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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, and Msh3 variations in B6 and CBy mice.A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the Msh2−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the Msh3 locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (Figure 1B). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (e.g.[10], [13], [15], [16]. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by Taq polymerase slippage [62], [63]. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the Msh3 locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) 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.
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pgen-1003280-g001: Representative CAG repeat distributions, and Msh3 variations in B6 and CBy mice.A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the Msh2−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the Msh3 locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (Figure 1B). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (e.g.[10], [13], [15], [16]. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by Taq polymerase slippage [62], [63]. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the Msh3 locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) 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.

Mentions: To assess CAG repeat instability in mice with different genetic backgrounds, we backcrossed B6CBA-Tg(HDexon1)61Gpb(R6/1) transgenic mice [10] to B6 and CBy inbred mice to obtain B6.Cg-Tg(HDexon1)61Gpb (B6.Cg-R6/1) and CBy.Cg-Tg(HDexon1)61Gpb (CBy.Cg-R6/1) congenic lines, respectively. These congenic lines were typed at each generation for the presence of the R6/1 transgene, thus after 10 backcross generations, it was predicted that 99.8% of the genome was homozygous for the inbred line (B6 or CBy), while the remaining 0.2% of the genome remained heterozygous. The B6.Cg-R6/1 and CBy.Cg-R6/1 congenic mice contained (CAG)98 and (CAG)94, respectively - so these mice and their progeny should be well matched for HD transgene effects with the same flanking cis-elements. Genome-wide SNP analysis confirmed that the HTT transgene had integrated into chromosome 3 [57] and showed minimal contamination of adjacent regions in the congenic strains (Figure S1A, Table S1). We analysed CAG instability by SP-PCR in liver, striatum, tail and heart from 20 week-old mice. B6.Cg-R6/1 mice showed a high level of somatic instability biased toward expansions in liver and striatum (Figure 1A), while the repeat was relatively stable in heart and tail, as previously described [10], [13]. Surprisingly, the CAG repeats were very stable in all of these four tissues from age-matched CBy.Cg-R6/1 mice, including in liver and striatum (Figure 1A). The stabilizing effect of the CBy background was as striking as the genetic deficiency of the MMR protein MSH2, as previously described [13]. Thus, the level of somatic CAG expansions can be dramatically different between B6.Cg-R6/1 and CBy.Cg-R6/1 mice, revealing that CAG expansions are affected by genetic background.


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, and Msh3 variations in B6 and CBy mice.A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the Msh2−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the Msh3 locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (Figure 1B). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (e.g.[10], [13], [15], [16]. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by Taq polymerase slippage [62], [63]. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the Msh3 locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) 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.
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Related In: Results  -  Collection

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pgen-1003280-g001: Representative CAG repeat distributions, and Msh3 variations in B6 and CBy mice.A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the Msh2−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the Msh3 locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (Figure 1B). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (e.g.[10], [13], [15], [16]. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by Taq polymerase slippage [62], [63]. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the Msh3 locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) 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.
Mentions: To assess CAG repeat instability in mice with different genetic backgrounds, we backcrossed B6CBA-Tg(HDexon1)61Gpb(R6/1) transgenic mice [10] to B6 and CBy inbred mice to obtain B6.Cg-Tg(HDexon1)61Gpb (B6.Cg-R6/1) and CBy.Cg-Tg(HDexon1)61Gpb (CBy.Cg-R6/1) congenic lines, respectively. These congenic lines were typed at each generation for the presence of the R6/1 transgene, thus after 10 backcross generations, it was predicted that 99.8% of the genome was homozygous for the inbred line (B6 or CBy), while the remaining 0.2% of the genome remained heterozygous. The B6.Cg-R6/1 and CBy.Cg-R6/1 congenic mice contained (CAG)98 and (CAG)94, respectively - so these mice and their progeny should be well matched for HD transgene effects with the same flanking cis-elements. Genome-wide SNP analysis confirmed that the HTT transgene had integrated into chromosome 3 [57] and showed minimal contamination of adjacent regions in the congenic strains (Figure S1A, Table S1). We analysed CAG instability by SP-PCR in liver, striatum, tail and heart from 20 week-old mice. B6.Cg-R6/1 mice showed a high level of somatic instability biased toward expansions in liver and striatum (Figure 1A), while the repeat was relatively stable in heart and tail, as previously described [10], [13]. Surprisingly, the CAG repeats were very stable in all of these four tissues from age-matched CBy.Cg-R6/1 mice, including in liver and striatum (Figure 1A). The stabilizing effect of the CBy background was as striking as the genetic deficiency of the MMR protein MSH2, as previously described [13]. Thus, the level of somatic CAG expansions can be dramatically different between B6.Cg-R6/1 and CBy.Cg-R6/1 mice, revealing that CAG expansions are affected by genetic background.

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