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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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Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (p = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.
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pgen-1003930-g008: Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (p = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.

Mentions: The highly polymorphic nature of the Mlh1 gene indicated that delineation of the functional polymorphism(s) that drives the difference in instability between B6 and 129 mice may well be complex. However, based on the above prediction that at least the F192I substitution may have a functional impact we tested the simplest hypothesis that the B6 and 129 versions of MLH1 have different levels of activity. As there is currently no good assay for MutLγ function, we performed cell-free assays using MutLα (MLH1-PMS2 complexes), known to be required to repair G-T mismatches and single repeat slip-outs of CAG/CTG tracts [49], [50], in order to provide the most sensitive test of B6 and 129 MLH1 function. We thus cloned and co-purified B6-like (mMLH1.B6-hPMS2) and 129-like (mMLH1.129-hPMS2) MutLα proteins (Figure S11) and assessed the ability of these proteins (containing all 4 amino acid differences; Figure 6A) to repair various DNA substrates using cell-free assays. The results revealed that B6 and 129 MLH1 proteins displayed no overt difference in their abilities to repair a G-T mismatch (Figure S12). In addition, the human MLH1 protein carrying the F192I mutation showed MMR activity comparable to that of wild-type human MLH1 (Figure S12). We then tested the ability of B6 and 129 MLH1 proteins to repair a single CTG slip-out (CAG)47•(CTG)48[50], [51], a potential intermediate in the expansion process, as requirements for processing of slipped-DNAs formed by trinucleotide repeats may more closely resemble those that ultimately result in CAG expansion in mice. As shown previously [50], complementation of MLH1- and PMS2-deficient HEK293T cells with wild-type human MutLα restored repair activity (Figure 8A). Complementation with mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 MutLα complexes also restored repair to similar efficiencies (Figure 8A). Titration of concentration of the B6-like and 129-like MutLα complexes confirmed similar repair efficiencies between the MLH1 protein from the two mouse strains at each concentration (2-tailed unpaired t-tests: 5 ng, p = 0.477; 25 ng, p = 0.885; 100 ng, p = 0.736), but also demonstrated a statistically significant MutLα dose dependency of CTG slip-out repair (linear regression: R2 = 0.557, p = 0.0004; Figure 8B). Together, these results demonstrate that B6 and 129 MLH1 proteins, in the context of the mixed-species MutLα complex, do not differ substantially in their G-T mismatch or CTG slip-out repair activities and that the F192I mutation in the human protein does not have a significant functional impact. This suggests that if Mlh1 gene variations are in fact the source of the CAG repeat instability differences between the B6 and 129 mouse strains in vivo, this is unlikely to be due to major differences in MLH1 protein activity within the context of the MutLα complex. However, the dose-dependence of the MutLα complex in the CTG slip-out repair assay indicated that differential MLH1 protein levels between the two strains may be relevant to their different levels of instability in vivo.


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)

Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (p = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814320&req=5

pgen-1003930-g008: Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (p = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.
Mentions: The highly polymorphic nature of the Mlh1 gene indicated that delineation of the functional polymorphism(s) that drives the difference in instability between B6 and 129 mice may well be complex. However, based on the above prediction that at least the F192I substitution may have a functional impact we tested the simplest hypothesis that the B6 and 129 versions of MLH1 have different levels of activity. As there is currently no good assay for MutLγ function, we performed cell-free assays using MutLα (MLH1-PMS2 complexes), known to be required to repair G-T mismatches and single repeat slip-outs of CAG/CTG tracts [49], [50], in order to provide the most sensitive test of B6 and 129 MLH1 function. We thus cloned and co-purified B6-like (mMLH1.B6-hPMS2) and 129-like (mMLH1.129-hPMS2) MutLα proteins (Figure S11) and assessed the ability of these proteins (containing all 4 amino acid differences; Figure 6A) to repair various DNA substrates using cell-free assays. The results revealed that B6 and 129 MLH1 proteins displayed no overt difference in their abilities to repair a G-T mismatch (Figure S12). In addition, the human MLH1 protein carrying the F192I mutation showed MMR activity comparable to that of wild-type human MLH1 (Figure S12). We then tested the ability of B6 and 129 MLH1 proteins to repair a single CTG slip-out (CAG)47•(CTG)48[50], [51], a potential intermediate in the expansion process, as requirements for processing of slipped-DNAs formed by trinucleotide repeats may more closely resemble those that ultimately result in CAG expansion in mice. As shown previously [50], complementation of MLH1- and PMS2-deficient HEK293T cells with wild-type human MutLα restored repair activity (Figure 8A). Complementation with mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 MutLα complexes also restored repair to similar efficiencies (Figure 8A). Titration of concentration of the B6-like and 129-like MutLα complexes confirmed similar repair efficiencies between the MLH1 protein from the two mouse strains at each concentration (2-tailed unpaired t-tests: 5 ng, p = 0.477; 25 ng, p = 0.885; 100 ng, p = 0.736), but also demonstrated a statistically significant MutLα dose dependency of CTG slip-out repair (linear regression: R2 = 0.557, p = 0.0004; Figure 8B). Together, these results demonstrate that B6 and 129 MLH1 proteins, in the context of the mixed-species MutLα complex, do not differ substantially in their G-T mismatch or CTG slip-out repair activities and that the F192I mutation in the human protein does not have a significant functional impact. This suggests that if Mlh1 gene variations are in fact the source of the CAG repeat instability differences between the B6 and 129 mouse strains in vivo, this is unlikely to be due to major differences in MLH1 protein activity within the context of the MutLα complex. However, the dose-dependence of the MutLα complex in the CTG slip-out repair assay indicated that differential MLH1 protein levels between the two strains may be relevant to their different levels of instability in vivo.

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