Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-lengt...Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene.The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy,in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7.In this study,we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer(ISS)sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length(FL)gene expression.It happens that there was a protospacer adjacent motif(PAM)at one end of the ISS sequence according to the design of sgRNA.The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells.Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction,in which deleting small fragments,inserting small amounts and mutation.Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8%(P<0.05).In the primary cultured chondrocytes of SMA mice,in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23%(P<0.05)and significantly improve SMN protein levels(P<0.05).CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases,but it is rarely reported in the treatment of SMA diseases.This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing,which can effectively promote the production of SMN2 FL gene expressions,in which there was an important clinical reference value.展开更多
Spinal muscular atrophy(SMA)is a rare hereditary neuromuscular disease with a high lethality rate in infants.Variants in the homologous genes survival of motor neuron(SMN)1 and SMN2 have been reported to be SMA pathog...Spinal muscular atrophy(SMA)is a rare hereditary neuromuscular disease with a high lethality rate in infants.Variants in the homologous genes survival of motor neuron(SMN)1 and SMN2 have been reported to be SMA pathogenic factors.Previous studies showed that a highinclusion rate of SMN2 exon 7 increased SMN expression,which in turn reduced the severity of SMA.The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues.Neuro-oncological ventral antigen(NOVA)is a splicing factor that is specifically and highly expressed in neurons.It plays a key role in nervous system development and in the induction of nervous system diseases.Howeve r,it remains unclear whether this splicing factor affects SMA.In this study,we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA(genotype smn^(-/-)SMN2^(2 tg/0))and litter mate controls(genotype smn^(+/-)SMN2^(2 tg/0)).We found that inclusion level of SMN2 exon 7 was high in the brain and spinal co rd tissue,and that NOVA1 was also highly expressed in nervous system tissues.In addition,SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system.We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1-7,and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed.We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression ofthe SMN2 protein in the U87 MG cell line,whereas the opposite was observed when NOVA1 was knocked down.Finally,point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7.Moreove r,CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif.Collectively,these findings indicate that NOVA1 intera cts with the UCAC motif in exon 7 of SMN2,there by enhancing inclusion of exon 7 in SMN2,which in turn increases expression of the SMN protein.展开更多
The homozygous loss of the survival motor neuron 1 （SMN1） gene is the primary cause of spinal muscular atrophy （SMA）, a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functional...The homozygous loss of the survival motor neuron 1 （SMN1） gene is the primary cause of spinal muscular atrophy （SMA）, a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functionally equivalent in all SMA patients, modifies the clinical SMA phenotypes. We analyzed the methylation levels of 4 CpG islands （CGIs） in SMN2 in 35 Chinese children with SMA by MassARRAY. We found that three CpG units located in CGI 1 （nucleotides （nt） -871, -735） and CGI 4 （nt ＋999） are significantly hypomethylated in SMA type III compared with type I or II children after receiving Bonferroni correction. In addition to the differentially methylated CpG unit of nt -871, the methylation level of the nt -290/-288/-285 unit was negatively correlated with the expression of SMN2 full-length transcripts （SMN2-fl）. In addition, the methylation level at nt ＋938 was inversely proportional to the ratio of SMN2-fl and lacking exon 7 transcripts （SMN2-A7, fl/A7）, and was not associated with the SMN2 transcript levels. Thus, we can conclude that SMN2 methylation may regulate the SMA disease phenotype by modulating its transcription.展开更多
Purpose: To compare the accuracy of a commercially available MLPA kit with a laboratory developed RT-PCR assay for the detection of SMN1 and SMN2 copy numbers in clinical samples. Methods: We developed and validated a...Purpose: To compare the accuracy of a commercially available MLPA kit with a laboratory developed RT-PCR assay for the detection of SMN1 and SMN2 copy numbers in clinical samples. Methods: We developed and validated a laboratory developed real time PCR based test capable of detecting SMN1 and SMN2 copy numbers in individuals. We also validated an MLPA kit purchased from MRC Holland for the same purpose. We then analyzed a series of 1027 anonymized samples using both technologies. When discrepant results were obtained, each sample was re-analyzed at least twice using both platforms. Results: Five samples did not yield results in either assay. For SMN1 copy number analysis, 2 RT-PCR assays revealed indeterminant results and all 1020 other samples were concordant for SMN1 copy number. There were 9 discrepancies in SMN2 copy number determination mostly due to a variability in MLPA analysis. Conclusion: Both MLPA and RTPCR assays give a reliable estimate of SMN1 copy number and are therefore appropriate for population based carrier screening for Spinal Muscular Atrophy Type 1. The MLPA kit has a low incidence (<1%) of underestimating the SMN2 copy number by 1 copy, but this inconsistency is of little clinical significance and can be overcome by replicate testing.展开更多
Spinal muscular atrophy （SMA） is a common Pautosomal recessive neuromuscular disorder （1in 6000 to 10 000 births） caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous del...Spinal muscular atrophy （SMA） is a common Pautosomal recessive neuromuscular disorder （1in 6000 to 10 000 births） caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1, which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN2. Based on nucleotide mismatches between SMN1 and SMN2, the following two DNA tests are usually performed： single-strand conformational polymorphism （SSCP） and polymerase chain reaction （PCR） followed by a restriction enzyme digestion.In this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography （DHPLC）, which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1.展开更多
基金Nantong Science and Technology Program,grant number(JC2018090)the Practice Innovation Training Program Projects for the Jiangsu College Students,grant number(201810304028z)the Scientific Innovation Research of College Graduates in Jiangsu Province,grant number(KYCX18-2415)。
文摘Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene.The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy,in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7.In this study,we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer(ISS)sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length(FL)gene expression.It happens that there was a protospacer adjacent motif(PAM)at one end of the ISS sequence according to the design of sgRNA.The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells.Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction,in which deleting small fragments,inserting small amounts and mutation.Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8%(P<0.05).In the primary cultured chondrocytes of SMA mice,in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23%(P<0.05)and significantly improve SMN protein levels(P<0.05).CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases,but it is rarely reported in the treatment of SMA diseases.This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing,which can effectively promote the production of SMN2 FL gene expressions,in which there was an important clinical reference value.
基金the National Natural Science Foundation of China,No.32000841(to JJS)a grant from Science and Technology Project of Nantong of Jiangsu Province,No.JC2018090(to LCW)a grant from Graduate Research and Innovation Project of Jiangsu Province,No.KYCX18-2415(to LLD)。
文摘Spinal muscular atrophy(SMA)is a rare hereditary neuromuscular disease with a high lethality rate in infants.Variants in the homologous genes survival of motor neuron(SMN)1 and SMN2 have been reported to be SMA pathogenic factors.Previous studies showed that a highinclusion rate of SMN2 exon 7 increased SMN expression,which in turn reduced the severity of SMA.The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues.Neuro-oncological ventral antigen(NOVA)is a splicing factor that is specifically and highly expressed in neurons.It plays a key role in nervous system development and in the induction of nervous system diseases.Howeve r,it remains unclear whether this splicing factor affects SMA.In this study,we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA(genotype smn^(-/-)SMN2^(2 tg/0))and litter mate controls(genotype smn^(+/-)SMN2^(2 tg/0)).We found that inclusion level of SMN2 exon 7 was high in the brain and spinal co rd tissue,and that NOVA1 was also highly expressed in nervous system tissues.In addition,SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system.We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1-7,and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed.We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression ofthe SMN2 protein in the U87 MG cell line,whereas the opposite was observed when NOVA1 was knocked down.Finally,point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7.Moreove r,CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif.Collectively,these findings indicate that NOVA1 intera cts with the UCAC motif in exon 7 of SMN2,there by enhancing inclusion of exon 7 in SMN2,which in turn increases expression of the SMN protein.
基金Project supported by the National Natural Science Foundation of China(Nos.81050034 and 81500979)the Research Foundation of the Capital Institute of Pediatrics(No.Fangxiang-2014-01)the Beijing Talents Fund(No.2014000021469G228)
文摘The homozygous loss of the survival motor neuron 1 （SMN1） gene is the primary cause of spinal muscular atrophy （SMA）, a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functionally equivalent in all SMA patients, modifies the clinical SMA phenotypes. We analyzed the methylation levels of 4 CpG islands （CGIs） in SMN2 in 35 Chinese children with SMA by MassARRAY. We found that three CpG units located in CGI 1 （nucleotides （nt） -871, -735） and CGI 4 （nt ＋999） are significantly hypomethylated in SMA type III compared with type I or II children after receiving Bonferroni correction. In addition to the differentially methylated CpG unit of nt -871, the methylation level of the nt -290/-288/-285 unit was negatively correlated with the expression of SMN2 full-length transcripts （SMN2-fl）. In addition, the methylation level at nt ＋938 was inversely proportional to the ratio of SMN2-fl and lacking exon 7 transcripts （SMN2-A7, fl/A7）, and was not associated with the SMN2 transcript levels. Thus, we can conclude that SMN2 methylation may regulate the SMA disease phenotype by modulating its transcription.
文摘Purpose: To compare the accuracy of a commercially available MLPA kit with a laboratory developed RT-PCR assay for the detection of SMN1 and SMN2 copy numbers in clinical samples. Methods: We developed and validated a laboratory developed real time PCR based test capable of detecting SMN1 and SMN2 copy numbers in individuals. We also validated an MLPA kit purchased from MRC Holland for the same purpose. We then analyzed a series of 1027 anonymized samples using both technologies. When discrepant results were obtained, each sample was re-analyzed at least twice using both platforms. Results: Five samples did not yield results in either assay. For SMN1 copy number analysis, 2 RT-PCR assays revealed indeterminant results and all 1020 other samples were concordant for SMN1 copy number. There were 9 discrepancies in SMN2 copy number determination mostly due to a variability in MLPA analysis. Conclusion: Both MLPA and RTPCR assays give a reliable estimate of SMN1 copy number and are therefore appropriate for population based carrier screening for Spinal Muscular Atrophy Type 1. The MLPA kit has a low incidence (<1%) of underestimating the SMN2 copy number by 1 copy, but this inconsistency is of little clinical significance and can be overcome by replicate testing.
基金This study was supported by grants from National 863 Program (No. 2002BA711A07-08) and National 973 Program (No.2001CB510302).
文摘Spinal muscular atrophy （SMA） is a common Pautosomal recessive neuromuscular disorder （1in 6000 to 10 000 births） caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1, which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN2. Based on nucleotide mismatches between SMN1 and SMN2, the following two DNA tests are usually performed： single-strand conformational polymorphism （SSCP） and polymerase chain reaction （PCR） followed by a restriction enzyme digestion.In this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography （DHPLC）, which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1.
