mutation and were presumed to have 2+0, and one (2.9%) was not tested. This study provides insight into the carrier mutational analysis of families with SMA disease manifestations in KSA. Further studies are needed to understand the burden and impact of SMA among the Saudi population.

Modern approaches to the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. The cause of the disease is the presence of mutations in the SMN1 gene, which leads to a decrease in the expression of the SMN protein. Decreased functional activity of the SMN protein contributes to the degeneration of motor neurons. Understanding the molecular genetic cause of spinal muscular atrophy made it possible to develop and start using Nusinersen, Onasemnogene Abeparvovec and Risdiplam drugs in clinical practice for the treatment of this disease. The main approaches to the treatment of SMA are either modifying the splicing of the SMN2 gene, which, due to a point mutation in exon 7, is unable to express the full-length SMN protein, or viral delivery of a functional copy of the SMN1 gene.
 Approved drugs differ in peculiarities of use, which are associated, in particular, with the route of administration, adverse reactions and restrictions of use, including restrictions associated with the socio-economic burden of patients with SMA.
 The development and use of domestic products for the treatment of SMA with a comparable clinical effect and at a lower price would reduce the cost of drug therapy for severe forms of the disease.

Evaluation of the possible association between higher than normal nuchal translucency with common mutations of SMN2 and PTPN11 genes in normal karyotype of pregnant women referred to Noor Comprehensive Genetics Center’s Clinic between 2019 and 2021

Evaluation of the possible association between higher than normal nuchal translucency with common mutations of SMN2 and PTPN11 genes in normal karyotype of pregnant women referred to Noor Comprehensive Genetics Center’s Clinic between 2019 and 2021

Spinal Muscular Atrophy.

This article provides a comprehensive overview of the diagnostic assessment and treatment of individuals with spinal muscular atrophy (SMA) due to homozygous deletions of SMN1 . In recent years, most states have incorporated SMA in their newborn screening panel. To provide the earliest diagnosis possible after symptom onset, vigilance is needed for births in states without newborn screening for SMA and when compound heterozygotes are missed by newborn screening programs. Supportive care for respiratory, nutritional, and orthopedic health impacts outcomes and is the cornerstone of care. Adaptive equipment, including assistive home technology, enables affected individuals to gain autonomy in their daily activities. Pharmacologic treatments approved by the US Food and Drug Administration (FDA) include three drugs that increase deficient survival motor neuron protein levels through SMN1 - or SMN2 - directed pathways: nusinersen, onasemnogene abeparvovec, and risdiplam. Efficacy for these trials was measured in event-free survival (survival without the need for permanent ventilation) and gains in functional motor outcomes. Earlier treatment is most effective across all treatments. The diagnostic and therapeutic landscapes for SMA have seen dramatic advancements in recent years, improving prognosis. Optimized supportive care remains essential, and vigilance is needed to define the new natural history of this disease.

Patient and Caregiver Outcomes After Onasemnogene Abeparvovec Treatment: Findings from the Cure SMA 2021 Membership Survey.

Onasemnogene abeparvovec (OA) is the only gene replacement therapy currently approved for spinal muscular atrophy (SMA) treatment. We sought to assess real-world patient and caregiver outcomes after OA treatment for SMA. Patients who received OA were identified from the 2021 Cure SMA Membership Survey. Those treated at 6-23months of age were matched to non-patients treated with OA on the basis of age at the time of survey and survival motor neuron2 gene copy number. Patient characteristics, motor milestones, and resource and supportive care use, as well as caregiver proxy-reported health-related quality of life (HRQOL), were described. Caregiver unmet needs and HRQOL were also assessed. Of the 614 patients in the survey, 64 received OA, and 17 were matched with 28 non-OA-treated patients. In general, a greater percentage of OA-treated patients achieved various motor milestones, including 100% sitting without support and 58.8% walking with assistance. OA-treated patients also had numerically lower rates of hospitalization and surgery. None required tracheostomy with a ventilator. The rate of using oxygen or a breathing machine for more than 16h was also lower for OA-treated patients. OA-treated patients had less frequent trouble swallowing. HRQOL was reported to be similar to non-OA-treated patients. Caregivers of OA-treated patients reported better patient mobility scores and less work impairment. The study suggests that treatment with OA is associated with greater rates of motor milestone achievements and less resource and supportive care use for patients with SMA treated at 6-23months of age in the real world. For caregivers, it may also potentially reduce unmet needs, improve HRQOL, and reduce work impairment.

B-263 Molecular Assay Evaluation to SMA and SCID Diagnosis in Newborn Dried Blood Spots (DBS)

Abstract Background Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Almost 95% of SMA cases are caused by the homozygous deletion involving exon 7 of the SMN1 gene. Its incidence is estimated in 1:6000–11 800 live births and it is the most common genetic cause of death in infancy. Symptoms of SMA include several cognitive disabilities leading to death, due to loss of motor functions, rapid denervation, and progressive weakening. In addition, another very common syndrome among newborns is Severe Combined Immunodeficiency (SCID). SCID is a genetic disease that may compromise the Lymphocytes T, B, and/or Natural Killers cells functions causing repeated and persistent infections. Low levels of the recombination excision circles of T and B cells (TREC and KREC, respectively) are indicative of SCID or other lymphopenias. Due to the early onset and rapid progression of SMA and SCID, accurate diagnosis of these syndromes is of utmost importance to perform therapeutic intervention before the onset of symptoms. Therefore, this study aims to describe the analytical and clinical validation of a kit for the implementation of a multiplex SCID/SMA real-time diagnoses routine in dried blood spots (DBS). Methods Commercial quantified positive control and samples were used for the analytical and clinical validation, respectively. The Clinical validation included 67 samples with the SMN1 gene and 7 samples with gene deletion. For the SCID evaluation, 60 samples with normal levels of TREC and KREC and 11 samples with reduced levels were included. DNA isolation was performed with the DNA Extract All Reagents Kit (Thermo Fisher, Massachusetts, EUA) from a 3.2mm disc of newborn DBS. The qPCR assay was performed by the TaqMan SCID/SMA Plus Assay (Thermo Fisher, Massachusetts, EUA) to target TREC, KREC, and SMN1 according to the manufacturer's instructions. The analysis parameters included: (i) Determination of assay efficiency; (ii) Analytical sensitivity (Limit of detection); (iii) Intra-assay and inter-assay precision; and (iv) Clinical validation. All reactions included the endogenous control RNase P. Results The assay demonstrated reaction efficiency >98% and the limit of detection was 30 copies per reaction for TREC, KREC, and SMN1 targets with a 95% confidence interval. The regression equations demonstrated good amplification conditions with a coefficient of determination (r2) = 0.99. The precision experiments demonstrated optimal repeatability and reproducibility. The results obtained in the clinical validation showed 100% agreement with the expected results for the SMN1 target. To the TREC and KREC targets, the assay demonstrated 95% and 100% agreement for normal and reduced levels, respectively. Conclusion An accurate and rapid diagnosis is essential to guide the clinical management of SCID and SMA in newborns. Here, we evaluated and described the TaqMan SCID/SMA Plus Assay performance, a molecular test for detecting SCID and SMA by Quantitative Real-time PCR (qPCR). This assay was highly efficient due offer of a multiplex detection method, allowing the rapid release of results. The use of this tool can help clinicians in rapid newborn screening, providing agility in clinical conduct.

Intrathecal delivery of AAV-NDNF ameliorates disease progression of ALS mice

Amyotrophic lateral sclerosis (ALS) is a uniformly lethal neurodegenerative disease characterized by progressive deterioration of motor neurons and neuromuscular denervation. Adeno-associated virus (AAV)-mediated delivery of trophic factors is being considered as a potential disease-modifying therapeutic avenue. Here we show a marked effect of AAV-mediated over-expression of neuron-derived neurotrophic factor (NDNF) on SOD1G93A ALS model mice. First, we adopt AAV-PHP.eB capsid to enable widespread expression of target proteins in the brain and spinal cord when delivered intrathecally. Then we tested the effects of AAV-NDNF on SOD1G93A mice at different stages of disease. Interestingly, AAV-NDNF markedly improved motor performance and alleviated weight loss when delivered at early post-symptomatic stage. Injection in the middle post-symptomatic stages still improved the locomotion ability, although it did not alleviate the loss of body weight. Injection in the late stage also extended the life span of SOD1G93A mice. Furthermore, NDNF expression promoted the survival of spinal motoneurons, reduced abnormal protein aggregation, and preserved the innervated neuromuscular functions. We further analyzed the signaling pathways of NDNF expression and found that it activates cell survival and growth-associated mammalian target of rapamycin signaling pathway and downregulates apoptosis-related pathways. Thus, intrathecally AAV-NDNF delivery has provided a potential strategy for the treatment of ALS.