| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2
Introduction
Results
Sequence analysis of genomic clones containing the SMN2 and SMN1 genes
Analysis of sequence differences between SMN2 and SMN1
Construction of SMN minigenes and splicing assays
Discussion
Materials And Methods
DNA sequencing
Database screening and sequence alignments
Polymorphism screen
SMN minigenes
Transfections and RT-PCR
Acknowledgements
References
A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2
Received April 21, 1999; Accepted April 23, 1999
Spinal muscular atrophy (SMA) is a recessive disorder characterized by loss of motor neurons in the spinal cord. It is caused by mutations in the telomeric survival motor neuron 1 (SMN1) gene. Alterations within an almost identical copy gene, the centromeric survival motor neuron 2 (SMN2) gene produce no known phenotypic effect. The exons of the two genes differ by just two nucleotides, neither of which alters the encoded amino acids. At the genomic level, only five nucleotides that differentiate the two genes from one another have been reported. The entire genomic sequence of the two genes has not been determined. Thus, differences which might explain why SMN1is the SMA gene are not readily apparent. In this study, we have completely sequenced and compared genomic clones containing the SMNgenes. The two genes show striking similarity, with the homology being unprecedented between two different yet functional genes. The only critical difference in an ~32 kb region between the two SMN genes is the C->T base change 6 bp inside exon 7. This alteration but not other variations in the SMN genes affects the splicing pattern of the genes. The majority of the transcript from the SMN1 locus is full length, whereas the majority of the transcript produced by the SMN2 locus lacks exon 7. We suggest that the exon 7 nucleotide change affects the activity of an exon splice enhancer. In SMA patients, the loss of SMN1 but the presence of SMN2results in low levels of full-length SMN transcript and therefore low SMN protein levels which causes SMA.
INTRODUCTION
Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by destruction of motor neurons in the anterior horn of the spinal cord. SMA has an estimated incidence of 1 in 10 000 live births, with a carrier frequency of ~1 in 50 people (1). Childhood onset SMA is classified into three groups on the basis of age at onset and clinical course (2); type I SMA (Werdnig-Hoffman disease) is the most severe form, with onset before the age of 6 months and death usually occurring within the first 2 years. Type II SMA is intermediate in severity. Onset occurs at ~18 months and patients never gain the ability to walk. Type III SMA (Kugelberg-Welander disease) is the mildest form of the disease with onset after 18 months. Type III patients are able to stand and walk.
All three forms of proximal SMA are due to mutations in the telomeric but not centromeric survival motor neuron (SMN) genes (3-11). The full-length cDNAs of the two genes are identical except for single nucleotide differences in exons 7 and 8, yet their transcriptional products are not the same. SMN1 produces a majority of the full-length cDNA; SMN2 produces mostly transcript lacking exon 7 (3). We have shown previously that promoter differences do not account for the different levels of full-length transcript from the two genes (12). Instead, the exon 7 difference between the two genes affects splicing, causing increased levels of full-length transcript from SMN1 as compared with SMN2 (13).
The SMN protein is a 38 kDa polypeptide which is ubiquitously expressed (14,15). It is found at especially high levels in the spinal motor neurons. The exact function of the protein remains unknown. However, recent studies have implicated its involvement in mRNA biogenesis. Specifically, SMN has been shown to be critical for spliceosomal snRNP assembly in the cytoplasm, most likely functioning in the regeneration/recycling of snRNPs and other splicing factors (16).
There is accumulating evidence to suggest that SMA is caused by mutations in SMN1 but not SMN2, because a majority of the full-length transcript, and thus SMN protein, derives from the former locus (3,13). It has now also been shown why SMN1 produces a majority of the full-length transcript (13). However, other regions of the two genes have not been examined to determine whether differences elsewhere might also influence splicing and/or expression levels of full-length SMN. To address this, we have completely sequenced genomic clones containing the two SMN genes. We show an unusual degree of similarity between them. Over an ~32 kb region there is >99% homology. All but five differences which have been reported previously turned out to be variants marking neither gene in particular. Thus, our study conclusively demonstrates for the first time that there is only one critical difference between the two SMN genes over their entire genomic sequence. We suggest that this difference is part of an exon splice enhancer (ESE) which determines why SMN1 but not SMN2 is the SMA gene. In addition, our study reports a degree of homology between two different, functional genes that is unprecedented.
RESULTS
Sequence analysis of genomic clones containing the SMN2 and SMN1 genes
The P1 clone 3996 contains the SMN1 gene while the PAC clone 215P15 contains the centromeric copy. Each clone was typed with respect to the differences between the SMN genes in exons 7 and 8. To ensure that each clone contained an intact SMN gene, Southern blots using various restriction enzymes were carried out and compared with similarly digested control DNA. The clones were then sequenced as described in Materials and Methods.
The P1 clone has an insert size of ~83 kb while PAC 215P15 contains an insert of ~130 kb. Thus, each clone contains not only its respective SMN gene, but also certain neuronal apoptosis inhibitory protein (NAIP) exons (17) as well as the XS2G3 (18) sequence (Fig. 1). P1 3996 contains the entire 28 kb SMN1 gene and extends downstream of it. However, the clone terminates short of exon 5 of the NAIP gene which distinguishes this copy of the gene (NAIP5) from one lacking it (NAIP[Delta]). Upstream of SMN exon 1 lies the CA dinucleotide marker Ag1CA/C272 (19) which is contained within P1 3996. However, the P1 insert ends ~250 bp 5[prime] of Ag1CA. PAC 215P15 contains the entireSMN2 gene and extends downstream of it into intron 13 of the NAIP[Delta] gene. Upstream of the SMN2gene, the PAC 215P15 insert extends almost 77 kb and contains the newly identified gene H4F5 (20). Thus, there is an overlap of 53.5 kb between the SMN1unit in P1 3996 and the SMN2unit in PAC 215P15. This 53.5 kb region is also contained within PAC 125D9 which previously has been shown to carry the SMN1 gene, the entire NAIP5 gene and the 3[prime] portion of the BTFp44gene(21).
Figure 1. Genomic organization of the SMN genes in the clones P1 3996 and PACs 125D9 and 215P15. Depicted are the exons of the various genes in each clone including the ESTs in PAC 215P15. The ESTs are: 1, EST having homology to a fetal liver 1NFLS S1 clone; 2, brain cadherin; 3, EST derived from a human parathyroid tumor; and 4, EST having homology to a human gene signature 3[prime]-directed cDNA sequence. The superscripts C and T after the H4F5 gene denote the centromeric and telomeric copies, respectively. The two copies of the NAIP gene are NAIP5 which has exon 5 and NAIP[Delta] lacking exon 5. B represents the BamHI sites in the clones. The broken arrows denote the direction of transcription of the SMN, NAIP, H4F5 and BTF2p44 genes.
Analysis by exon trapping and using BLAST and exon prediction programs identified no additional genes within P1 3996. A similar search for genes in PAC 215P15 did reveal homology to numerous expressed sequence tags (ESTs), most notably the expressed brain cadherin pseudogene (22). However, it is unlikely that any of these hits represent intact genes as the ESTs are not split into separate exons in genomic DNA. Moreover, this region previously has been shown to contain numerous pseudogenes(18,22). An exception is the H4F5gene whose 3[prime] end lies 6.5 kb upstream of the SMN2gene in PAC 215P15 (20). Exon 3b of the telomeric copy of this gene is also present within PAC 125D9.
The 53.5 kb region of overlap between P1 3996 and PAC 215P15 is also contained within PAC 125D9 which has been sequencedpreviously (21). We have used this sequence, as well as the sequence we obtained from P1 3996, and compared it with the insert from PAC 215P15 in order to identify differences which might explain why SMN1but not SMN2is the SMA gene. Our analysis shows that there is a remarkably high homology (>99%) between the telomeric and centromeric regions examined. An alignment of the three clones, using either BLAST or Clustal, showed that there are 132 differences in the overlap region. Of these, 62 are consistent between P1 3996 and PAC 125D9 but different in PAC 215P15. It was concluded, therefore, that the remaining 70 are polymorphisms.
Analysis of sequence differences between SMN2 and SMN1
The essential components of the SMN gene, including the promoter, lie on an ~32 kb fragment whose 5[prime] end is 3.4 kb upstream of the transcriptional start site. Since this fragment is sufficient to mimic expression of the endogenous gene in transgenic mice (unpublished data), differences between the SMN genes in this region were analyzed extensively. A straightforward alignment between the two PAC clones in the promoter region and all three clones in the remaining 28 kb revealed 35 differences, excluding differences in poly(A/T) and poly(CA/GT) tracts (Table 1). Except for one difference, a polymorphic variant between the two SMN1 clones in exon 3, all other differences were consistent between P1 3996 and PAC 125D9 but differed in PAC 215P15. To rule out sequencing errors, all 35 differences were confirmed by amplification from the clones and re-sequencing. The differences were then analyzed to determine whether they were polymorphisms. A control population of 15 individuals was used to carry out this analysis. Five were type I SMA patients completely deleted for SMN1, five were unaffected individuals lacking SMN2, and the remaining fivecarried both genes. Each variant identified was analyzed as described in Materials and Methods. If one of the sequence variants marked the SMN1 gene then it should be absent in the type I SMA individuals and present in the unaffected individuals lacking SMN2. The sequence differences found are listed in Table 1. Of the 35 differences, 12 lie in the promoter, one in intron 1, one in exon 3, 17 in intron 6, one in exon 7, two in intron 7 and one in exon 8. The majority of these variants are single nucleotide polymorphisms (SNPs), two are 3 bp deletions in the centromeric copy and one a 5 bp deletion in the telomeric copy. In our control population, only 11 of the 35 changes were found consistently to distinguish SMN1 from SMN2. Five of these have been reportedpreviously (3,6,11). The remaining six all lie in intron 6, in an ~1 kb region 5[prime] of exon 7 containing numerous repeat elements. One of these six differences is a telomeric-specific 5 bp deletion. To further assess whether the six non-polymorphic differences influenced disease development, an additional 17 type I SMA patients were screened. The nucleotide at the variant +26 156 position (GSMN1/ASMN2) was SMN2-derived in 29 alleles and SMN1-derived in six alleles. Variant +26 236 (absence of AGGCASMN1/presence of AGGCASMN2) was SMN2-derived in 27 alleles and SMN1-derived in seven alleles. Variant +26 287 (ASMN1/CSMN2) was SMN2-derived in 27 alleles and SMN1-derived in seven alleles. Variant +26 587 (GSMN1/ASMN2) was SMN2-derived in 29 alleles and SMN1-derived in five alleles. Variant +26 658 (TSMN1/CSMN2) was SMN2-derived in 28 alleles and SMN1-derived in six alleles. Variant +26 769 (CSMN1/ASMN2) was SMN2-derived in 24 alleles andSMN1-derived in eight alleles. Variant +26 236 was analyzed in seven additional unaffected individuals who lacked the SMN2 gene, and in all cases these individuals had only the telomeric form of the gene. In addition, analysis of SMA type II and III patients showed that none of these changes could be correlated to phenotypic severity of SMA. Taken together, these results demonstrate that the telomeric variants could occur in the SMN2 gene and that the telomeric or centromeric version of these changes are not sufficient to critically affect the SMN gene.
Table 1. Nucleotide differences between the SMN1 and SMN2 genes in the genomic clones P1 3996, PAC 215P15 and PAC 125D9
| Position | P1 3996 | PAC 125D9 | PAC 215P15 |
| Promoter | |||
| -3366 | N/A | - | G |
| -2052 | N/A | C | A |
| -2020 | N/A | C | T |
| -1990 | N/A | C | T |
| -1805 | N/A | C | G |
| -1438 | N/A | A | T |
| -1427 | N/A | C | G |
| -1317 | N/A | C | G |
| -1155 | N/A | G | A |
| -893 | N/A | A | G |
| -769 | N/A | GAG | - |
| -318 | N/A | GCC | - |
| Intron 1 | |||
| +8451 | T | T | C |
| Exon 3 | |||
| +17 739 | G | A | G |
| Intron 6 | |||
| +21 851 | G | G | T |
| +22 872 | A | A | G |
| +23 117 | G | G | A |
| +23 505 | A | A | G |
| +25 239 | T | T | C |
| +25 379 | G | G | A |
| +25 381 | T | T | C |
| +25 519 | G | G | A |
| +25 683 | G | G | A |
| +25 729 | C | C | G |
| +26 156 | G | G | A |
| +26 236 | - | - | AGGCA |
| +26 287 | A | A | C |
| +26 587 | G | G | A |
| +26 658 | T | T | C |
| +26 769 | C | C | A |
| +27 092 | G | G | A |
| Exon 7 | |||
| +27 141 | C | C | T |
| Intron 7 | |||
| +27 289 | A | A | G |
| +27 404 | A | A | G |
| Exon 8 | |||
| +27 869 | G | G | A |
Construction of SMN minigenes and splicing assays
Previous studies have demonstrated chimeric SMN genes with SMN2exon 7 fused to SMN1exon 8 in SMA patients (6,11,23). These chimeric genes that have SMN2-derived exon 7 sequences are SMA alleles. This argues strongly that differences between the SMN genes that result in SMA alleles lie in or upstream of exon 7. Furthermore, these differences most likely affect promoter activity or splicing, as it has been shown that the majority of the full-length SMN transcript derives from SMN1 (3). In a previous study, we ruled out promoter differences as a likely explanation for SMN1 being the disease-determining gene. On the other hand, Lorson et al. (13) have shown that one of the differences between the SMN genes, the C->T base change in exon 7, does indeed affect splicing; however, the six novel SMN differences in intron 6 were not taken into account. To determine whether the newly described differences affected SMN exon 7 RNA splicing, a series of SMNhybrid minigenes were constructed and assessed for their effects upon exon 7 splicing. Exon 7 splicing could be envisaged to be influenced by any of the six variants, based upon their proximity to exon 7. SMN1andSMN2 minigenes were constructed (see Materials and Methods) and derivatives of these constructs were then made by introducing the six SMN2differences lying on a PmlI-BclI fragment into an SMN1background or vice versa. Finally, these derivatives were mutated by site-directed mutagenesis converting the exon 7 difference from the centromeric thymine to the telomeric cytosine and vice versa (Fig. 2A). Transfection of these constructs into cultured cells followed by RT-PCR clearly showed that the six intron 6 differences did not affect splicing. Indeed, none of the nucleotide differences between SMN2and SMN1that flank exon 7 affects its splicing. It is the presence of either a thymine or a cytosine in exon 7 that determines the efficiency with which exon 7 is retained in the final transcript (Fig. 2B).
Figure 2. SMN minigenes and their transcriptional products. (A) Diagrammatic representation of the SMN2, SMN1 and chimeric minigenes used to assay the effect of the exon 7 nucleotide change and six of the intron 6 differences on splicing. Open boxes denote SMN1 while filled boxes denote SMN2sequence. Relative locations of the exon and intron differences are depicted by crosses. The letters P and B refer to the PmlI and BclI sites in intron 6 used to make the chimeric minigenes. The letters C and T in exon 7 refer to the nucleotides cytosine and thymine, respectively. (B) Results of the RT-PCR assay following transfection of the minigenes shown in (A). In every case, presence/absence of exon 7 in the transcript ultimately depends on the nucleotide difference in exon 7. Lanes are numbered according to which minigene construct depicted in (A) was used for the transfection. Lane 9 is a mock transfection using no DNA, lane 10 is the 100 bp ladder (Gibco BRL).
DISCUSSION
Although the cDNAs of the SMN genes differ by just two translationally silent nucleotides, only SMN1 causes SMA. Thus, the exact reason(s) why SMN1 but not SMN2 is the disease gene have not been determined. Lorson et al. (13) have demonstrated recently that the exon 7 nucleotide difference between the SMN genes affects exon 7 splicing. They also showed that a single base change in intron 7 predicted to disrupt the consensus exon 7 splice donor motif resulted in levels of full-length transcript similar to those from SMN2. This, together with earlier studies using semi-quantitative PCR to analyze levels of full-length transcript from the two genes, clearly indicates an important reason why SMN1 is the SMA gene. However, other potential sequence differences in the remaining ~32 kb which might affect splicing/levels of full-length SMN were not examined.
Our study has addressed the above point and presents a detailed comparison of the two SMN genes along their entire genomic sequence. We show that the critical difference in the 32 kb region examined is indeed the single base change in exon 7 which in the SMN1 gene enhances inclusion of exon 7 into the transcript. Exon 7 has been shown to affect SMN oligomerization and the ability of SMN to perform its function in splicing (24).
Single nucleotide changes within exons have previously been shown to alter splicing(25,26). Indeed, alterations of sequences known as ESEs have been implicated in a wide variety of human diseases (27 and references therein). We suggest that the nucleotide difference in exon 7 between the two SMN genes constitutes part of an exon splice enhancer. Substitution of the telomeric cytosine with the centromeric thymine reduces the activity of the enhancer, resulting in the majority of transcript lacking exon 7. In this regard, it is worth noting that in the mouse Smn gene which produces only the full-length transcript (28), the sequence at the putative ESE exactly matches that of human SMN1 over a 15 bp stretch. It would be interesting to determine the effect on splicing of site-directed mutagenesis that converts the cytosine in mouse Smn exon 7 to thymine. SMA patients who have only the SMN2 gene (thymine in the exon 7 ESE) produce only low levels of full-length transcript, with the amount of full-length transcript dependent on the number of intact SMN2 genes(7,29,30). It has been shown that there is a correlation of SMN protein levels and SMA phenotype, with type I SMA patients showing low SMN levels in particular in spinal motor neurons(14,15). This would indicate that the decreased levels of full-length SMN transcript result in low levels of SMN protein, which causes SMA.
This study has also revealed surprisingly high homology between the two SMN genes. Previous studies of unusual similarity between different genes include those on the genes for 21-hydroxylase, CYP21 and CYP21 (31); polycystic kidney disease, PKD1 and its homolog (32); and Gaucher's disease, [beta]-glucocerebrosidase and its homolog (33). However, in each of these instances, the copy gene is a pseudogene that either lacks exons or intronic segments or contains numerous nonsense mutations and is therefore non-functional. The similarity between the SMN genes that we have presented here is therefore unprecedented. To our knowledge, it is the first example of two almost identical yet distinct, functional genes only one of which determines a disease phenotype. Our study opens up the exciting prospect of converting SMN2 to SMN1 by targeting the exon 7 nucleotide in a manner similar to that involving the correction of the mutation responsible for sickle cell anemia (34). It also suggests a strategy for creating a mouse model of SMA by introducing the human SMN2 gene onto a null (Smn-/Smn-) background.
MATERIALS AND METHODS
DNA sequencing
P1 clone 3996 and PAC 215P15 have previously been shown to contain the SMN1 and SMN2 genes, respectively. Large-scale preparation of the clones was carried out using the alkaline lysis method followed by banding on a CsCl gradient with minor modifications; twice the recommended volume of solutions I, II and III were used. Sequencing was carried out at the Washington University Genome Sequencing Center. Isolated DNA was sheared by sonication, sized on a 1% agarose gel and fragments in the 1.5 kb range cloned into dephosphorylated M13mp8 and pUC18 vectors. Sequencing was performed by the cycle sequencing method using Thermosequenase (Amersham, Piscataway, NJ) and energy transfer dye-labeled primers (four color; Applied Biosystems, Foster City, CA). Sequence products were separated on an ABI 377 sequencer and contigs assembled using the base calling (PHRED) and assembly (PHRAP) software programs.
Database screening and sequence alignments
The inserts from P1 3996 and PAC 215P15 were filtered for repetitive sequence using RepeatMasker (http://genome.washington.edu/UWGC/analysistools/repeatmask.html ) and the remaining sequence used to screen the NCBI EST database (http://www.ncbi.nlm.nih.gov/BLAST/ ). Positive ESTs were mapped onto the P1 and PAC clones using the DNAStar software program (DNAStar, Madison, WI). In addition, sequences from both clones were analyzed for the presence of putative genes and exons using GRAIL 1.3, Genie and HEXON, all of which are available at the Baylor College of Medicine server (http://dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html ). PAC 125D9 has been sequenced previously. A detailed comparison between its insert and those of P1 3996 and PAC 215P15 was carried out using BLAST and the DNAStar alignment program.
Polymorphism screen
Genomic DNA was amplified using primers flanking the variants (all primers are available on request). The genomic DNA was derived from five type I SMA patients lacking SMN1 and with two copies of SMN2, five unaffected individuals lacking the SMN2 gene and five individuals with both SMN2 and SMN1. The amplified product was then analyzed for the presence or absence of the variant by single strand conformation polymorphism, direct sequencing of the PCR product or selective base-specific enzyme digestion.
SMN minigenes
SMN minigenes were created using the Expand High Fidelity PCR System (Boehringer Mannheim, Indianapolis, IN). A 250 ng aliquot of human genomic DNA was amplified using the primers Ex6FX (5[prime]-CGATCTCGAGATAATTCCCCCACCACCTC-3[prime]) and Ex8RS (5[prime]-GCTACCCGGGCACATACGCCTCACATACA-3[prime]). Products were digested with XhoI and SmaI and cloned directionally into the appropriate sites of the vector pCI (Promega, Madison, WI). The clones were then sequenced to identify which of these were telomeric and which were centromeric. Single nucleotide conversions were introduced by oligonucleotide site-directed mutagenesis using Thermo Pol Vent polymerase (New England Biolabs, Beverly, MA): pSMN1/pSMN2[Delta]Ex7 (5[prime]-GATTTTGTCTG/AAAACCCTGTAAG-3[prime]; 5[prime]-CTTACAGGGTTTC/TAGACAAAATC-3[prime]). Identical primer sets were used to generate the nucleotide substitutions except for appropriate SMN1- or SMN2-derived nucleotides as indicated in bold. Constructs containing intron 6 hybrids were created by cloning the PmlI-BclI intron 6 fragment of SMN1 or SMN2into the background ofSMN2 or SMN1, respectively, as indicated (Fig. 2A).
Transfections and RT-PCR
The SMN minigenes, SMN1, SMN2 and their derivatives were transfected into C33a cells using Lipofectamine (Gibco BRL, Bethesda, MD) and 2 µg of DNA according to the manufacturer's recommendations. Total cellular RNA was then isolated over a CsCl gradient 48 h post-transfection. A 1 µg aliquot of total RNA was used to generate first strand cDNA using oligo(dT) and Super Script II reverse transcriptase (Gibco BRL). Plasmid-derived cDNAs were amplified using the primer set pCIFwd (5[prime]-GCTAACGCAGTCAGTGCTTC-3[prime]); pCIRev (5[prime]-GTATCTTATCATGTCTGCTCG-3[prime]). Amplification was carried out in 50 µl with 20 pmol of each primer, 200 µM dNTP and 1 U of Thermo Pol Vent polymerase (New England Biolabs). Cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 56°C for 1.5 min, 72°C for 1 min and a final extension of 72°C for 10 min. Reaction products were resolved by electrophoresis in a 1.9% agarose gel and visualized by ethidium bromide staining. Products were cloned and sequenced separately.
ACKNOWLEDGEMENTS
We thank Patty McAndrew for providing us with patient DNA samples and SMN copy number data. Support for this work came from Families of SMA and the Muscular Dystrophy Association of America, and NIH grant NS38650. The authors wish to thank the genome sequencing center, Washington University, St Louis for communication of DNA sequence prior to publication.
REFERENCES
*To whom correspondence should be addressed at: Department of Medical Biochemistry, 363 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA. Tel: +1 614 688 4710; Fax: +1 614 292 4118; Email: burghes.1{at}osu.edu
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H. Tadesse, J. Deschenes-Furry, S. Boisvenue, and J. Cote KH-type splicing regulatory protein interacts with survival motor neuron protein and is misregulated in spinal muscular atrophy Hum. Mol. Genet., February 14, 2008; 17(4): 506 - 524. [Abstract] [Full Text] [PDF]
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 O. Biondi, C. Grondard, S. Lecolle, S. Deforges, C. Pariset, P. Lopes, C. Cifuentes-Diaz, H. Li, B. della Gaspera, C. Chanoine, et al. Exercise-Induced Activation of NMDA Receptor Promotes Motor Unit Development and Survival in a Type 2 Spinal Muscular Atrophy Model Mouse J. Neurosci., January 23, 2008; 28(4): 953 - 962. [Abstract] [Full Text] [PDF]
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T. Kashima, N. Rao, C. J. David, and J. L. Manley hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing Hum. Mol. Genet., December 15, 2007; 16(24): 3149 - 3159. [Abstract] [Full Text] [PDF]
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 A. Nadeau, G. D'Anjou, G. Debray, Y. Robitaille, L. R. Simard, and M. Vanasse A Newborn With Spinal Muscular Atrophy Type 0 Presenting With a Clinicopathological Picture Suggestive of Myotubular Myopathy J Child Neurol, November 1, 2007; 22(11): 1301 - 1304. [Abstract] [PDF]
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 R. E. Pyatt, D. C. Mihal, and T. W. Prior Assessment of Liquid Microbead Arrays for the Screening of Newborns for Spinal Muscular Atrophy Clin. Chem., November 1, 2007; 53(11): 1879 - 1885. [Abstract] [Full Text] [PDF]
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 S. Jablonka, M. Beck, B. D. Lechner, C. Mayer, and M. Sendtner Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy J. Cell Biol., October 8, 2007; 179(1): 139 - 149. [Abstract] [Full Text] [PDF]
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C. J. Sumner Molecular Mechanisms of Spinal Muscular Atrophy J Child Neurol, August 1, 2007; 22(8): 979 - 989. [Abstract] [PDF]
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 J. van Bergeijk, K. Rydel-Konecke, C. Grothe, and P. Claus The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus FASEB J, May 1, 2007; 21(7): 1492 - 1502. [Abstract] [Full Text] [PDF]
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C.-H. Ting, C.-W. Lin, S.-L. Wen, H.-M. Hsieh-Li, and H. Li Stat5 constitutive activation rescues defects in spinal muscular atrophy Hum. Mol. Genet., March 1, 2007; 16(5): 499 - 514. [Abstract] [Full Text] [PDF]
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 T. Kashima, N. Rao, and J. L. Manley An intronic element contributes to splicing repression in spinal muscular atrophy PNAS, February 27, 2007; 104(9): 3426 - 3431. [Abstract] [Full Text] [PDF]
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V. Setola, M. Terao, D. Locatelli, S. Bassanini, E. Garattini, and G. Battaglia Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis PNAS, February 6, 2007; 104(6): 1959 - 1964. [Abstract] [Full Text] [PDF]
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 N. N. Singh, R. N. Singh, and E. J. Androphy Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes Nucleic Acids Res., January 28, 2007; 35(2): 371 - 389. [Abstract] [Full Text] [PDF]
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L. Gangwani Deficiency of the Zinc Finger Protein ZPR1 Causes Defects in Transcription and Cell Cycle Progression J. Biol. Chem., December 29, 2006; 281(52): 40330 - 40340. [Abstract] [Full Text] [PDF]
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T. L. Carrel, M. L. McWhorter, E. Workman, H. Zhang, E. C. Wolstencroft, C. Lorson, G. J. Bassell, A. H. M. Burghes, and C. E. Beattie Survival Motor Neuron Function in Motor Axons Is Independent of Functions Required for Small Nuclear Ribonucleoprotein Biogenesis J. Neurosci., October 25, 2006; 26(43): 11014 - 11022. [Abstract] [Full Text] [PDF]
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C. J. Sumner, S. J. Kolb, G. G. Harmison, N. O. Jeffries, K. Schadt, R. S. Finkel, G. Dreyfuss, and K. H. Fischbeck SMN mRNA and protein levels in peripheral blood: Biomarkers for SMA clinical trials Neurology, April 11, 2006; 66(7): 1067 - 1073. [Abstract] [Full Text] [PDF]
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H.-Y. Kao, Y.-N. Su, H.-K. Liao, M. S. Liu, and Y.-J. Chen Determination of SMN1/SMN2 Gene Dosage by a Quantitative Genotyping Platform Combining Capillary Electrophoresis and MALDI-TOF Mass Spectrometry Clin. Chem., March 1, 2006; 52(3): 361 - 369. [Abstract] [Full Text] [PDF]
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N. K. Singh, N. N. Singh, E. J. Androphy, and R. N. Singh Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron Mol. Cell. Biol., February 15, 2006; 26(4): 1333 - 1346. [Abstract] [Full Text] [PDF]
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S. Jablonka, K. Karle, B. Sandner, C. Andreassi, K. von Au, and M. Sendtner Distinct and overlapping alterations in motor and sensory neurons in a mouse model of spinal muscular atrophy Hum. Mol. Genet., February 1, 2006; 15(3): 511 - 518. [Abstract] [Full Text] [PDF]
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A. Botta, A. Tacconelli, I. Bagni, E. Giardina, E. Bonifazi, A. Pietropolli, M. Clementi, and G. Novelli Transmission ratio distortion in the spinal muscular atrophy locus: Data from 314 prenatal tests Neurology, November 22, 2005; 65(10): 1631 - 1635. [Abstract] [Full Text] [PDF]
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J. Jarecki, X. Chen, A. Bernardino, D. D. Coovert, M. Whitney, A. Burghes, J. Stack, and B. A. Pollok Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy Hum. Mol. Genet., July 15, 2005; 14(14): 2003 - 2018. [Abstract] [Full Text] [PDF]
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L. E. Kernochan, M. L. Russo, N. S. Woodling, T. N. Huynh, A. M. Avila, K. H. Fischbeck, and C. J. Sumner The role of histone acetylation in SMN gene expression Hum. Mol. Genet., May 1, 2005; 14(9): 1171 - 1182. [Abstract] [Full Text] [PDF]
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E. C. Wolstencroft, V. Mattis, A. A. Bajer, P. J. Young, and C. L. Lorson A non-sequence-specific requirement for SMN protein activity: the role of aminoglycosides in inducing elevated SMN protein levels Hum. Mol. Genet., May 1, 2005; 14(9): 1199 - 1210. [Abstract] [Full Text] [PDF]
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L. Gangwani, R. A. Flavell, and R. J. Davis ZPR1 Is Essential for Survival and Is Required for Localization of the Survival Motor Neurons (SMN) Protein to Cajal Bodies Mol. Cell. Biol., April 1, 2005; 25(7): 2744 - 2756. [Abstract] [Full Text] [PDF]
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N. N. SINGH, E. J. ANDROPHY, and R. N. SINGH In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes RNA, August 1, 2004; 10(8): 1291 - 1305. [Abstract] [Full Text] [PDF]
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S. Majumder, S. Varadharaj, K. Ghoshal, U. Monani, A. H. M. Burghes, and S. T. Jacob Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene J. Biol. Chem., April 9, 2004; 279(15): 14803 - 14811. [Abstract] [Full Text] [PDF]
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J. Zou, F. Barahmand-pour, M. L. Blackburn, Y. Matsui, H. A. Chansky, and L. Yang Survival Motor Neuron (SMN) Protein Interacts with Transcription Corepressor mSin3A J. Biol. Chem., April 9, 2004; 279(15): 14922 - 14928. [Abstract] [Full Text] [PDF]
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A. Mankodi and T. Ashizawa Echo of silence: Silent mutations, RNA splicing, and neuromuscular diseases Neurology, November 25, 2003; 61(10): 1330 - 1331. [Full Text] [PDF]
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W. Rossoll, S. Jablonka, C. Andreassi, A.-K. Kroning, K. Karle, U. R. Monani, and M. Sendtner Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of {beta}-actin mRNA in growth cones of motoneurons J. Cell Biol., November 24, 2003; 163(4): 801 - 812. [Abstract] [Full Text] [PDF]
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M. L. McWhorter, U. R. Monani, A. H.M. Burghes, and C. E. Beattie Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding J. Cell Biol., September 1, 2003; 162(5): 919 - 932. [Abstract] [Full Text] [PDF]
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Y. B. Chan, I. Miguel-Aliaga, C. Franks, N. Thomas, B. Trulzsch, D. B. Sattelle, K. E. Davies, and M. van den Heuvel Neuromuscular defects in a Drosophila survival motor neuron gene mutant Hum. Mol. Genet., June 15, 2003; 12(12): 1367 - 1376. [Abstract] [Full Text] [PDF]
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L. A. Skordis, M. G. Dunckley, B. Yue, I. C. Eperon, and F. Muntoni Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts PNAS, April 1, 2003; 100(7): 4114 - 4119. [Abstract] [Full Text] [PDF]
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U. R. Monani, M. T. Pastore, T. O. Gavrilina, S. Jablonka, T. T. Le, C. Andreassi, J. M. DiCocco, C. Lorson, E. J. Androphy, M. Sendtner, et al. A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy J. Cell Biol., January 2, 2003; 160(1): 41 - 52. [Abstract] [Full Text] [PDF]
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 S. Ogino and R. B. Wilson Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis J. Mol. Diagn., November 1, 2002; 4(4): 185 - 190. [Abstract] [Full Text] [PDF]
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S. Vyas, C. Bechade, B. Riveau, J. Downward, and A. Triller Involvement of survival motor neuron (SMN) protein in cell death Hum. Mol. Genet., October 15, 2002; 11(22): 2751 - 2764. [Abstract] [Full Text] [PDF]
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 S. T. Iannaccone and and the American Spinal Muscular Atrophy Randomize Outcome Measures for Pediatric Spinal Muscular Atrophy Arch Neurol, September 1, 2002; 59(9): 1445 - 1450. [Abstract] [Full Text] [PDF]
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Y. Hofmann and B. Wirth hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-{beta}1 Hum. Mol. Genet., August 15, 2002; 11(17): 2037 - 2049. [Abstract] [Full Text] [PDF]
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L. Fan and L. R. Simard Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development Hum. Mol. Genet., July 1, 2002; 11(14): 1605 - 1614. [Abstract] [Full Text] [PDF]
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S. Lefebvre, P. Burlet, L. Viollet, S. Bertrandy, C. Huber, C. Belser, and A. Munnich A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy Hum. Mol. Genet., May 1, 2002; 11(9): 1017 - 1027. [Abstract] [Full Text] [PDF]
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P. J. Young, C. J. DiDonato, D. Hu, R. Kothary, E. J. Androphy, and C. L. Lorson SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2{beta}1 Hum. Mol. Genet., March 1, 2002; 11(5): 577 - 587. [Abstract] [Full Text] [PDF]
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S. Ogino, D. G. B. Leonard, H. Rennert, and R. B. Wilson Spinal Muscular Atrophy Genetic Testing Experience at an Academic Medical Center J. Mol. Diagn., February 1, 2002; 4(1): 53 - 58. [Abstract] [Full Text] [PDF]
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C. J. DiDonato, C. L. Lorson, Y. De Repentigny, L. Simard, C. Chartrand, E. J. Androphy, and R. Kothary Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing Hum. Mol. Genet., November 1, 2001; 10(23): 2727 - 2736. [Abstract] [Full Text] [PDF]
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C. Andreassi, J. Jarecki, J. Zhou, D. D. Coovert, U. R. Monani, X. Chen, M. Whitney, B. Pollok, M. Zhang, E. Androphy, et al. Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients Hum. Mol. Genet., November 1, 2001; 10(24): 2841 - 2849. [Abstract] [Full Text] [PDF]
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S. Ferrari, S. Giliani, A. Insalaco, A. Al-Ghonaium, A. R. Soresina, M. Loubser, M. A. Avanzini, M. Marconi, R. Badolato, A. G. Ugazio, et al. Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM PNAS, October 23, 2001; 98(22): 12614 - 12619. [Abstract] [Full Text] [PDF]
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J.-G. Chang, H.-M. Hsieh-Li, Y.-J. Jong, N. M. Wang, C.-H. Tsai, and H. Li Treatment of spinal muscular atrophy by sodium butyrate PNAS, August 14, 2001; 98(17): 9808 - 9813. [Abstract] [Full Text] [PDF]
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R. W. Orrell and D. A. Figlewicz Clinical implications of the genetics of ALS and other motor neuron diseases Neurology, July 10, 2001; 57(1): 9 - 17. [Abstract] [Full Text] [PDF]
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V. Wong and V. Chan Molecular Genetic Study of a Childhood Form of Spinal Muscular Atrophy J Child Neurol, April 1, 2001; 16(4): 291 - 294. [Abstract] [PDF]
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P. J. Young, N. t. Man, C. L. Lorson, T. T. Le, E. J. Androphy, A. H.M. Burghes, and G. E. Morris The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding Hum. Mol. Genet., November 1, 2000; 9(19): 2869 - 2877. [Abstract] [Full Text] [PDF]
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U. R. Monani, D. D. Coovert, and A. H.M. Burghes Animal models of spinal muscular atrophy Hum. Mol. Genet., October 1, 2000; 9(16): 2451 - 2457. [Abstract] [Full Text] [PDF]
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R. Gotz, C. Karch, M. R. Digby, J. Troppmair, U. R. Rapp, and M. Sendtner The neuronal apoptosis inhibitory protein suppresses neuronal differentiation and apoptosis in PC12 cells Hum. Mol. Genet., October 1, 2000; 9(17): 2479 - 2489. [Abstract] [Full Text] [PDF]
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Y. Hofmann, C. L. Lorson, S. Stamm, E. J. Androphy, and B. Wirth Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2) PNAS, August 6, 2000; (2000) 160181697. [Abstract] [Full Text]
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U. R. Monani, M. Sendtner, D. D. Coovert, D. W. Parsons, C. Andreassi, T. T. Le, S. Jablonka, B. Schrank, W. Rossol, T. W. Prior, et al. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn-/- mice and results in a mouse with spinal muscular atrophy Hum. Mol. Genet., February 12, 2000; 9(3): 333 - 339. [Abstract] [Full Text] [PDF]
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S. Jablonka, B. Schrank, M. Kralewski, W. Rossoll, and M. Sendtner Reduced survival motor neuron (Smn) gene dose in mice leads to motor neuron degeneration: an animal model for spinal muscular atrophy type III Hum. Mol. Genet., February 12, 2000; 9(3): 341 - 346. [Abstract] [Full Text] [PDF]
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C. L. Lorson and E. J. Androphy An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN Hum. Mol. Genet., January 22, 2000; 9(2): 259 - 265. [Abstract] [Full Text] [PDF]
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S. R. Lim and K. J. Hertel Modulation of Survival Motor Neuron Pre-mRNA Splicing by Inhibition of Alternative 3' Splice Site Pairing J. Biol. Chem., November 21, 2001; 276(48): 45476 - 45483. [Abstract] [Full Text] [PDF]
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P. J. Young, P. M. Day, J. Zhou, E. J. Androphy, G. E. Morris, and C. L. Lorson A Direct Interaction between the Survival Motor Neuron Protein and p53 and Its Relationship to Spinal Muscular Atrophy J. Biol. Chem., January 18, 2002; 277(4): 2852 - 2859. [Abstract] [Full Text] [PDF]
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Y. Hofmann, C. L. Lorson, S. Stamm, E. J. Androphy, and B. Wirth Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2) PNAS, August 15, 2000; 97(17): 9618 - 9623. [Abstract] [Full Text] [PDF]
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