The Experts below are selected from a list of 3003 Experts worldwide ranked by ideXlab platform
Brunhilde Wirth - One of the best experts on this subject based on the ideXlab platform.
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infants diagnosed with spinal muscular atrophy and 4 SMN2 copies through newborn screening opportunity or burden
Journal of neuromuscular diseases, 2020Co-Authors: W Mullerfelber, Brunhilde Wirth, Katharina Vill, Oliver Schwartz, Dieter Glaser, Uta Nennstiel, Siegfried Burggraf, Wulf Roschinger, Marc Becker, Jurgen DurnerAbstract:Although the value of newborn screening (NBS) for early detection and treatment opportunity in SMA patients is generally accepted, there is still an ongoing discussion about the best strategy in children with 4 and more copies of the SMN2 gene. This gene is known to be the most important but not the only disease modifier.In our SMA-NBS pilot project in Germany comprising 278,970 infants screened between January 2018 and November 2019 were 38 positive cases with a homozygous SMN1 deletion. 40% of them had 4 or more SMN2 copies. The incidence for homozygous SMN1 deletion was 1 : 7350, which is within the known range of SMA incidence in Germany.Of the 15 SMA children with 4 SMN2 copies, one child developed physical signs of SMA by the age of 8 months. Reanalysis of the SMN2 copy number by a different test method revealed 3 copies. Two children had affected siblings with SMA Type III, who were diagnosed only after detection of the index patient in the NBS. One had a positive family history with an affected aunt (onset of disease at the age of 3 years). Three families were lost to medical follow up; two because of socioeconomic reasons and one to avoid the psychological stress associated with the appointments.Decisions on how to handle patients with 4 SMN2 copies are discussed in the light of the experience gathered from our NBS pilot SMA program.
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discrepancy in redetermination of SMN2 copy numbers in children with sma
Neurology, 2019Co-Authors: David Schorling, Brunhilde Wirth, Jutta Becker, Astrid Pechmann, Thorsten Langer, Janbernd KirschnerAbstract:Spinal muscular atrophy (SMA) is a rare autosomal recessive inherited neuromuscular disease with an incidence of about 1:6,000 to 1:10,000 in newborns. The clinical spectrum of severity is broad and ranges from early and severe weakness with respiratory insufficiency (type 1) to milder phenotypes with onset during childhood or adolescence (types 2–3).1 SMA is caused by deletions and less commonly by point mutations in SMN1 (survival of motor neuron) on chromosome 5q. The paralogous SMN2 gene is present in a variable copy number and differs from SMN1 by few nucleotides. A single nucleotide change disrupts an exonic splicing enhancer and creates a new exonic splicing silencer resulting in splice modification and exclusion of exon 7, so that a mainly truncated and unstable SMN∆7 protein is produced.2 While being of no importance in healthy individuals, SMN2 is crucial in patients with SMA, where smaller amounts of functional SMN protein (estimated to be around 10%) can be produced by each SMN2 copy . Therefore, the number of SMN2 copies inversely correlates with disease severity.3,4 SMN2 is also targeted by antisense-oligonucleotide therapy, which leads to higher amounts of functional SMN protein by modifying splicing of exon 7.5
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the benzamide m344 a novel histone deacetylase inhibitor significantly increases SMN2 rna protein levels in spinal muscular atrophy cells
Human Genetics, 2006Co-Authors: Markus Riessland, Eric Hahnen, Lars Brichta, Brunhilde WirthAbstract:Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder causing infant death in half of all patients. Homozygous loss of the survival motor neuron 1 (SMN1) gene causes SMA, whereas the number of the SMN2 copy genes modulates the severity of the disease. Due to a silent mutation within an exonic splicing enhancer, SMN2 mainly produces alternatively spliced transcripts lacking exon 7 and only ∼ 10% of a full-length protein identical to SMN1. However, SMN2 represents a promising target for an SMA therapy. The correct splicing of SMN2 can be efficiently restored by over-expression of the splicing factor Htra2-β1 as well as by exogenous factors like drugs that inhibit histone deacetylases (HDACs). Here we show that the novel benzamide M344, an HDAC inhibitor, up-regulates SMN2 protein expression in fibroblast cells derived from SMA patients up to 7-fold after 64 h of treatment. Moreover, M344 significantly raises the total number of gems/nucleus as well as the number of nuclei that contain gems. This is the strongest in vitro effect of a drug on the SMN protein level reported so far. The reversion of Δ7-SMN2 into FL-SMN2 transcripts as demonstrated by quantitative RT-PCR is most likely facilitated by elevated levels of Htra2-β1. Investigations of the cytotoxicity of M344 using an MTT assay revealed toxic cell effects only at very high concentrations. In conclusion, M344 can be considered as highly potent HDAC inhibitor which is active at low doses and therefore represents a promising candidate for a causal therapy of SMA.
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The benzamide M344, a novel histone deacetylase inhibitor, significantly increases SMN2 RNA/protein levels in spinal muscular atrophy cells.
Human Genetics, 2006Co-Authors: Markus Riessland, Eric Hahnen, Lars Brichta, Brunhilde WirthAbstract:Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder causing infant death in half of all patients. Homozygous loss of the survival motor neuron 1 (SMN1) gene causes SMA, whereas the number of the SMN2 copy genes modulates the severity of the disease. Due to a silent mutation within an exonic splicing enhancer, SMN2 mainly produces alternatively spliced transcripts lacking exon 7 and only ∼ 10% of a full-length protein identical to SMN1. However, SMN2 represents a promising target for an SMA therapy. The correct splicing of SMN2 can be efficiently restored by over-expression of the splicing factor Htra2-β1 as well as by exogenous factors like drugs that inhibit histone deacetylases (HDACs). Here we show that the novel benzamide M344, an HDAC inhibitor, up-regulates SMN2 protein expression in fibroblast cells derived from SMA patients up to 7-fold after 64 h of treatment. Moreover, M344 significantly raises the total number of gems/nucleus as well as the number of nuclei that contain gems. This is the strongest in vitro effect of a drug on the SMN protein level reported so far. The reversion of Δ7-SMN2 into FL-SMN2 transcripts as demonstrated by quantitative RT-PCR is most likely facilitated by elevated levels of Htra2-β1. Investigations of the cytotoxicity of M344 using an MTT assay revealed toxic cell effects only at very high concentrations. In conclusion, M344 can be considered as highly potent HDAC inhibitor which is active at low doses and therefore represents a promising candidate for a causal therapy of SMA.
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mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number
Human Genetics, 2006Co-Authors: Brunhilde Wirth, Lars Brichta, Bertold Schrank, Hanns Lochmuller, S Blick, A Baasner, Raoul HellerAbstract:Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by loss of the SMN1 gene. The clinical distinction between SMA type I to IV reflects different age of onset and disease severity. SMN2, a nearly identical copy gene of SMN1, produces only 10% of full-length SMN RNA/protein and is an excellent target for a potential therapy. Several clinical trials with drugs that increase the SMN2 expression such as valproic acid and phenylbutyrate are in progress. Solid natural history data for SMA are crucial to enable a correlation between genotype and phenotype as well as the outcome of therapy. We provide genotypic and phenotypic data from 115 SMA patients with type IIIa (age of onset 3 years) and rare type IV (onset >30 years). While 62% of type IIIa patients carry two or three SMN2 copies, 65% of type IIIb patients carry four or five SMN2 copies. Three type IV SMA patients had four and one had six SMN2 copies. Our data support the disease-modifying role of SMN2 leading to later onset and a better prognosis. A statistically significant correlation for ≥4 SMN2 copies with SMA type IIIb or a milder phenotype suggests that SMN2 copy number can be used as a clinical prognostic indicator in SMA patients. The additional case of a foetus with homozygous SMN1 deletion and postnatal measurement of five SMN2 copies illustrates the role of genotypic information in making informed decisions on the management and therapy of such patients.
Christian L. Lorson - One of the best experts on this subject based on the ideXlab platform.
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Optimization of SMN Trans-Splicing Through the Analysis of SMN Introns
Journal of Molecular Neuroscience, 2012Co-Authors: Monir Shababi, Christian L. LorsonAbstract:Spinal muscular atrophy (SMA), a neurodegenerative disease, is the leading genetic cause of infantile death and is caused by the loss of survival motor neuron 1 ( SMN1 ). Humans carry a duplicated copy gene, SMN2 , which produces very low levels of functional protein due to an alternative splicing event. This splicing difference is the reason that SMN2 cannot prevent SMA development when SMN1 is deleted. SMN2 generates a transcript lacking exon 7 and consequently gives rise to an unstable truncated SMN protein that cannot protect from SMA. To increase full-length SMN protein, we utilize a strategy referred to as trans -splicing. This strategy relies upon pre-mRNA splicing occurring between two separate molecules: (1) the endogenous target RNA and (2) the therapeutic RNA that provides the correct RNA sequence via a trans -splicing event. The initial trans -splicing RNA targeted intron 6 and replaced exon 7 with the SMN1 exon 7 in SMN2 pre-mRNA. To determine the most efficient intron for SMN trans -splicing event, a panel of trans -splicing RNA molecules was constructed. Each trans -splicing RNA molecule targets a specific intron within the SMN2 pre-mRNA and based on the target intron, replaces the downstream exons including exon 7. These constructs were examined by RT-PCR, immunofluorescence, and Western blotting. We have identified intron 3 as the most efficient intron to support trans -splicing in cellular assays. The intron 3 trans -splicing construct targets intron 3 and replaces exons 4–7 and was distinguished based on its ability to produce the highest level of the trans -spliced RNA and full-length SMN protein in SMA patient fibroblasts. The efficiency of the intron 3 construct was further improved by addition of an antisense that blocks the 3′ splice site at the intron 4/exon 5 junction. Most importantly, intracerebroventricular injection of the Int3 construct into SMNΔ7 mice elevated the SMN protein levels in the central nervous system. This research demonstrates an alternative platform to correct genetic defects, including SMN expression and examines the molecular basis for trans -splicing.
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decreasing disease severity in symptomatic smn SMN2 spinal muscular atrophy mice following scaav9 smn delivery
Human Gene Therapy, 2012Co-Authors: Jacqueline J Glascock, Monir Shababi, Erkan Y Osman, Mary J Wetz, Megan M Krogman, Christian L. LorsonAbstract:Abstract Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C-to-T transition at the 5’ end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene-replacement strategies. Recently, self-complementary adeno-associated virus (scAAV) vectors have been used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. In this study, we examine a severe model of SMA, Smn–/–;SMN2+/+, to determine whether gene replacement is viable in a model in which disease development begins in utero. Using two delivery paradigms, intracerebroventricular injections and intravenous injections, we delivered sc...
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stimulating full length SMN2 expression by delivering bifunctional rnas via a viral vector
Molecular Therapy, 2006Co-Authors: Travis D Baughan, Monir Shababi, Tristan H Coady, Alexa M Dickson, Gregory E Tullis, Christian L. LorsonAbstract:Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. SMN2 is retained in all SMA patients and encodes an identical protein compared to SMN1. However, a single silent nucleotide difference in SMN2 exon 7 results in the production of a spliced isoform (called SMN7) that encodes a nonfunctional protein. The presence of SMN2 represents a unique therapeutic target since SMN2 has the capacity to encode a fully functional protein. Here we describe an in vivo delivery system for short bifunctional RNAs that modulate SMN2 splicing. Bifunctional RNAs derive their name from the presence of two domains: an antisense RNA sequence specific to a target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. Plasmid-based and recombinant adeno-associated virus vectors were developed that expressed bifunctional RNAs that stimulated SMN2 exon 7 inclusion and full-length SMN protein in patient fibroblasts. These experiments provide a mechanism to modulate splicing from a variety of genetic contexts and demonstrate directly a novel therapeutic approach for SMA.
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253. AAV Delivery of a Trans-Splicing RNA Re-Directs SMN2 Splicing and Results in Increased Full-Length SMN
Molecular Therapy, 2006Co-Authors: Tristan H Coady, Monir Shababi, Christian L. LorsonAbstract:Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients. SMN2 produces low levels of functional protein, while nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein that lacks the final coding exon (exon 7). While SMN is ubiquitously expressed, motor neurons are especially sensitive to the low levels of SMN and this heightened sensitivity leads to the development of SMA. Even though the majority of SMN2-derived transcripts are alternatively spliced and encode a protein that is truncated and unstable, the presence of SMN2 represents a unique therapeutic target because the SMN2 gene has the capacity to encode a fully functional protein. SMN1 and SMN2 differ by 10 nucleotides, however, none of these non-polymorphic differences alter the SMN coding sequence. Rather, due to a subtle disruption of a critical pre-mRNA processing regulatory element, SMN2 transcripts are alternatively spliced. Therefore, by increasing the inclusion of SMN2 exon 7 at the pre- mRNA splicing step, this would result in the production of increased levels of full-length SMN transcript, and in turn, fully functional SMN protein.
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an in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mrna potential therapy of sma
Gene Therapy, 2001Co-Authors: M L Zhang, Christian L. Lorson, Elliot J Androphy, Jianhua ZhouAbstract:Spinal muscular atrophy (SMA) is a degenerative motor neuron disorder resulting from homozygous loss of the SMN1 gene. SMN2, a nearly identical copy gene, is preserved in SMA patients. A single nucleotide difference between SMN1 and SMN2 causes exon 7 skipping in the majority of SMN2 mRNA. Gene therapy through modulation of SMN2 gene transcription in SMA patients may be possible. We constructed a series of SMN mini-genes comprised of SMN exon 6 to exon 8 sequences fused to green fluorescence protein (GFP) or luciferase reporters, to monitor SMN exon 7 splicing. These reporters recapitulated the splicing patterns of the endogenous SMN gene in stable cell lines. The SMN1-luciferase reporter was approximately 3.5-fold more active than SMN2-luciferase and SMN1-GFP intensities were visually distinguishable from SMN2-GFP. We have screened chemical inducers and inhibitors of kinase pathways using stable SMN-reporter lines and found that the phosphatase inhibitor sodium vanadate specifically stimulated exon 7 inclusion within SMN2 mRNAs. This is the first compound identified that can stimulate exon 7 inclusion into transcripts derived from the endogenous SMN2 gene. These results demonstrate that this system can be utilized to identify small molecules that regulate the splicing of SMN exon 7.
Yuhjyh Jong - One of the best experts on this subject based on the ideXlab platform.
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Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells
Biomedicine & Pharmacotherapy, 2017Co-Authors: Yu-chia Chen, Yuhjyh Jong, Jangowth Chang, Wei-lin ChengAbstract: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.
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high expression level of tra2 β1 is responsible for increased SMN2 exon 7 inclusion in the testis of sma mice
PLOS ONE, 2015Co-Authors: Yu-chia Chen, Yuhjyh Jong, Jangowth ChangAbstract:Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.
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identification of bidirectional gene conversion between smn1 and SMN2 by simultaneous analysis of smn dosage and hybrid genes in a chinese population
Journal of the Neurological Sciences, 2011Co-Authors: Taiheng Chen, Chunchi Wang, Jangowth Chang, Shoumei Wu, Chingcherng Tzeng, Sannan Yang, Chihhsing Hung, Yuhjyh JongAbstract:Abstract Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by programmed motoneuron death. The survival motor neuron 1 ( SMN1 ) gene is an SMA-determining gene and SMN2 represents an SMA-modifying gene. Here, we applied capillary electrophoresis to quantify the SMN gene dosage in 163 normal individuals, 94 SMA patients and 138 of their parents. We further quantified exons 7 and 8 in SMN1 and SMN2 . We found that the SMA patients carried the highest SMN2 copies, which was inversely correlated with disease severity among its three subtypes. Increased SMN1 was significantly associated with decreased SMN2 in the normal group. We also observed that parents of type I SMA patients had significantly fewer SMN2 copies than those of types II and III patients. The hybrid SMN genes were detected in two normal individuals and one patient and her mother. These results imply that increased SMN2 copies in SMA patient group might be derived from SMN1 -to- SMN2 conversion, whereas the trend that normal individuals with higher SMN1 copies simultaneously carry fewer SMN2 copies suggested a reverse conversion, SMN2 -to- SMN1 . Together with the identification of hybrid SMN genes, our data provided additional evidence to support that SMN1 and SMN2 gene loci are interchangeable between population groups.
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universal fluorescent multiplex pcr and capillary electrophoresis for evaluation of gene conversion between smn1 and SMN2 in spinal muscular atrophy
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Chunchi Wang, Yuhjyh Jong, Jangowth Chang, Yenling Chen, Shoumei WuAbstract:We have developed a capillary electrophoresis (CE) method with universal fluorescent multiplex PCR to simultaneously detect the SMN1 and SMN2 genes in exons 7 and 8. Spinal muscular atrophy (SMA) is a very frequent inherited disease caused by the absence of the SMN1 gene in approximately 94% of patients. Those patients have deletion of the SMN1 gene or gene conversion between SMN1 and SMN2. However, most methods only focus on the analysis of whole gene deletion, and ignore gene conversion. Simultaneous quantification of SMN1 and SMN2 in exons 7 and 8 is a good strategy for estimating SMN1 deletion or SMN1 to SMN2 gene conversion. This study established a CE separation allowing differentiation of all copy ratios of SMN1 to SMN2 in exons 7 and 8. Among 212 detected individuals, there were 23 SMA patients, 45 carriers, and 144 normal subjects. Three individuals had different ratios of SMN1 to SMN2 in two exons, including an SMA patient having two SMN2 copies in exon 7 but one SMN1 copy in exon 8. This method could provide more information about SMN1 deletion or SMN1 to SMN2 gene conversion for SMA genotyping and diagnosis.
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universal multiplex pcr and ce for quantification of smn1 SMN2 genes in spinal muscular atrophy
Electrophoresis, 2009Co-Authors: Chunchi Wang, Yuhjyh Jong, Jangowth Chang, Shoumei WuAbstract:We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.
Adrian R Krainer - One of the best experts on this subject based on the ideXlab platform.
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a 44g transition in SMN2 intron 6 protects patients with spinal muscular atrophy
Human Molecular Genetics, 2017Co-Authors: Xingxing Wu, Adrian R Krainer, Shuhuei Wang, Thomas W. PriorAbstract:Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced expression of survival of motor neuron (SMN), a protein expressed in humans by two paralogous genes, SMN1 and SMN2. These genes are nearly identical, except for 10 single-nucleotide differences and a 5-nucleotide insertion in SMN2. SMA is subdivided into four main types, with type I being the most severe. SMN2 copy number is a key positive modifier of the disease, but it is not always inversely correlated with clinical severity. We previously reported the c.859G>C variant in SMN2 exon 7 as a positive modifier in several patients. We have now identified A-44G as an additional positive disease modifier, present in a group of patients carrying 3 SMN2 copies but displaying milder clinical phenotypes than other patients with the same SMN2 copy number. One of the three SMN2 copies appears to have been converted from SMN1, but except for the C6T transition, no other changes were detected. Analyzed with minigenes, SMN1C6T displayed a approximately 20% increase in exon 7 inclusion, compared to SMN2. Through systematic mutagenesis, we found that the improvement in exon 7 splicing is mainly attributable to the A-44G transition in intron 6. Using RNA-affinity chromatography and mass spectrometry, we further uncovered binding of the RNA-binding protein HuR to the -44 region, where it acts as a splicing repressor. The A-44G change markedly decreases the binding affinity of HuR, resulting in a moderate increase in exon 7 inclusion.
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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
Genomics, 2015Co-Authors: John F Staropoli, Adrian R Krainer, Huo Li, Seung J Chun, Norm Allaire, Patrick Cullen, Alice Thai, Christina Fleet, Frank C Bennett, Doug KerrAbstract: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 intron 7 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.
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a positive modifier of spinal muscular atrophy in the SMN2 gene
American Journal of Human Genetics, 2009Co-Authors: Thomas W. Prior, Arthur H M Burghes, Kathryn J Swoboda, Adrian R Krainer, Pamela C Snyder, Scott J Bridgeman, John T KisselAbstract:Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new exonic splicing enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.
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differential 3 splice site recognition of smn1 and SMN2 transcripts by u2af and u2 snrnp
RNA, 2009Co-Authors: Martins De Araujo M, Michelle L Hastings, Adrian R Krainer, Sophie Bonnal, Juan ValcarcelAbstract:Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF65 with the 3′ splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF65 crosslinking. Furthermore, blocking recognition of a Tra2-β1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF65 crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.
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Differential 3′ splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP
RNA, 2009Co-Authors: Martins De Araujo M, Michelle L Hastings, Adrian R Krainer, Sophie Bonnal, Juan ValcarcelAbstract:Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF65 with the 3′ splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF65 crosslinking. Furthermore, blocking recognition of a Tra2-β1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF65 crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.
Thomas W. Prior - One of the best experts on this subject based on the ideXlab platform.
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complete sequencing of the SMN2 gene in sma patients detects smn gene deletion junctions and variants in SMN2 that modify the sma phenotype
Human Genetics, 2019Co-Authors: Corey Ruhno, Thomas W. Prior, Vicki L Mcgovern, Matthew R Avenarius, Pamela J Snyder, Flavia C Nery, Abdurrahman W Muhtaseb, Jennifer Roggenbuck, John T Kissel, Valeria SansoneAbstract:Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21 bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.
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a 44g transition in SMN2 intron 6 protects patients with spinal muscular atrophy
Human Molecular Genetics, 2017Co-Authors: Xingxing Wu, Adrian R Krainer, Shuhuei Wang, Thomas W. PriorAbstract:Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced expression of survival of motor neuron (SMN), a protein expressed in humans by two paralogous genes, SMN1 and SMN2. These genes are nearly identical, except for 10 single-nucleotide differences and a 5-nucleotide insertion in SMN2. SMA is subdivided into four main types, with type I being the most severe. SMN2 copy number is a key positive modifier of the disease, but it is not always inversely correlated with clinical severity. We previously reported the c.859G>C variant in SMN2 exon 7 as a positive modifier in several patients. We have now identified A-44G as an additional positive disease modifier, present in a group of patients carrying 3 SMN2 copies but displaying milder clinical phenotypes than other patients with the same SMN2 copy number. One of the three SMN2 copies appears to have been converted from SMN1, but except for the C6T transition, no other changes were detected. Analyzed with minigenes, SMN1C6T displayed a approximately 20% increase in exon 7 inclusion, compared to SMN2. Through systematic mutagenesis, we found that the improvement in exon 7 splicing is mainly attributable to the A-44G transition in intron 6. Using RNA-affinity chromatography and mass spectrometry, we further uncovered binding of the RNA-binding protein HuR to the -44 region, where it acts as a splicing repressor. The A-44G change markedly decreases the binding affinity of HuR, resulting in a moderate increase in exon 7 inclusion.
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hydroxyurea enhances SMN2 gene expression in spinal muscular atrophy cells
Annals of Neurology, 2010Co-Authors: Susanna M Grzeschik, Thomas W. Prior, Madhuri Ganta, William D Heavlin, Ching H WangAbstract:Spinal muscular atrophy (SMA) is a motor neuron disease caused by dysfunction of the survival motor neuron (SMN) gene. Human SMN gene is present in duplicated copies: SMN1 and SMN2. More than 95% of patients with SMA lack a functional SMN1 but retain at least one copy of SMN2. Unlike SMN1, SMN2 is primarily transcribed into truncated messenger RNA and produces low levels of SMN protein. We tested a therapeutic strategy by treating cultured lymphocytes from patients with SMA with hydroxyurea to modify SMN2 gene expression and to increase the production of SMN protein. Twenty lymphoblastoid cell lines (15 SMA and 5 control lines) were treated with hydroxyurea at 5 concentrations (0.5, 5, 50, 500, and 5,000μg/ml) and 3 time points (24, 48, and 72 hours). SMN2 gene copy numbers were determined using real-time quantitative polymerase chain reaction. Hydroxyurea treatment resulted in a time-related and dose-dependent increase in the ratio of full-length to truncated SMN messenger RNA. SMN protein levels and intranuclear gems also were significantly increased in these hydroxyurea-treated cells. The SMN2 gene copy number correlated inversely with the SMA phenotypic severity. This study provides the first evidence for a therapeutic indication of hydroxyurea in SMA. Ann Neurol 2005;58:194–202
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a positive modifier of spinal muscular atrophy in the SMN2 gene
American Journal of Human Genetics, 2009Co-Authors: Thomas W. Prior, Arthur H M Burghes, Kathryn J Swoboda, Adrian R Krainer, Pamela C Snyder, Scott J Bridgeman, John T KisselAbstract:Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new exonic splicing enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.
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homozygous smn1 deletions in unaffected family members and modification of the phenotype by SMN2
American Journal of Medical Genetics Part A, 2004Co-Authors: Thomas W. Prior, Kathryn J Swoboda, Denman H Scott, Ashley Q HejmanowskiAbstract:Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by the homozygous loss of the SMN1 gene. The absence of the SMN1 gene has been shown to occur in all types of SMA, childhood and adult forms. In rare cases, asymptomatic family members have also been found with homozygous mutations in the SMN1 gene, suggesting a role for phenotypic modifiers. We describe three unrelated asymptomatic individuals, with family histories of SMA, who were shown to have the homozygous SMN1 deletion. Quantitative studies indicated that the three individuals all had increased SMN2 copy numbers. These cases not only support the role of SMN2 in modifying the phenotype, but our data also demonstrate that expression levels consistent with five copies of the SMN2 genes maybe enough to compensate for the absence of the SMN1 gene. Lastly, in cases similar to the ones described, the measurement of the SMN2 gene copy number may provide valuable prognostic information.
