The Experts below are selected from a list of 12282 Experts worldwide ranked by ideXlab platform
Shoumei Wu - One of the best experts on this subject based on the ideXlab platform.
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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, Shoumei Wu, Chingcherng Tzeng, Sannan Yang, Chihhsing Hung, Jangowth Chang, 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, Yenling Chen, Yuhjyh Jong, Jangowth Chang, 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.
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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.
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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.
Yuhjyh Jong - One of the best experts on this subject based on the ideXlab platform.
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Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy.
Analytica Chimica Acta, 2014Co-Authors: Chunchi Wang, Chi-jen Shih, Yuhjyh JongAbstract:Abstract This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1 / SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 ( SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16 ± 0.37 ( n = 3). The r values of SMN1 and SMN2 regression curves over a range of 1–4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.
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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, Shoumei Wu, Chingcherng Tzeng, Sannan Yang, Chihhsing Hung, Jangowth Chang, 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, Yenling Chen, Yuhjyh Jong, Jangowth Chang, 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.
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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.
Chunchi Wang - One of the best experts on this subject based on the ideXlab platform.
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Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy.
Analytica Chimica Acta, 2014Co-Authors: Chunchi Wang, Chi-jen Shih, Yuhjyh JongAbstract:Abstract This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1 / SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 ( SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16 ± 0.37 ( n = 3). The r values of SMN1 and SMN2 regression curves over a range of 1–4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.
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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, Shoumei Wu, Chingcherng Tzeng, Sannan Yang, Chihhsing Hung, Jangowth Chang, 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, Yenling Chen, Yuhjyh Jong, Jangowth Chang, 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.
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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.
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, Vicki L. Mcgovern, Pamela J Snyder, Abdurrahman W Muhtaseb, Fernanda Carlota Nery, Thomas W. Prior, Jennifer Roggenbuck, Matthew R Avenarius, 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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single dose gene replacement therapy for spinal muscular atrophy
The New England Journal of Medicine, 2017Co-Authors: Jerry R Mendell, Thomas W. Prior, Samiah Alzaidy, Richard Shell, Dave W Arnold, Louise R Rodinoklapac, Linda Lowes, L Alfano, Katherine Berry, Kathleen ChurchAbstract:BackgroundSpinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. MethodsFifteen patients with SMA1 received a single dose of intravenous adeno-associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×1013 vg per kilogram of body weight), and 12 received a high dose (2.0×1014 vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating be...
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carrier screening for spinal muscular atrophy
Genetics in Medicine, 2008Co-Authors: Thomas W. PriorAbstract:The autosomal recessive disorder proximal spinal muscular atrophy (SMA, MIM #253300) is a severe neuromuscular disease characterized by degeneration of alpha motor neurons in the spinal cord, which results in progressive proximal muscle weakness and paralysis. SMA is the second most common fatal autosomal recessive disorder after cystic fibrosis, with an esti.mated prevalence of 1 in 10,000 live births and a carrier frequency of 1/40Ð1/60. Childhood SMA is subdivided into three clinical groups on the basis of age of onset and clinical course: type I SMA (Werdnig-Hoffmann) is characterized by severe, generalized muscle weakness and hypotonia at birth or within the first 3 months. Death from respiratory failure usually oc.curs within the first 2 years. Children with type II are able to sit, although they cannot stand or walk unaided and survive beyond 4 years. Type III SMA (Kugelberg-Welander) is a milder form, with onset during infancy or youth: patients learn to walk unaided. The survival motor neuron (SMN) gene comprises nine exons and has been shown to be the primary SMA determining gene. Two almost identical SMN genes are present on 5q13: the telomeric or SMN1 gene, which is the SMA-determining gene, and the centromeric or SMN2 gene. The SMN1 gene exon 7 is homozygously absent in approximately 95% of affected patients, with few exceptions, the remainder are heterozygous for the exon 7 deletion and a small more subtle mutation in the other allele (compound heterozygotes). Although abnormalities of the SMN1 gene are observed in the majority of patients, no phenotypeÐgenotype correlation was observed because SMN1 exon 7 is absent in the majority of patients independent of the type of SMA. This is because routine diagnostic methods do not distinguish between a deletion of SMN1 and a conversion event whereby SMN1 is replaced by a copy of SMN2. There have now been several studies that have shown that the SMN2 copy number influences the severity of the disease. The copy number varies from zero to three copies in the normal population, with approximately 15% of normal individuals having no SMN2. However, milder patients with type II or III have been shown to have more copies of SMN2 than do type I patients. It has been proposed that the extra SMN2 in the more mildly affected patients arise through gene conversions, whereby the SMN2 gene is copied either partially or totally into the telomeric locus. Five base pair changes exist between SMN1 and SMN2 transcripts, and none of these differences change amino acids. Because virtually all SMA individuals have at least one SMN2 gene copy, it was initially not understood why individuals with SMN1 mutations have a SMA phenotype. It has now been shown that the SMN1 gene produces predominately full-length transcript, whereas the SMN2 copy produces predominately an alternatively transcribed (exon 7 deleted) product. The inclusion of exon 7 in SMN1 transcripts and exclusion of this exon in SMN2 transcripts is caused by a single nucleotide difference at +6in SMN exon 7. Although the C to T change in SMN2 exon 7 does not change an amino acid, it does disrupt an exonic splicing enhancer that results in the majority of SMN2 transcripts lacking exon 7. Therefore, SMA arises because the SMN2 gene cannot fully compensate for the lack of SMN1 expression when SMN1 is mutated. However, the small amount of full length transcripts generated by SMN2 is able to produce a milder type II or III phenotype when the copy number of SMN2 is increased. The molecular diagnosis of the SMA consists of the detection of the absence of exon 7 of the SMN1 gene. The homozygous absence of detectable SMN1 in SMA patients is being used as a powerful diagnostic test for SMA. Although the targeted mutation analysis has an excellent sensitivity of approximately 95% in identifying affected homozygotes, it cannot detect SMA carriers who have heterozygous deletions of SMN1. Rather, SMN1 gene dosage analysis is required to detect carriers and is highly accurate when performed in an experienced laboratory. Because SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1/40Ð1/60, direct carrier dosage testing has been beneficial to many families with affected children. A number of quantitative polymerase chain reaction assays have been used for the identification of SMA carriers. There are two limitations of the carrier test. First, approximately 2% of SMA cases arise as the result of de novo mutation events, which is high when compared with most autosomal recessive disorders. The high rate of de novo mutations in SMN1 may account for the high carrier frequency in the general population despite the genetic lethality of the type I disease. The large number of repeated sequences around the SMN1 and SMN2 locus likely predispose this region to unequal crossovers and recombination events and results in the high de novo mutation rate. The de novo mutations have been shown to occur primarily during paternal meiosis. Second, the copy number of SMN1 can vary on a chromosome; we have observed that approximately 5% of the normal population possess three copies of SMN1. It is therefore possible for a carrier to possess one chromosome with two copies and a second chromosome with zero copies. The finding of two SMN1 genes on a single chromosome has serious genetic counseling implications, because a carrier with two SMN1 genes on one chromosome and a SMN1 deletion on the other chromosome will have the same dosage result as a noncarrier with one SMN1 gene on each chromosome 5. Thus, the finding of normal two SMN1 copy dosage significantly reduces the risk of being a carrier; however, there is still a residual risk of being a carrier and subsequently a small recurrence risk of future affected offspring for individuals with 2 SMN1 gene copies. Risk assessment calculations using Bayesian analysis are essential for the proper genetic counseling of SMA families. Currently, only individuals with a family history of SMA are routinely being offered carrier testing. However, more broad-based population carrier screening is currently recommended for a number of other genetic disorders with similar carrier frequencies. The prototype for heterozygote screening was testing for Tay-Sachs disease in the Ashkenazi Jewish population, where carrier testing has been offered since 1969. Carrier screening, followed by prenatal diagnosis when indicated, has resulted in a dramatic decrease in the incidence of Tay-Sachs disease in the Jewish population. It is generally accepted that the following criteria should be met for a screening program to be successful: (1) disorder is clinically severe, (2) high frequency of carriers in the screened population, (3) availability of a reliable test with a high specificity and sensitivity, (4) availability of prenatal diagnosis, and (5) access to genetic counseling. SMA fits the criteria for population-based genetic screening. Carrier screening is recommended upon the availability of educational material that can be utilized by patients and providers. The goal of population based SMA carrier screening is to identify couples at risk for having a child with SMA. Preconception carrier screening allows carrier couples to consider the fullest range of reproductive options. The choice to have a SMA carrier test should be made by an informed decision. Educational brochures are available and provide information about SMA and the inheritance patterns. It is important for couples to understand the dosage testing. Because, SMA is the result of a common single deletion event in 95% of the cases, the carrier test is very sensitive (.90% detection rate). However, the molecular testing does not identify all carriers and therefore false-negatives can occur. Approximately 5% of affected patients are compound heterozygotes, exhibiting a deletion and a point mutation. The dosage testing does not identify such point mutation carriers. It is well known that a false-negative result in SMA carriers occurs when the carrier has two SMN1 genes in cis on the one chromosome 5. Further, approximately 2% of affected individuals have a de novo mutation. Therefore, genetic counseling addressing specifically the possibility of false-negative results must be provided for individuals choosing carrier testing.
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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, Denman H Scott, Kathryn J Swoboda, 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.
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Clinical Report Homozygous SMN1 Deletions in Unaffected Family Members and Modification of the Phenotype by SMN2
2004Co-Authors: Thomas W. Prior, H. Denman Scott, Kathryn J Swoboda, Ashley Q HejmanowskiAbstract:Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by the homozygous loss of theSMN1 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. 2004 Wiley-Liss, Inc.
Jangowth Chang - One of the best experts on this subject based on the ideXlab platform.
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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, Shoumei Wu, Chingcherng Tzeng, Sannan Yang, Chihhsing Hung, Jangowth Chang, 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, Yenling Chen, Yuhjyh Jong, Jangowth Chang, 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.
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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.
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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.
