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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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Western blot analysis of MMR and DHFR protein levels.MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods. B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
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pgen-1003280-g003: Western blot analysis of MMR and DHFR protein levels.MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods. B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.

Mentions: To test the possibility that Msh3 polymorphisms may affect the expression of MMR proteins, which subsequently lead to variable levels of CAG instability between mouse strains, we assessed MMR protein levels in mouse tissues by Western blotting [38], [42], [45]. In liver, the levels of MSH2 and MSH6 were similar between all mouse strains (Figure 3A). However, the level of MSH3 protein varied widely between mice, with high expression in mice carrying the B6 Msh3 gene, and undetectable levels in mice carrying the CBy Msh3 gene (Figure 3A). An intermediate level of MSH3 was reproducibly observed in mice heterozygous for the B6 and CBy Msh3 genes, on both B6 and CBy genetic backgrounds, thus indicating a gene dosage effect between Msh3 variant alleles. This pattern did not vary with age (Figure 3A; compare 4 weeks with 16 weeks). The same MSH3 expression patterns were observed using a MSH3-specific antibody alone (Figure 3B, right panel). The striatum displayed the same strain-specific MSH3 expression pattern, where mice homozygous for the B6 Msh3 gene showed the highest levels of MSH3 protein, while mice homozygous for the CBy Msh3 gene expressed the lowest level, and mice heterozygous for the Msh3 allele displayed intermediate MSH3 protein expression (Figure 3B, right panel). It is notable that MSH3 levels varied in a manner that depended on the Msh3 variant and was independent of mouse strain background. The spleen, thymus, cortex and cerebellum also showed a similar Msh3 gene variant-specific pattern of MSH3 protein expression (Figure S3). Towards ensuring that the apparent expression variations were not due to differential ability of the antibody to recognize its epitope, we analyzed MSH3 protein expression using an independent monoclonal MSH3 antibody (5A5, which recognizes an epitope within exon 4 compared to 2F11 which recognizes an epitope in exon 1, neither of which have amino acid differences between B6 and CBy mice), as described by [65]. We observed the same expression patterns, suggesting that the MSH3 levels observed in tissues are independent of the binding site of the antibody on MSH3 (Figure S4). Thus, regardless of genetic background, the level of MSH3 protein expression depended upon whether the mouse carried the B6 Msh3 variant (high) or the CBy Msh3 variant (low).


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)

Western blot analysis of MMR and DHFR protein levels.MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods. B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
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pgen-1003280-g003: Western blot analysis of MMR and DHFR protein levels.MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods. B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
Mentions: To test the possibility that Msh3 polymorphisms may affect the expression of MMR proteins, which subsequently lead to variable levels of CAG instability between mouse strains, we assessed MMR protein levels in mouse tissues by Western blotting [38], [42], [45]. In liver, the levels of MSH2 and MSH6 were similar between all mouse strains (Figure 3A). However, the level of MSH3 protein varied widely between mice, with high expression in mice carrying the B6 Msh3 gene, and undetectable levels in mice carrying the CBy Msh3 gene (Figure 3A). An intermediate level of MSH3 was reproducibly observed in mice heterozygous for the B6 and CBy Msh3 genes, on both B6 and CBy genetic backgrounds, thus indicating a gene dosage effect between Msh3 variant alleles. This pattern did not vary with age (Figure 3A; compare 4 weeks with 16 weeks). The same MSH3 expression patterns were observed using a MSH3-specific antibody alone (Figure 3B, right panel). The striatum displayed the same strain-specific MSH3 expression pattern, where mice homozygous for the B6 Msh3 gene showed the highest levels of MSH3 protein, while mice homozygous for the CBy Msh3 gene expressed the lowest level, and mice heterozygous for the Msh3 allele displayed intermediate MSH3 protein expression (Figure 3B, right panel). It is notable that MSH3 levels varied in a manner that depended on the Msh3 variant and was independent of mouse strain background. The spleen, thymus, cortex and cerebellum also showed a similar Msh3 gene variant-specific pattern of MSH3 protein expression (Figure S3). Towards ensuring that the apparent expression variations were not due to differential ability of the antibody to recognize its epitope, we analyzed MSH3 protein expression using an independent monoclonal MSH3 antibody (5A5, which recognizes an epitope within exon 4 compared to 2F11 which recognizes an epitope in exon 1, neither of which have amino acid differences between B6 and CBy mice), as described by [65]. We observed the same expression patterns, suggesting that the MSH3 levels observed in tissues are independent of the binding site of the antibody on MSH3 (Figure S4). Thus, regardless of genetic background, the level of MSH3 protein expression depended upon whether the mouse carried the B6 Msh3 variant (high) or the CBy Msh3 variant (low).

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