Full-length SMN Protein Research Articles

P.376 - A series of case reports from JEWELFISH, an open-label study to investigate the safety, tolerability, and pharmacokinetics/ pharmacodynamics of RG7916 in adult and pediatric patients with spinal muscular atrophy who previously participated in a study with another SMN2-targeting therapy

Spinal muscular atrophy (SMA) is a rare genetic disease caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. SMA is characterized by progressive degeneration of spinal motor neurons, leading to muscle weakness and atrophy. Humans carry two, almost identical copies of SMN genes: SMN1 and SMN2. While SMN1 expresses full-length SMN protein, exon 7 is mostly skipped from SMN2 transcripts. RG7916 is a highly selective, orally available, investigational small molecule designed to modify the splicing of the SMN2 gene, resulting in the production of full-length SMN2 mRNA. JEWELFISH is a multicenter, open-label study evaluating daily oral administration of RG7916 (3 mg) in patients with SMA aged 12–55 years who have previously participated in a study with another SMN2-targeting therapy. A series of case reports from patients enrolled in this study will be presented. Data will include available safety and pharmacodynamics data. Together with the ongoing SUNFISH (SMA Type 2 and 3) and FIREFISH (SMA Type 1) studies, JEWELFISH will provide valuable insights into the safety, tolerability, and pharmacokinetics/pharmacodynamics of daily oral RG7916 treatment in SMA patients who have previously participated in a study with another SMN2-targeting therapy (i.e. RG7800 or nusinersen).

Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-β1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-β1 gene transcription, indicating a post-transcriptional mechanism for Tra2-β1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease.

Normalization of Patient-Identified Plasma Biomarkers in SMNΔ7 Mice following Postnatal SMN Restoration.

Introduction and ObjectiveSpinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the SMN1 gene and retention of the SMN2 gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers.MethodsSMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from SMN2 or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90.ResultsOf the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO.ConclusionThis study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.

A critical examination of the recently reported crystal structures of the human SMN protein.

A recent publication by Seng et al. in this journal reports the crystallographic structure of refolded, full-length SMN protein and two disease-relevant derivatives thereof. Here, we would like to suggest that at least two of the structures reported in that study are incorrect. We present evidence that one of the associated crystallographic datasets is derived from a crystal of the bacterial Sm-like protein Hfq and that a second dataset is derived from a crystal of the bacterial Gab protein. Both proteins are frequent contaminants of bacterially overexpressed proteins which might have been co-purified during metal affinity chromatography. A third structure presented in the Seng et al. paper cannot be examined further because neither the atomic coordinates, nor the diffraction intensities were made publicly available. The Tudor domain protein SMN has been shown to be a component of the SMN complex, which mediates the assembly of RNA-protein complexes of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). Importantly, this activity is reduced in SMA patients, raising the possibility that the aetiology of SMA is linked to RNA metabolism. Structural studies on diverse components of the SMN complex, including fragments of SMN itself have contributed greatly to our understanding of the cellular UsnRNP assembly machinery. Yet full-length SMN has so far evaded structural elucidation. The Seng et al. study claimed to have closed this gap, but based on the results presented here, the only conclusion that can be drawn is that the Seng et al. study is largely invalid and should be retracted from the literature.

Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy

Proximal spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder caused by deletion or mutation of the survival of motor neuron 1 (SMN1). Here, we studied SMA molecular pathology in 653 Chinese patients and found approximately 88.2% with homozygous SMN1 exon 7 deletion and 6.3% with heterozygous exon 7 loss using multiplex ligation-dependent probe amplification. SMN1 variants were detected in 34 patients with heterozygous SMN1 loss by clone sequencing. In 27 of them, 15 variants were identified: five were unreported novel variants [c.-7_9del(p.0), p.Tyr109Cys, p.Ile249Tyrfs*16, p.Tyr272Trpfs*35, and c.835-5T>G], five were previously found only in Chinese patients (p.Ser8Lysfs*23, p.Gln14*, p.Val19Glyfs*21, p.Leu228*, and p.Tyr277Cys), and five were reported in other populations [p.Ala2Gly, p.Gln15*, p.Glu134Lys, p.Ser230Leu, and c.863G>T (r.835_*3del, p.Gly279Glufs*5)]. Variants p.Ser8Lysfs*23 and p.Leu228* were the most common in Chinese SMA. Five variants (p.Ser8Lysfs*23, p.Gln14*, p.Gln15*, p.Val19Glyfs*21, and p.Leu228*) resulted in premature stop codons, likely causing SMN1 mRNA nonsense-mediated decay. The novel variant c.-7_9del (p.0) caused deletion of the translation start codon (AUG), resulting in full-length SMN protein loss. The novel variant c.835-5T>G, located in a splice site, resulted in 90% exon 7 skipping. Our study could facilitate early diagnosis for SMA patients in mutation detection and revealed the specific mutation spectrum ofSMN1 in Chinese SMA and high genetic heterogeneity in subtle variants observed between patients from China and Caucasians.

Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine To Treat Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.

Cell-type-specific miR-431 dysregulation in a motor neuron model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal-recessive pediatric neurodegenerative disease characterized by selective loss of spinal motor neurons. It is caused by mutation in the survival of motor neuron 1, SMN1, gene and leads to loss of function of the full-length SMN protein. microRNAs (miRNAs) are small RNAs that are involved in post-transcriptional regulation of gene expression. Prior studies have implicated miRNAs in the pathogenesis of motor neuron disease. We hypothesized that motor neuron-specific miRNA expression changes are involved in their selective vulnerability in SMA. Therefore, we sought to determine the effect of SMN loss on miRNAs and their target mRNAs in spinal motor neurons. We used microarray and RNAseq to profile both miRNA and mRNA expression in primary spinal motor neuron cultures after acute SMN knockdown. By integrating the miRNA:mRNA profiles, a number of dysregulated miRNAs were identified with enrichment in differentially expressed putative mRNA targets. miR-431 expression was highly increased, and a number of its putative mRNA targets were significantly downregulated in motor neurons after SMN loss. Further, we found that miR-431 regulates motor neuron neurite length by targeting several molecules previously identified to play a role in motor neuron axon outgrowth, including chondrolectin. Together, our findings indicate that cell-type-specific dysregulation of miR-431 plays a role in the SMA motor neuron phenotype.

Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1) gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a) regulation of SMN gene expression and (b) degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms.

The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN.

SMN expression is required in motor neurons to rescue electrophysiological deficits in the SMNΔ7 mouse model of SMA.

Proximal spinal muscular atrophy (SMA) is the most frequent cause of hereditary infant mortality. SMA is an autosomal recessive neuromuscular disorder that results from the loss of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The SMN2 gene produces an insufficient amount of full-length SMN protein that results in loss of motor neurons in the spinal cord and subsequent muscle paralysis. Previously we have shown that overexpression of human SMN in neurons in the SMA mouse ameliorates the SMA phenotype while overexpression of human SMN in skeletal muscle had no effect. Using Cre recombinase, here we show that either deletion or replacement of Smn in motor neurons (ChAT-Cre) significantly alters the functional output of the motor unit as measured with compound muscle action potential and motor unit number estimation. However ChAT-Cre alone did not alter the survival of SMA mice by replacement and did not appreciably affect survival when used to deplete SMN. However replacement of Smn in both neurons and glia in addition to the motor neuron (Nestin-Cre and ChAT-Cre) resulted in the greatest improvement in survival of the mouse and in some instances complete rescue was achieved. These findings demonstrate that high expression of SMN in the motor neuron is both necessary and sufficient for proper function of the motor unit. Furthermore, in the mouse high expression of SMN in neurons and glia, in addition to motor neurons, has a major impact on survival.

Developing therapies for spinal muscular atrophy.

Spinal muscular atrophy is an autosomal-recessive pediatric neurodegenerative disease characterized by loss of spinal motor neurons. It is caused by mutation in the gene survival of motor neuron 1 (SMN1), leading to loss of function of the full-length SMN protein. SMN has a number of functions in neurons, including RNA splicing and snRNP biogenesis in the nucleus, and RNA trafficking in neurites. The expression level of full-length SMN protein from the SMN2 locus modifies disease severity. Increasing full-length SMN protein by a small amount can lead to significant improvements in the neurological phenotype. Currently available interventions for spinal muscular atrophy patients are physical therapy and orthopedic, nutritional, and pulmonary interventions; these are palliative or supportive measures and do not address the etiology of the disease. In the past decade, there has been a push for developing therapeutics to improve motor phenotypes and increase life span of spinal muscular atrophy patients. These therapies are aimed primarily at restoration of full-length SMN protein levels, but other neuroprotective treatments have been investigated as well. Here, we discuss recent advances in basic and clinical studies toward finding safe and effective treatments of spinal muscular atrophy using gene therapy, antisense oligonucleotides, and other small molecule modulators of SMN expression.

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Detection of survival motor neuron protein in buccal cells through electrochemiluminescence-based assay.

Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30 pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.

ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets.

Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK), which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myoblasts, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice.

Solving the puzzle of spinal muscular atrophy: What are the missing pieces?

Spinal muscular atrophy (SMA) is an autosomal recessive, lower motor neuron disease. Clinical heterogeneity is pervasive: three infantile (type I-III) and one adult-onset (type IV) forms are recognized. Type I SMA is the most common genetic cause of death in infancy and accounts for about 50% of all patients with SMA. Most forms of SMA are caused by mutations of the survival motor neuron (SMN1) gene. A second gene that is 99% identical to SMN1 (SMN2) is located in the same region. The only functionally relevant difference between the two genes identified to date is a C → T transition in exon 7 of SMN2, which determines an alternative spliced isoform that predominantly excludes exon 7. Thus, SMN2 genes do not produce sufficient full length SMN protein to prevent the onset of the disease. Since the identification of the causative mutation, biomedical research of SMA has progressed by leaps and bounds: from clues on the function of SMN protein, to the development of different models of the disease, to the identification of potential treatments, some of which are currently in human trials. The aim of this review is to elucidate the current state of knowledge, emphasizing how close we are to the solution of the puzzle that is SMA, and, more importantly, to highlight the missing pieces of this puzzle. Filling in these gaps in our knowledge will likely accelerate the development and delivery of efficient treatments for SMA patients and be a prerequisite towards achieving our final goal, the cure of SMA.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug‐like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA.

Anesthesia and spinal muscle atrophy

Spinal muscle atrophy (SMA) is autosomal recessive and one of the most common inherited lethal diseases in childhood. The spectrum of symptoms of SMA is continuous and varies from neonatal death to progressive symmetrical muscle weakness first appearing in adulthood. The disease is produced by degeneration of spinal motor neurons and can be described in three or more categories: SMA I with onset of symptoms before 6months of age; SMAII with onset between 6 and 18months and SMA III, which presents later in childhood. Genetics: The disease is in more than 95% of cases caused by a homozygous deletion in survival motor neuron gene 1 (SMN1). The loss of full-length functioning SMN protein leads to a degeneration of anterior spinal motor neurons which causes muscle weakness. Anesthetic risks: Airway: Tracheal intubation can be difficult. Respiration: Infants with SMA I almost always need postoperative respiratory support. Patients with SMA II sometimes need support, while SMA III patients seldom need support. Circulation: Circulatory problems during anesthesia are rare. Anesthetic drugs: Neuromuscular blockers: Patients with SMA may display increased sensitivity to and prolonged effect of nondepolarizing neuromuscular blockers. Intubation without muscle relaxation should be considered. Succinylcholine should be avoided. Opioids: These should be titrated carefully. Anesthetic techniques: All types of anesthetic technique have been used. Although none is absolutely contraindicated, none is perfect: anesthesia must be individualized. The perioperative risks can be considerable and are mainly related to the respiratory system, from respiratory failure to difficult/impossible intubation.

Therapeutic Strategies For The Treatment Of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease that results in progressive dysfunction of motor neurons of the anterior horn of the spinal cord. SMA is caused by the loss of full-length protein expression from the survival of motor neuron 1 (SMN1) gene. The disease has a unique genetic profile as it is autosomal recessive for the loss of SMN1, but a nearly identical homolog, SMN2, acts as a disease modifier whose expression is inversely correlated to clinical severity. Targeted therapeutic approaches primarily focus on increasing the levels of full-length SMN protein, through either gene replacement or regulation of SMN2 expression. There is currently no US FDA approved treatment for SMA. This is an exciting time as multiple efforts from academic and industrial laboratories are reaching the preclinical and clinical testing stages.

Candidate Proteins, Metabolites and Transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

BackgroundSpinal Muscular Atrophy (SMA) is a neurodegenerative motor neuron disorder resulting from a homozygous mutation of the survival of motor neuron 1 (SMN1) gene. The gene product, SMN protein, functions in RNA biosynthesis in all tissues. In humans, a nearly identical gene, SMN2, rescues an otherwise lethal phenotype by producing a small amount of full-length SMN protein. SMN2 copy number inversely correlates with disease severity. Identifying other novel biomarkers could inform clinical trial design and identify novel therapeutic targets.Objective:To identify novel candidate biomarkers associated with disease severity in SMA using unbiased proteomic, metabolomic and transcriptomic approaches.Materials and Methods:A cross-sectional single evaluation was performed in 108 children with genetically confirmed SMA, aged 2–12 years, manifesting a broad range of disease severity and selected to distinguish factors associated with SMA type and present functional ability independent of age. Blood and urine specimens from these and 22 age-matched healthy controls were interrogated using proteomic, metabolomic and transcriptomic discovery platforms. Analyte associations were evaluated against a primary measure of disease severity, the Modified Hammersmith Functional Motor Scale (MHFMS) and to a number of secondary clinical measures.ResultsA total of 200 candidate biomarkers correlate with MHFMS scores: 97 plasma proteins, 59 plasma metabolites (9 amino acids, 10 free fatty acids, 12 lipids and 28 GC/MS metabolites) and 44 urine metabolites. No transcripts correlated with MHFMS.DiscussionIn this cross-sectional study, “BforSMA” (Biomarkers for SMA), candidate protein and metabolite markers were identified. No transcript biomarker candidates were identified. Additional mining of this rich dataset may yield important insights into relevant SMA-related pathophysiology and biological network associations. Additional prospective studies are needed to confirm these findings, demonstrate sensitivity to change with disease progression, and assess potential impact on clinical trial design.Trial Registry Clinicaltrials.gov NCT00756821.