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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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Representative CAG repeat distributions from reciprocal Msh3 congenic lines of mice.Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the Msh3 locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.
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pgen-1003280-g002: Representative CAG repeat distributions from reciprocal Msh3 congenic lines of mice.Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the Msh3 locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.

Mentions: In order to test the potential role of MSH3 protein variants on CAG instability, we created Msh3-locus reciprocal congenic mice carrying the B6 Msh3 variant on a CBy genetic background (CBy.B6-msh3B6/B6), and a CBy Msh3 variant in the B6 genetic background (B6.CBy-msh3CBy/CBy). Each line was backcrossed to the recipient strain 10 times as in the creation of the R6/1 congenic lines. Next they were inter-crossed as appropriate with the R6/1 congenic lines to create mice CBy homozygous at the Msh3 locus on a B6 genetic background and hemizygous for the R6/1 transgene (B6.CBy-msh3CBy/CBy, R6/1) and mice B6 homozygous at the Msh3 locus on a CBy genetic background and hemizygous for the R6/1 transgene (CBy.B6-msh3B6/B6, R6/1). With these mice we could better isolate the effect of each Msh3 variant on both mouse backgrounds on CAG stability. Genome-wide SNP genotyping revealed minimal donor haplotype contamination in the reciprocal congenic strains B6.CBy-msh3CBy/CBy and CBy.B6-msh3B6/B6 and their corresponding R6/1 congenic strains B6.Cg-R6/1 and CBy.Cg-R6/1 (Figure S1B, Table S1). Outside of the genomic region flanking chromosome 3 integration site of the R6/1 transgene [57], there appears to be no contamination of donor DNA in the CBy background line, and only minor areas of residual heterozygosity in the B6 background lines on chromosomes 6, 15 and 17. The contaminating regions linked to the Msh3 gene in the reciprocal congenics contain a limited number of genes (Figure S7), none of which have an obvious or documented role in CAG repeat instability. The regions linked to the Msh3 gene in the CBy.B6-Msh3 R6/1 reciprocal congenic mice span 43 Mbp and include 314 genes, of which 233 are protein-coding (Figure S7). In the B6.CBy-Msh3 R6/1 strain, the linked genes cover a region of approximately 22 Mbp, which lies within the 43 Mbp region of the CBy.B6-Msh3 R6/1 strain. A total of 151 genes are found within this region with 104-protein coding transcripts (Figure S7). Therefore, differences in CAG instability between and within the strains were interpreted to be a consequence of the introgressed Msh3 allele variants. At 16–20 weeks of age, a high level of CAG expansion was present in the liver from mice containing the B6 Msh3 gene for both B6 and CBy backgrounds. This instability was evident as a broad bimodal distribution profile whereas the liver DNA from mice with the CBy Msh3 gene showed a low level of instability with a unimodal distribution (Figure 2A). A similar pattern of CAG instability in the striatum further indicated greater levels of CAG instability in mice with the B6 Msh3 gene than those with the CBy Msh3 gene (Figure 2B). The striking differences in the levels of instability between mice harbouring B6 Msh3 compared to CBy Msh3, regardless of background, supports the concept that the B6 Msh3 gene variant drives CAG expansions to a greater degree than does the CBy Msh3 gene variant.


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)

Representative CAG repeat distributions from reciprocal Msh3 congenic lines of mice.Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the Msh3 locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585117&req=5

pgen-1003280-g002: Representative CAG repeat distributions from reciprocal Msh3 congenic lines of mice.Typical GeneScan traces for sizing of the CAG repeat as outlined in Figure 1B. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the Msh3 locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.
Mentions: In order to test the potential role of MSH3 protein variants on CAG instability, we created Msh3-locus reciprocal congenic mice carrying the B6 Msh3 variant on a CBy genetic background (CBy.B6-msh3B6/B6), and a CBy Msh3 variant in the B6 genetic background (B6.CBy-msh3CBy/CBy). Each line was backcrossed to the recipient strain 10 times as in the creation of the R6/1 congenic lines. Next they were inter-crossed as appropriate with the R6/1 congenic lines to create mice CBy homozygous at the Msh3 locus on a B6 genetic background and hemizygous for the R6/1 transgene (B6.CBy-msh3CBy/CBy, R6/1) and mice B6 homozygous at the Msh3 locus on a CBy genetic background and hemizygous for the R6/1 transgene (CBy.B6-msh3B6/B6, R6/1). With these mice we could better isolate the effect of each Msh3 variant on both mouse backgrounds on CAG stability. Genome-wide SNP genotyping revealed minimal donor haplotype contamination in the reciprocal congenic strains B6.CBy-msh3CBy/CBy and CBy.B6-msh3B6/B6 and their corresponding R6/1 congenic strains B6.Cg-R6/1 and CBy.Cg-R6/1 (Figure S1B, Table S1). Outside of the genomic region flanking chromosome 3 integration site of the R6/1 transgene [57], there appears to be no contamination of donor DNA in the CBy background line, and only minor areas of residual heterozygosity in the B6 background lines on chromosomes 6, 15 and 17. The contaminating regions linked to the Msh3 gene in the reciprocal congenics contain a limited number of genes (Figure S7), none of which have an obvious or documented role in CAG repeat instability. The regions linked to the Msh3 gene in the CBy.B6-Msh3 R6/1 reciprocal congenic mice span 43 Mbp and include 314 genes, of which 233 are protein-coding (Figure S7). In the B6.CBy-Msh3 R6/1 strain, the linked genes cover a region of approximately 22 Mbp, which lies within the 43 Mbp region of the CBy.B6-Msh3 R6/1 strain. A total of 151 genes are found within this region with 104-protein coding transcripts (Figure S7). Therefore, differences in CAG instability between and within the strains were interpreted to be a consequence of the introgressed Msh3 allele variants. At 16–20 weeks of age, a high level of CAG expansion was present in the liver from mice containing the B6 Msh3 gene for both B6 and CBy backgrounds. This instability was evident as a broad bimodal distribution profile whereas the liver DNA from mice with the CBy Msh3 gene showed a low level of instability with a unimodal distribution (Figure 2A). A similar pattern of CAG instability in the striatum further indicated greater levels of CAG instability in mice with the B6 Msh3 gene than those with the CBy Msh3 gene (Figure 2B). The striking differences in the levels of instability between mice harbouring B6 Msh3 compared to CBy Msh3, regardless of background, supports the concept that the B6 Msh3 gene variant drives CAG expansions to a greater degree than does the CBy Msh3 gene variant.

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