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Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

Pinto RM, Dragileva E, Kirby A, Lloret A, Lopez E, St Claire J, Panigrahi GB, Hou C, Holloway K, Gillis T, Guide JR, Cohen PE, Li GM, Pearson CE, Daly MJ, Wheeler VC - PLoS Genet. (2013)

Bottom Line: Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3).The Mlh1 locus is highly polymorphic between B6 and 129 strains.Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

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Reduced MLH1 expression in 129 versus B6 mice.Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.Mlh1+/+, 129.Mlh1+/+ and B6.Mlh1+/− 10-week-old mice (n = 3). (A) Striatal Mlh1 mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.Mlh1+/+ mice were significantly reduced by approximately 50% when compared to B6.Mlh1+/+ (p<0.05), and were comparable to levels in B6.Mlh1+/− mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.Mlh1+/+ striata compared to B6.Mlh1+/+ striata. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01.
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pgen-1003930-g009: Reduced MLH1 expression in 129 versus B6 mice.Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.Mlh1+/+, 129.Mlh1+/+ and B6.Mlh1+/− 10-week-old mice (n = 3). (A) Striatal Mlh1 mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.Mlh1+/+ mice were significantly reduced by approximately 50% when compared to B6.Mlh1+/+ (p<0.05), and were comparable to levels in B6.Mlh1+/− mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.Mlh1+/+ striata compared to B6.Mlh1+/+ striata. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01.

Mentions: The cell-free CTG slip-out repair assays suggested that levels of MLH1 may impact the ability of MutL complexes to execute a repair process that results in CAG expansion in vivo. We therefore assessed whether Mlh1 expression levels differed between the B6 and 129 strains that exhibit comparatively high and low HTT CAG instability, respectively. Striatal Mlh1 mRNA amount was significantly reduced in 129 mice to 54% of that in B6 mice (2-tailed unpaired t-test: p = 0.017), reaching approximately the same mRNA level as that in B6.Mlh1+/− mice (Figure 9A). Striatal Mlh1 mRNA levels were consistently reduced in 129 mice across 3 distinct regions of the primary Mlh1 transcript (exons 4–5, 11–12, and 18–19), and in various other tissues (cerebellum, liver, jejunum and ileum) to between 25% and 50% of B6 levels (Figure S13). Analysis of MLH1 protein by western blot showed similarly reduced protein levels in 129 compared to B6 striata (Figure 9B, C). In contrast to the mRNA, however, the MLH1 protein level in B6.Mlh1+/− mice was intermediate between that in B6 (Mlh1+/+) and 129 striata (Figure 9B, C). We were unable to detect any evidence for novel isoforms or truncation products in the 129 mice (Figure S14).


Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

Pinto RM, Dragileva E, Kirby A, Lloret A, Lopez E, St Claire J, Panigrahi GB, Hou C, Holloway K, Gillis T, Guide JR, Cohen PE, Li GM, Pearson CE, Daly MJ, Wheeler VC - PLoS Genet. (2013)

Reduced MLH1 expression in 129 versus B6 mice.Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.Mlh1+/+, 129.Mlh1+/+ and B6.Mlh1+/− 10-week-old mice (n = 3). (A) Striatal Mlh1 mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.Mlh1+/+ mice were significantly reduced by approximately 50% when compared to B6.Mlh1+/+ (p<0.05), and were comparable to levels in B6.Mlh1+/− mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.Mlh1+/+ striata compared to B6.Mlh1+/+ striata. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01.
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pgen-1003930-g009: Reduced MLH1 expression in 129 versus B6 mice.Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.Mlh1+/+, 129.Mlh1+/+ and B6.Mlh1+/− 10-week-old mice (n = 3). (A) Striatal Mlh1 mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.Mlh1+/+ mice were significantly reduced by approximately 50% when compared to B6.Mlh1+/+ (p<0.05), and were comparable to levels in B6.Mlh1+/− mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.Mlh1+/+ striata compared to B6.Mlh1+/+ striata. Bar graphs represent mean ±SD. *, p<0.05; **, p<0.01.
Mentions: The cell-free CTG slip-out repair assays suggested that levels of MLH1 may impact the ability of MutL complexes to execute a repair process that results in CAG expansion in vivo. We therefore assessed whether Mlh1 expression levels differed between the B6 and 129 strains that exhibit comparatively high and low HTT CAG instability, respectively. Striatal Mlh1 mRNA amount was significantly reduced in 129 mice to 54% of that in B6 mice (2-tailed unpaired t-test: p = 0.017), reaching approximately the same mRNA level as that in B6.Mlh1+/− mice (Figure 9A). Striatal Mlh1 mRNA levels were consistently reduced in 129 mice across 3 distinct regions of the primary Mlh1 transcript (exons 4–5, 11–12, and 18–19), and in various other tissues (cerebellum, liver, jejunum and ileum) to between 25% and 50% of B6 levels (Figure S13). Analysis of MLH1 protein by western blot showed similarly reduced protein levels in 129 compared to B6 striata (Figure 9B, C). In contrast to the mRNA, however, the MLH1 protein level in B6.Mlh1+/− mice was intermediate between that in B6 (Mlh1+/+) and 129 striata (Figure 9B, C). We were unable to detect any evidence for novel isoforms or truncation products in the 129 mice (Figure S14).

Bottom Line: Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3).The Mlh1 locus is highly polymorphic between B6 and 129 strains.Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

Show MeSH
Related in: MedlinePlus