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MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE - PLoS Genet. (2013)

Bottom Line: The CAG stabilization was as dramatic as genetic deficiency of Msh2.B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability.Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

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MSH3 coding polymorphisms and protein expression in different mouse strains.A) Msh3 polymorphisms in Msh3 gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in dbSNP by sequencing the Msh3 gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in Table S2. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies [65]. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 [65]. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.
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pgen-1003280-g004: MSH3 coding polymorphisms and protein expression in different mouse strains.A) Msh3 polymorphisms in Msh3 gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in dbSNP by sequencing the Msh3 gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in Table S2. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies [65]. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 [65]. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.

Mentions: The higher levels of MSH3 in the B6 variant may be due to stabilizing amino acid sequences or alternatively, the lower levels of MSH3 in the CBy variant may be due to destabilizing amino acid sequences. Since the levels of MSH2 were consistent between the congenic and reciprocal congenics we presume that the contribution of MSH2 variants upon MSH3 levels is less than that of MSH3. Towards identifying Msh3 gene polymorphisms that may affect MSH3 protein levels, we sequenced the Msh3 gene from 12 other inbred mouse lines for promoter and exon 2, 3, 7, 8 and 10 variations (A, AKR, C3H, CBA, FVB, DBA/2, 129P2, 129S1, 129S2, 129S6, 129T2, & 129X1). These mouse lines contained variant amino acids similar to either CBy or B6 (Figure 4A, Table S2). We next assessed the MMR protein levels in various strains that harboured the B6 and CBy Msh3 gene coding polymorphisms (Figure 4B). MSH3 expression varied between strains. MSH3 was barely detectable in CBy and was the highest in B6 and C3H/HEJ (Figure 4B). These MSH3 levels are similar to the lower and higher levels observed in our reciprocal congenic mice with the CBy- and B6-Msh3 alleles, respectively. MSH3 is highest in B6 and C3H/HEJ mice, which share alleles in exon 3, exon 7 and exon 10 suggesting that these may contribute positively to MSH3 levels. MSH3 levels were intermediate in DBA/2J, CBA/J and 129/S1 (Figure 4B), and these all share the B6 variants at exon 10, which provides additional support for a stabilizing association of exon 10. This is further supported by the higher MSH3 expression in DBA/2 than CBy since DBA/2 differs from CBy by two polymorphisms in exon 10 (Figure 4A). Our results indicate that polymorphisms within exon 3, exon 7 and exon 10 may modulate the level of MSH3 protein in mouse tissue.


MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE - PLoS Genet. (2013)

MSH3 coding polymorphisms and protein expression in different mouse strains.A) Msh3 polymorphisms in Msh3 gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in dbSNP by sequencing the Msh3 gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in Table S2. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies [65]. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 [65]. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.
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Related In: Results  -  Collection

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pgen-1003280-g004: MSH3 coding polymorphisms and protein expression in different mouse strains.A) Msh3 polymorphisms in Msh3 gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in dbSNP by sequencing the Msh3 gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in Table S2. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies [65]. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 [65]. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.
Mentions: The higher levels of MSH3 in the B6 variant may be due to stabilizing amino acid sequences or alternatively, the lower levels of MSH3 in the CBy variant may be due to destabilizing amino acid sequences. Since the levels of MSH2 were consistent between the congenic and reciprocal congenics we presume that the contribution of MSH2 variants upon MSH3 levels is less than that of MSH3. Towards identifying Msh3 gene polymorphisms that may affect MSH3 protein levels, we sequenced the Msh3 gene from 12 other inbred mouse lines for promoter and exon 2, 3, 7, 8 and 10 variations (A, AKR, C3H, CBA, FVB, DBA/2, 129P2, 129S1, 129S2, 129S6, 129T2, & 129X1). These mouse lines contained variant amino acids similar to either CBy or B6 (Figure 4A, Table S2). We next assessed the MMR protein levels in various strains that harboured the B6 and CBy Msh3 gene coding polymorphisms (Figure 4B). MSH3 expression varied between strains. MSH3 was barely detectable in CBy and was the highest in B6 and C3H/HEJ (Figure 4B). These MSH3 levels are similar to the lower and higher levels observed in our reciprocal congenic mice with the CBy- and B6-Msh3 alleles, respectively. MSH3 is highest in B6 and C3H/HEJ mice, which share alleles in exon 3, exon 7 and exon 10 suggesting that these may contribute positively to MSH3 levels. MSH3 levels were intermediate in DBA/2J, CBA/J and 129/S1 (Figure 4B), and these all share the B6 variants at exon 10, which provides additional support for a stabilizing association of exon 10. This is further supported by the higher MSH3 expression in DBA/2 than CBy since DBA/2 differs from CBy by two polymorphisms in exon 10 (Figure 4A). Our results indicate that polymorphisms within exon 3, exon 7 and exon 10 may modulate the level of MSH3 protein in mouse tissue.

Bottom Line: The CAG stabilization was as dramatic as genetic deficiency of Msh2.B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability.Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

Show MeSH
Related in: MedlinePlus