Real-world evidence: Risdiplam in a patient with spinal muscular atrophy type I with a novel splicing mutation and one SMN2 copy.

Spinal muscular atrophy (SMA), which results from the deletion or/and mutation in the SMN1 gene, is an autosomal recessive neuromuscular disorder that leads to weakness and muscle atrophy. SMN2 is a paralogous gene of SMN1. SMN2 copy number affects the severity of SMA, but its role in patients treated with disease modifying therapies is unclear. The most appropriate individualized treatment for SMA has not yet been determined. Here, we reported a case of SMA type I with normal breathing and swallowing function. We genetically confirmed that this patient had a compound heterozygous variant: one deleted SMN1 allele and a novel splice mutation c.628-3T>G in the retained allele, with one SMN2 copy. Patient-derived sequencing of 4 SMN1 cDNA clones showed that this intronic single transversion mutation results in an alternative exon (e)5 3' splice site, which leads to an additional 2 nucleotides (AG) at the 5' end of e5, thereby explaining why the patient with only one copy of SMN2 had a mild clinical phenotype. Additionally, a minigene assay of wild type and mutant SMN1 in HEK293T cells also demonstrated that this transversion mutation induced e5 skipping. Considering treatment cost and goals of avoiding pain caused by injections and starting treatment as early as possible, risdiplam was prescribed for this patient. However, the patient showed remarkable clinical improvements after treatment with risdiplam for 7 months despite carrying only one copy of SMN2. This study is the first report on the treatment of risdiplam in a patient with one SMN2 copy in a real-world setting. These findings expand the mutation spectrum of SMA and provide accurate genetic counseling information, as well as clarify the molecular mechanism of careful genotype-phenotype correlation of the patient.

Epigenetic regulation of plastin 3 expression by the macrosatellite DXZ4 and the transcriptional regulator CHD4.

Dysregulated Plastin 3 (PLS3) levels associate with a wide range of skeletal and neuromuscular disorders and the most common types of solid and hematopoietic cancer. Most importantly, PLS3 overexpression protects against spinal muscular atrophy. Despite its crucial role in F-actin dynamics in healthy cells and its involvement in many diseases, the mechanisms that regulate PLS3 expression are unknown. Interestingly, PLS3 is an X-linked gene and all asymptomatic SMN1-deleted individuals in SMA-discordant families who exhibit PLS3 upregulation are female, suggesting that PLS3 may escape X chromosome inactivation. To elucidate mechanisms contributing to PLS3 regulation, we performed a multi-omics analysis in two SMA-discordant families using lymphoblastoid cell lines and iPSC-derived spinal motor neurons originated from fibroblasts. We show that PLS3 tissue-specifically escapes X-inactivation. PLS3 is located ∼500 kb proximal to the DXZ4 macrosatellite, which is essential for X chromosome inactivation. By applying molecular combing in a total of 25 lymphoblastoid cell lines (asymptomatic individuals, individuals with SMA, control subjects) with variable PLS3 expression, we found a significant correlation between the copy number of DXZ4 monomers and PLS3 levels. Additionally, we identified chromodomain helicase DNA binding protein 4 (CHD4) as an epigenetic transcriptional regulator of PLS3 and validated co-regulation of the two genes by siRNA-mediated knock-down and overexpression of CHD4. We show that CHD4 binds the PLS3 promoter by performing chromatin immunoprecipitation and that CHD4/NuRD activates the transcription of PLS3 by dual-luciferase promoter assays. Thus, we provide evidence for a multilevel epigenetic regulation of PLS3 that may help to understand the protective or disease-associated PLS3 dysregulation.

Screening of Spinal Muscular Atrophy Carriers and Prenatal Diagnosis in Pregnant Women in Yancheng, China.

Spinal muscular atrophy (SMA) is a neuromuscular disorder with an autosomal recessive inheritance pattern. Patients with severe symptoms may suffer respiratory failure, leading to death. The homozygous deletion of exon 7 in the SMN1 gene accounts for nearly 95% of all cases. Population carrier screening for SMA and prenatal diagnosis by amniocentesis for high-risk couples can assist in identifying the risk of fetal disease. We provided the SMA carrier screening process to 55,447 pregnant women in Yancheng from October 2020 to December 2022. Among them, 8185 participated in this process, with a participation rate of around 14.76% (95% CI 14.47-15.06%). Quantitative real-time polymerase chain reaction (qPCR) was used to detect deletions of SMN1 exons 7 and 8 (E7, E8) in screened pregnant women. 127 were identified as carriers (111 cases of E7 and E8 heterozygous deletions, 15 cases of E7 heterozygous deletions, and 1 case of E7 heterozygous deletions and E8 homozygous deletions), resulting in a carrying rate of around 1.55% (95% CI 1.30-1.84%). After genetic counseling, 114 spouses of pregnant women who tested positive underwent SMA carrier screening; three of them were screened as SMA carriers. Multiplexed ligation-dependent probe amplification (MLPA) was used for the prenatal diagnosis of the fetuses of high-risk couples. Two of them exhibited two copies of SMN1 exon 7 (normal), and the pregnancy was continued; one exhibited no copies of SMN1 exon 7 and exon 8 (SMA patient), and the pregnancy was terminated. Analyzing SMN1 mutations in Yancheng and provide clinical evidence for SMA genetic counseling and birth defect prevention. Interventional prenatal diagnosis for high-risk families can promote informed reproductive selection and prepare for the fetus's early treatment.

In Search of Spinal Muscular Atrophy Disease Modifiers.

The 5q Spinal Muscular Atrophy (SMA) is a hereditary autosomal recessive disease caused by defects in the survival motor neuron (SMN1) gene encoding survival motor neuron (SMN) protein. Currently, it is the leading cause of infantile mortality worldwide. SMA is a progressive neurodegenerative disease with "continuum of clinical severity", which can be modulated by genetic and epigenetic factors known as disease modifiers (DMs). Individuals (even siblings) with the same defects in SMN1 gene might have strikingly different types of SMA, supposedly due to the impact of DMs. There are several therapeutic options for SMA, all of them focusing on the restoration of the SMN protein levels to normal. Determining DMs and the pathways in which they are involved might aid in enhancing existing curative approaches. Furthermore, DMs might become novel therapeutic targets or prognostic biomarkers of the disease. This narrative review provides a brief overview of the genetics and pathobiology of SMA, and its bona fide modifiers. We describe novel, emerging DMs, approaches and tools used to identify them, as well as their potential mechanisms of action and impact on disease severity. We also propose several disease-modifying molecular mechanisms which could provide a partial explanation of the staggering variability of SMA phenotypes.

Engineered U1 snRNAs to modulate alternatively spliced exons.

Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community.

Moxifloxacin rescues SMA phenotypes in patient-derived cells and animal model.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models.

Survival of motor neuron (SMN) deficiency causes spinal muscular atrophy (SMA), but the pathogenesis mechanisms remain elusive. Restoring SMN in motor neurons only partially rescues SMA in mouse models, although it is thought to be therapeutically essential. Here, we address the relative importance of SMN restoration in the central nervous system (CNS) versus peripheral tissues in mouse models using a therapeutic splice-switching antisense oligonucleotide to restore SMN and a complementary decoy oligonucleotide to neutralize its effects in the CNS. Increasing SMN exclusively in peripheral tissues completely rescued necrosis in mild SMA mice and robustly extended survival in severe SMA mice, with significant improvements in vulnerable tissues and motor function. Our data demonstrate a critical role of peripheral pathology in the mortality of SMA mice and indicate that peripheral SMN restoration compensates for its deficiency in the CNS and preserves motor neurons. Thus, SMA is not a cell-autonomous defect of motor neurons in SMA mice.

Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects.

For SMA patients with only two SMN2 copies, available therapies might be insufficient to counteract lifelong motor neuron (MN) dysfunction. Therefore, additional SMN-independent compounds, supporting SMN-dependent therapies, might be beneficial. Neurocalcin delta (NCALD) reduction, an SMA protective genetic modifier, ameliorates SMA across species. In a low-dose SMN-ASO-treated severe SMA mouse model, presymptomatic intracerebroventricular (i.c.v.) injection of Ncald-ASO at postnatal day 2 (PND2) significantly ameliorates histological and electrophysiological SMA hallmarks at PND21. However, contrary to SMN-ASOs, Ncald-ASOs show a shorter duration of action limiting a long-term benefit. Here, we investigated the longer-term effect of Ncald-ASOs by additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in the brain and spinal cord and well tolerated. Next, we performed a double-blinded preclinical study combining low-dose SMN-ASO (PND1) with 2× i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and NMJ denervation at 2 months. Moreover, we developed and identified a non-toxic and highly efficient human NCALD-ASO that significantly reduced NCALD in hiPSC-derived MNs. This improved both neuronal activity and growth cone maturation of SMA MNs, emphasizing the additional protective effect of NCALD-ASO treatment.

Spinal Muscular Atrophy: The Past, Present, and Future of Diagnosis and Treatment.

Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance. The first cases of SMA were reported by Werdnig in 1891. Although the phenotypic variation of SMA led to controversy regarding the clinical entity of the disease, the genetic homogeneity of SMA was proved in 1990. Five years later, in 1995, the gene responsible for SMA, SMN1, was identified. Genetic testing of SMN1 has enabled precise epidemiological studies, revealing that SMA occurs in 1 of 10,000 to 20,000 live births and that more than 95% of affected patients are homozygous for SMN1 deletion. In 2016, nusinersen was the first drug approved for treatment of SMA in the United States. Two other drugs were subsequently approved: onasemnogene abeparvovec and risdiplam. Clinical trials with these drugs targeting patients with pre-symptomatic SMA (those who were diagnosed by genetic testing but showed no symptoms) revealed that such patients could achieve the milestones of independent sitting and/or walking. Following the great success of these trials, population-based newborn screening programs for SMA (more precisely, SMN1-deleted SMA) have been increasingly implemented worldwide. Early detection by newborn screening and early treatment with new drugs are expected to soon become the standards in the field of SMA.

Combinatorial ASO-mediated therapy with low dose SMN and the protective modifier Chp1 is not sufficient to ameliorate SMA pathology hallmarks.

Spinal muscular atrophy (SMA) is a devastating genetically inherited neuromuscular disorder characterized by the progressive loss of motor neurons in the spinal cord, leading to muscle atrophy and weakness. Although SMA is caused by homozygous mutations in SMN1, the disease severity is mainly determined by the copy number of SMN2, an almost identical gene that produces ~10% correctly spliced SMN transcripts. Recently, three FDA- and EMA-approved therapies that either increase correctly spliced SMN2 transcripts (nusinersen and risdiplam) or replace SMN1 (onasemnogen abeparvovec-xioi) have revolutionized the clinical outcome in SMA patients. However, for severely affected SMA individuals carrying only two SMN2 copies even a presymptomatic therapy might be insufficient to fully counteract disease development. Therefore, SMN-independent compounds supporting SMN-dependent therapies represent a promising therapeutic approach. Recently, we have shown a significant amelioration of SMA disease hallmarks in a severely affected SMA mouse carrying a mutant Chp1 allele when combined with low-dose of SMN antisense oligonucleotide (ASO) treatment. CHP1 is a direct interacting partner of PLS3, a strong protective modifier of SMA. Both proteins ameliorate impaired endocytosis in SMA and significantly restore pathological hallmarks in mice. Here, we aimed to pharmacologically reduce CHP1 levels in an ASO-based combinatorial therapy targeting SMN and Chp1. Chp1 modulation is a major challenge since its genetic reduction to ~50% has shown to ameliorate SMA pathology, while the downregulation below that level causes cerebellar ataxia. Efficacy and tolerability studies determined that a single injection of 30 µg Chp1-ASO4 in the CNS is a safe dosage that significantly reduced CHP1 levels to ~50% at postnatal day (PND)14. Unfortunately, neither electrophysiological predictors such as compound muscle action potential (CMAP) or motor unit number estimation (MUNE) nor histological hallmarks of SMA in neuromuscular junction (NMJ), spinal cord or muscle were ameliorated in SMA mice treated with Chp1-ASO4 compared to CTRL-ASO at PND21. Surprisingly, CHP1 levels were almost at control level 4-weeks post injection, indicating a rather short-term effect of the ASO. Therefore, we re-administrated Chp1-ASO4 by i.c.v. bolus injection at PND28. However, no significant improvement of SMA hallmarks were seen at 2 month-of-age either. In conclusion, in contrast to the protective effect of genetically-induced Chp1 reduction on SMA, combinatorial therapy with Chp1- and SMN-ASOs failed to significantly ameliorate the SMA pathology. Chp1-ASOs compared to SMN-ASO proved to have rather short-term effect and even reinjection had no significant impact on SMA progression, suggesting that further optimization of the ASO may be required to fully explore the combination.

NCALD Antisense Oligonucleotide Therapy in Addition to Nusinersen further Ameliorates Spinal Muscular Atrophy in Mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease causing the most frequent genetic childhood lethality. Recently, nusinersen, an antisense oligonucleotide (ASO) that corrects SMN2 splicing and thereby increases full-length SMN protein, has been approved by the FDA and EMA for SMA therapy. However, the administration of nusinersen in severe and/or post-symptomatic SMA-affected individuals is insufficient to counteract the disease. Therefore, additional SMN-independent therapies are needed to support the function of motoneurons and neuromuscular junctions. We recently identified asymptomatic SMN1-deleted individuals who were protected against SMA by reduced expression of neurocalcin delta (NCALD). NCALD reduction is proven to be a protective modifier of SMA across species, including worm, zebrafish, and mice. Here, we identified Ncald-ASO3-out of 450 developed Ncald ASOs-as the most efficient and non-toxic ASO for the CNS, by applying a stepwise screening strategy in cortical neurons and adult and neonatal mice. In a randomized-blinded preclinical study, a single subcutaneous low-dose SMN-ASO and a single intracerebroventricular Ncald-ASO3 or control-ASO injection were presymptomatically administered in a severe SMA mouse model. NCALD reduction of >70% persisted for about 1 month. While low-dose SMN-ASO rescues multiorgan impairment, additional NCALD reduction significantly ameliorated SMA pathology including electrophysiological and histological properties of neuromuscular junctions and muscle at P21 and motoric deficits at 3 months. The present study shows the additional benefit of a combinatorial SMN-dependent and SMN-independent ASO-based therapy for SMA. This work illustrates how a modifying gene, identified in some asymptomatic individuals, helps to develop a therapy for all SMA-affected individuals.

Revealing diverse alternative splicing variants of the highly homologous SMN1 and SMN2 genes by targeted long-read sequencing.

The survival of motor neuron (SMN) genes, SMN1 and SMN2, are two highly homologous genes related to spinal muscular atrophy (SMA). Different patterns of alternative splicing have been observed in the SMN genes. In this study, the long-read sequencing technique for distinguishing SMN1 and SMN2 without any assembly were developed and applied to reveal multiple alternative splicing patterns and to comprehensively identify transcript variants of the SMN genes. In total, 36 types of transcript variants were identified, with an equal number of variants generated from both SMN1 and SMN2. Of these, 18 were novel SMN transcripts that have never been reported. The structures of SMN transcripts were revealed to be much more complicated and diverse than previously discovered. These novel transcripts were derived from diverse splicing events, including skipping of one or more exons, intron retention, and exon shortening or addition. SMN1 mainly produces FL-SMN1, SMN1Δ7, SMN1Δ5 and SMN1Δ3. The distribution of SMN2 transcripts was significantly different from those of SMN1, with the majority transcripts to be SMN2Δ7, followed by FL-SMN2, SMN2Δ3,5 and SMN2Δ5,7. Targeted long-read sequencing approach could accurately distinguish sequences of SMN1 from those of SMN2. Our study comprehensively addressed naturally occurring SMN1 and SMN2 transcript variants and splicing patterns in peripheral blood mononuclear cells (PBMCs). The novel transcripts identified in our study expanded knowledge of the diversity of transcript variants generated from the SMN genes and showed a much more comprehensive profile of the SMN splicing spectrum. Results in our study will provide valuable information for the study of low expression level of SMN proteins and SMA pathogenesis based on transcript levels.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a ß-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of ß-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype−phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype−phenotype correlations and improve prognostic outcomes.

Mitochondrial Dysfunction in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by recessive mutations in the SMN1 gene, globally affecting ~8-14 newborns per 100,000. The severity of the disease depends on the residual levels of functional survival of motor neuron protein, SMN. SMN is a ubiquitously expressed RNA binding protein involved in a plethora of cellular processes. In this review, we discuss the effects of SMN loss on mitochondrial functions in the neuronal and muscular systems that are the most affected in patients with spinal muscular atrophy. Our aim is to highlight how mitochondrial defects may contribute to disease progression and how restoring mitochondrial functionality may be a promising approach to develop new therapies. We also collected from previous studies a list of transcripts encoding mitochondrial proteins affected in various SMA models. Moreover, we speculate that in adulthood, when motor neurons require only very low SMN levels, the natural deterioration of mitochondria associated with aging may be a crucial triggering factor for adult spinal muscular atrophy, and this requires particular attention for therapeutic strategies.

[Spinal muscular atrophy]. / Spinale Muskelatrophie.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by biallelic mutations in the SMN1 (survival motor neuron 1) gene on chromosome 5q13.2, which leads to a progressive degeneration of alpha motor neurons in the spinal cord and in motor nerve nuclei in the caudal brainstem. It is characterized by progressive proximally accentuated muscle weakness with loss of already acquired motor skills, areflexia and, depending on the phenotype, varying degrees of weakness of the respiratory and bulbar muscles, although the facial muscles and eye muscles are not affected. The previously purely symptom-oriented treatment has undergone a significant expansion since 2017 with the approval of three drugs (nusinersen, onasemnogene abeparvovec and risdiplam) that modify the course of the disease at the gene expression level and have led to a change in the natural disease course of SMA. The effect of these new forms of treatment can only be fully assessed in the coming years. New aspects and challenges in this context are discussed in this article.

[Newborn screening program for spinal muscular atrophy]. / Neugeborenenscreeningprogramm für die spinale Muskelatrophie.

BACKGROUND: The introduction of a comprehensive newborn screening program for spinal muscular atrophy (SMA), specifically for 5q-SMA, is planned for the end of 2021 in Germany. Several targeted treatment options have become available for all patients with SMA. MATERIAL AND METHODS: Newborn screening for 5q-SMA is based on the detection of a homozygous deletion of exon 7 in the SMN1 gene by molecular genetic analysis from the dried blood card. In all cases a second blood sample must be drawn as a part of confirmation diagnostics including the determination of the SMN2 copy numbers. RESULTS: Insights from pilot projects performed in parts of Germany are presented. Advantages and disadvantages of the screening project are discussed. CONCLUSION: Consultation and treatment should be carried out in a department of neuropediatrics with experience in the treatment of children with 5q-SMA, which is able to provide all current treatment options for the child, so that, when necessary, the treatment can be started within the first month of life.

Identification of SRSF10 as a regulator of SMN2 ISS-N1.

Understanding the splicing code can be challenging as several splicing factors bind to many splicing-regulatory elements. The SMN1 and SMN2 silencer element ISS-N1 is the target of the antisense oligonucleotide drug, Spinraza, which is the treatment against spinal muscular atrophy. However, limited knowledge about the nature of the splicing factors that bind to ISS-N1 and inhibit splicing exists. It is likely that the effect of Spinraza comes from blocking binding of these factors, but so far, an unbiased characterization has not been performed and only members of the hnRNP A1/A2 family have been identified by Western blot analysis and nuclear magnetic resonance to bind to this silencer. Employing an MS/MS-based approach and surface plasmon resonance imaging, we show for the first time that splicing factor SRSF10 binds to ISS-N1. Furthermore, using splice-switching oligonucleotides we modulated the splicing of the SRSF10 isoforms generating either the long or the short protein isoform of SRSF10 to regulate endogenous SMN2 exon 7 inclusion. We demonstrate that the isoforms of SRSF10 regulate SMN1 and SMN2 splicing with different strength correlating with the length of their RS domain. Our results suggest that the ratio between the SRSF10 isoforms is important for splicing regulation.

Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients.

Spinal muscular atrophy (SMA) is caused by bi-allelic loss or pathogenic variants in the SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish robust genotype-phenotype correlations and predict disease evolution, to stratify patients for clinical trials, as well as to define those eligible for treatment. Discordant genotype-phenotype correlations are not uncommon in SMA, some of which are due to intragenic SMN2 variants that may influence the amount of complete SMN transcripts and, therefore, of full-length SMN protein. Detection of these variants is crucial to predict SMA phenotypes in the present scenario of therapeutic advances and with the perspective of SMA neonatal screening and early diagnosis to start treatments. Here, we present a novel, affordable, and versatile method for complete sequencing of the SMN2 gene based on long-range polymerase chain reaction and next-generation sequencing. The method was validated by analyzing samples from 53 SMA patients who lack SMN1, allowing to characterize paralogous, rare variants, and single-nucleotide polymorphisms of SMN2 as well as SMN2-SMN1 hybrid genes. The method identifies partial deletions and can be adapted to determine rare pathogenic variants in patients with at least one SMN1 copy.

Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR.

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1-SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.