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A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy.

Monani UR, Pastore MT, Gavrilina TO, Jablonka S, Le TT, Andreassi C, DiCocco JM, Lorson C, Androphy EJ, Sendtner M, Podell M, Burghes AH - J. Cell Biol. (2003)

Bottom Line: We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions.Animals homozygous for the mutant transgene are less severely affected than heterozygotes.This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ohio State University, Columbus, OH 43210, USA. monani.2@osu.edu

ABSTRACT
5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

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Generation and characterization of mild SMA mice. (A) Schematic representation of the 4.9-kb SMN A2G mutant transgene used to create transgenic mice. (B) Transgene copy number using Southern blot analysis. The blot was simultaneously probed with fragments that recognize the transgene and murine Smn, respectively. The latter, which is present in two copies per genome, serves as an internal control. Lane 1, DNA from a nontransgenic Smn+/+ animal; lane 2, line 2023; lane 3, line 2002. (C) Southern blot analysis after BamHI digestion of tail DNA from mice from line 2023. Presence/absence of the wild-type murine Smn and Smn knockout (KO) allele was determined using a probe in murine Smn intron 1. Lane 1, type I SMA mouse control; lane 2, Smn+/+ normal mouse; lane 3, SMN A2G;SMN2;Smn+/− female mouse; lane 4, SMN2;Smn+/− male lacking the A2G transgene; lanes 5 and 6, type III SMA pups of mice from lanes 3 and 4. Note that after genotyping for murine wild-type Smn and the KO alleles, the blot was stripped and reprobed to check for the presence of the A2G transgene. Genotyping for SMN2 was done by PCR (not depicted). 1-mo-old type III SMA mouse and a normal littermate showing a difference in size (D) and muscle weakness (E), as displayed by a hindlimb clasping reflex (arrow) in the former. Four of four type III SMA animals heterozygous for the A2G transgene displayed this reflex, compared with one out of four type III SMA animals homozygous for the mutant transgene and one of five normal littermates. (F) Weight curves of mild SMA mice (n = 5) heterozygous for the A2G transgene and SMN2 and normal littermates (n = 5). Error bars indicate standard deviation. An asterisk indicates that there was a significant difference (P < 0.05, t test) in weights measured on the given days. As adults (33 d of age), mild SMA mice were significantly smaller (14.16 ± 0.52 g; n = 5) than normal littermates (18.02 ± 1.08 g; n = 5).
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fig1: Generation and characterization of mild SMA mice. (A) Schematic representation of the 4.9-kb SMN A2G mutant transgene used to create transgenic mice. (B) Transgene copy number using Southern blot analysis. The blot was simultaneously probed with fragments that recognize the transgene and murine Smn, respectively. The latter, which is present in two copies per genome, serves as an internal control. Lane 1, DNA from a nontransgenic Smn+/+ animal; lane 2, line 2023; lane 3, line 2002. (C) Southern blot analysis after BamHI digestion of tail DNA from mice from line 2023. Presence/absence of the wild-type murine Smn and Smn knockout (KO) allele was determined using a probe in murine Smn intron 1. Lane 1, type I SMA mouse control; lane 2, Smn+/+ normal mouse; lane 3, SMN A2G;SMN2;Smn+/− female mouse; lane 4, SMN2;Smn+/− male lacking the A2G transgene; lanes 5 and 6, type III SMA pups of mice from lanes 3 and 4. Note that after genotyping for murine wild-type Smn and the KO alleles, the blot was stripped and reprobed to check for the presence of the A2G transgene. Genotyping for SMN2 was done by PCR (not depicted). 1-mo-old type III SMA mouse and a normal littermate showing a difference in size (D) and muscle weakness (E), as displayed by a hindlimb clasping reflex (arrow) in the former. Four of four type III SMA animals heterozygous for the A2G transgene displayed this reflex, compared with one out of four type III SMA animals homozygous for the mutant transgene and one of five normal littermates. (F) Weight curves of mild SMA mice (n = 5) heterozygous for the A2G transgene and SMN2 and normal littermates (n = 5). Error bars indicate standard deviation. An asterisk indicates that there was a significant difference (P < 0.05, t test) in weights measured on the given days. As adults (33 d of age), mild SMA mice were significantly smaller (14.16 ± 0.52 g; n = 5) than normal littermates (18.02 ± 1.08 g; n = 5).

Mentions: We previously identified three unrelated SMA patients (two with type III SMA and one with type II SMA) carrying a single SMN2 allele and an SMN A2G missense mutation (Parsons et al., 1998). A cDNA carrying the A2G mutation under a 3.4-kb SMN promoter fragment (Monani et al., 1999b) (Fig. 1 A) was microinjected into fertilized mouse oocytes. Of the four founders obtained, the two transmitting lines, 2002 and 2023, were first analyzed for transgene copy number and then mated to low copy SMN2;Smn+/− mice (Monani et al., 2000). To assess the number of copies of the SMN A2G allele in each of the lines, Southern blot analysis of tail DNA using probes that bound SMN A2G and murine Smn, respectively, was performed. Relative to the single copy murine Smn gene, line 2002 was found to have 15 copies of the transgene whereas line 2023 carried 11 copies (Fig. 1 B). Double transgenic SMN A2G;SMN2;Smn+/− mice were interbred or crossed to SMN2;Smn+/− animals, resulting in mice carrying one or two copies of SMN2 and the mutant transgene but completely lacking mouse Smn (SMN A2G;SMN2;Smn−/−) (Fig. 1 C). Mice of this genotype from line 2002 were no different from the severe type I SMA mice we have previously described. On Western blot analysis, this line was found not to express the A2G transgene (unpublished data), possibly due to a site-specific integration effect. However, SMN2;Smn−/− mice carrying the A2G transgene from line 2023 display all of the characteristics of type III SMA. Smn−/− animals with a single copy of SMN2 and heterozygous for the A2G transgene are 20–40% smaller (1.14 ± 0.06; n = 5) than their normal littermates (1.62 ± 0.18; n = 11) at birth and continue to remain so during development and into adulthood (Fig. 1, D and F). At 3 wk of age, these mice are less active than normal sibs. In addition, they begin to display signs of muscle weakness, as evidenced by a tendency to clasp the hind limbs when suspended by the tail (Fig. 1 E). SMN2;Smn−/− mice that are homozygous for the A2G transgene have a considerably milder phenotype and are indistinguishable from normal littermates often into adulthood. This is consistent with previous reports indicating a tight correlation between SMN2 copy number/protein levels and disease severity (Lefebvre et al., 1997; Coovert et al., 1997). Both heterozygous and homozygous A2G SMN2;Smn−/− mice breed, although the former are less efficient and live ≤1 yr. Mean survival of heterozygous A2G SMN2;Smn−/− mice is 227 d (n = 14) versus 5.16 d for type I SMA mice (n = 57) and 736 d for Smn+/− mice (n = 12) (see Fig. S1, available online at http://www.jcb.org/cgi/content/full/jcb.200208079/ DC1). Toward the end of their lives, type III SMA mice heterozygous for the A2G transgene exhibit very little activity, short, shallow breathing, lose considerable weight, and fail to groom efficiently. In contrast, the oldest SMN2;Smn−/− mice homozygous for the A2G transgene are currently 15 mo of age, healthy, and the males continue to breed well.


A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy.

Monani UR, Pastore MT, Gavrilina TO, Jablonka S, Le TT, Andreassi C, DiCocco JM, Lorson C, Androphy EJ, Sendtner M, Podell M, Burghes AH - J. Cell Biol. (2003)

Generation and characterization of mild SMA mice. (A) Schematic representation of the 4.9-kb SMN A2G mutant transgene used to create transgenic mice. (B) Transgene copy number using Southern blot analysis. The blot was simultaneously probed with fragments that recognize the transgene and murine Smn, respectively. The latter, which is present in two copies per genome, serves as an internal control. Lane 1, DNA from a nontransgenic Smn+/+ animal; lane 2, line 2023; lane 3, line 2002. (C) Southern blot analysis after BamHI digestion of tail DNA from mice from line 2023. Presence/absence of the wild-type murine Smn and Smn knockout (KO) allele was determined using a probe in murine Smn intron 1. Lane 1, type I SMA mouse control; lane 2, Smn+/+ normal mouse; lane 3, SMN A2G;SMN2;Smn+/− female mouse; lane 4, SMN2;Smn+/− male lacking the A2G transgene; lanes 5 and 6, type III SMA pups of mice from lanes 3 and 4. Note that after genotyping for murine wild-type Smn and the KO alleles, the blot was stripped and reprobed to check for the presence of the A2G transgene. Genotyping for SMN2 was done by PCR (not depicted). 1-mo-old type III SMA mouse and a normal littermate showing a difference in size (D) and muscle weakness (E), as displayed by a hindlimb clasping reflex (arrow) in the former. Four of four type III SMA animals heterozygous for the A2G transgene displayed this reflex, compared with one out of four type III SMA animals homozygous for the mutant transgene and one of five normal littermates. (F) Weight curves of mild SMA mice (n = 5) heterozygous for the A2G transgene and SMN2 and normal littermates (n = 5). Error bars indicate standard deviation. An asterisk indicates that there was a significant difference (P < 0.05, t test) in weights measured on the given days. As adults (33 d of age), mild SMA mice were significantly smaller (14.16 ± 0.52 g; n = 5) than normal littermates (18.02 ± 1.08 g; n = 5).
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fig1: Generation and characterization of mild SMA mice. (A) Schematic representation of the 4.9-kb SMN A2G mutant transgene used to create transgenic mice. (B) Transgene copy number using Southern blot analysis. The blot was simultaneously probed with fragments that recognize the transgene and murine Smn, respectively. The latter, which is present in two copies per genome, serves as an internal control. Lane 1, DNA from a nontransgenic Smn+/+ animal; lane 2, line 2023; lane 3, line 2002. (C) Southern blot analysis after BamHI digestion of tail DNA from mice from line 2023. Presence/absence of the wild-type murine Smn and Smn knockout (KO) allele was determined using a probe in murine Smn intron 1. Lane 1, type I SMA mouse control; lane 2, Smn+/+ normal mouse; lane 3, SMN A2G;SMN2;Smn+/− female mouse; lane 4, SMN2;Smn+/− male lacking the A2G transgene; lanes 5 and 6, type III SMA pups of mice from lanes 3 and 4. Note that after genotyping for murine wild-type Smn and the KO alleles, the blot was stripped and reprobed to check for the presence of the A2G transgene. Genotyping for SMN2 was done by PCR (not depicted). 1-mo-old type III SMA mouse and a normal littermate showing a difference in size (D) and muscle weakness (E), as displayed by a hindlimb clasping reflex (arrow) in the former. Four of four type III SMA animals heterozygous for the A2G transgene displayed this reflex, compared with one out of four type III SMA animals homozygous for the mutant transgene and one of five normal littermates. (F) Weight curves of mild SMA mice (n = 5) heterozygous for the A2G transgene and SMN2 and normal littermates (n = 5). Error bars indicate standard deviation. An asterisk indicates that there was a significant difference (P < 0.05, t test) in weights measured on the given days. As adults (33 d of age), mild SMA mice were significantly smaller (14.16 ± 0.52 g; n = 5) than normal littermates (18.02 ± 1.08 g; n = 5).
Mentions: We previously identified three unrelated SMA patients (two with type III SMA and one with type II SMA) carrying a single SMN2 allele and an SMN A2G missense mutation (Parsons et al., 1998). A cDNA carrying the A2G mutation under a 3.4-kb SMN promoter fragment (Monani et al., 1999b) (Fig. 1 A) was microinjected into fertilized mouse oocytes. Of the four founders obtained, the two transmitting lines, 2002 and 2023, were first analyzed for transgene copy number and then mated to low copy SMN2;Smn+/− mice (Monani et al., 2000). To assess the number of copies of the SMN A2G allele in each of the lines, Southern blot analysis of tail DNA using probes that bound SMN A2G and murine Smn, respectively, was performed. Relative to the single copy murine Smn gene, line 2002 was found to have 15 copies of the transgene whereas line 2023 carried 11 copies (Fig. 1 B). Double transgenic SMN A2G;SMN2;Smn+/− mice were interbred or crossed to SMN2;Smn+/− animals, resulting in mice carrying one or two copies of SMN2 and the mutant transgene but completely lacking mouse Smn (SMN A2G;SMN2;Smn−/−) (Fig. 1 C). Mice of this genotype from line 2002 were no different from the severe type I SMA mice we have previously described. On Western blot analysis, this line was found not to express the A2G transgene (unpublished data), possibly due to a site-specific integration effect. However, SMN2;Smn−/− mice carrying the A2G transgene from line 2023 display all of the characteristics of type III SMA. Smn−/− animals with a single copy of SMN2 and heterozygous for the A2G transgene are 20–40% smaller (1.14 ± 0.06; n = 5) than their normal littermates (1.62 ± 0.18; n = 11) at birth and continue to remain so during development and into adulthood (Fig. 1, D and F). At 3 wk of age, these mice are less active than normal sibs. In addition, they begin to display signs of muscle weakness, as evidenced by a tendency to clasp the hind limbs when suspended by the tail (Fig. 1 E). SMN2;Smn−/− mice that are homozygous for the A2G transgene have a considerably milder phenotype and are indistinguishable from normal littermates often into adulthood. This is consistent with previous reports indicating a tight correlation between SMN2 copy number/protein levels and disease severity (Lefebvre et al., 1997; Coovert et al., 1997). Both heterozygous and homozygous A2G SMN2;Smn−/− mice breed, although the former are less efficient and live ≤1 yr. Mean survival of heterozygous A2G SMN2;Smn−/− mice is 227 d (n = 14) versus 5.16 d for type I SMA mice (n = 57) and 736 d for Smn+/− mice (n = 12) (see Fig. S1, available online at http://www.jcb.org/cgi/content/full/jcb.200208079/ DC1). Toward the end of their lives, type III SMA mice heterozygous for the A2G transgene exhibit very little activity, short, shallow breathing, lose considerable weight, and fail to groom efficiently. In contrast, the oldest SMN2;Smn−/− mice homozygous for the A2G transgene are currently 15 mo of age, healthy, and the males continue to breed well.

Bottom Line: We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions.Animals homozygous for the mutant transgene are less severely affected than heterozygotes.This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ohio State University, Columbus, OH 43210, USA. monani.2@osu.edu

ABSTRACT
5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

Show MeSH
Related in: MedlinePlus