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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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Mlh3 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ mice on different Mlh3 genetic backgrounds. Mlh3+/+, CAG103; Mlh3+/−, CAG101; Mlh3−/−, CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3. Mlh3+/+, CAG103.3±1.5SD, n = 3; Mlh3+/−, CAG101.3±0.5SD, n = 4; Mlh3−/−, CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.
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pgen-1003930-g006: Mlh3 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ mice on different Mlh3 genetic backgrounds. Mlh3+/+, CAG103; Mlh3+/−, CAG101; Mlh3−/−, CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3. Mlh3+/+, CAG103.3±1.5SD, n = 3; Mlh3+/−, CAG101.3±0.5SD, n = 4; Mlh3−/−, CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.

Mentions: Given the critical role of MLH1 in somatic HTT CAG expansion we were interested in investigating further this MLH1-mediated pathway. It is known that MLH1 is an obligate subunit of three MutL complexes: MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1) and MutLγ (MLH1–MLH3) (reviewed in [37], [38]). These MutL heterodimers are essential downstream factors in MMR and are recruited to the MMR reaction following the binding of mismatched DNA by MutSα (MSH2–MSH6) or MutSβ (MSH2–MSH3). Outside of its role in meiotic recombination [39], MutLγ appears to function predominantly with MutSβ both in somatic and germ cells [40], [41]. Given the specific requirement for MutSβ in somatic CAG expansion in HdhQ111 mice [19] and other mouse models of CAG/CTG disease [22], [25], [26], we hypothesized that MLH3 may also play a major role in somatic expansion. A role for MLH3 had also been suggested from findings in a mouse model of myotonic dystrophy type 1 in which knockout of Pms2, encoding MLH1's major binding partner, reduced the rate of somatic CTG expansion by ∼50%, but did not eliminate somatic expansions [24]. We therefore crossed B6.HdhQ111 with Mlh3 mice (B6) [39] and quantified HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ animals on Mlh3+/+, Mlh3+/− and Mlh3−/− genetic backgrounds (Figure 6). Slightly reduced striatum- and liver-specific CAG instability was observed in Mlh3+/− mice when compared to Mlh3+/+ animals (2-tailed unpaired t-tests: striatum, p = 0.06; liver, p = 0.03). Interestingly, no instability was present in Mlh3−/− striatum or liver (2 tailed unpaired t-tests: p<0.0001 for both tissues compared to Mlh3+/+), demonstrating, as for MLH1, that MLH3 is absolutely required for somatic HTT CAG instability in B6.HdhQ111 mice, and implying that MutLγ dimers act in this process. The slight reduction of instability in Mlh3+/− mice (Figure 6), not apparent in Mlh1+/− mice (Figure 4) suggests that Mlh3 may be a limiting factor in somatic expansion, as previously reported for Msh3[19], [26]. The relatively strong impacts of heterozygous loss of Mlh3 and Msh3 compared to heterozygous loss of the Mlh1 and Msh2 genes encoding their respective binding partners may be explained in part by the lower levels of MSH3 compared to MSH2 and of MLH3 compared to MLH1 [42], [43].


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)

Mlh3 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ mice on different Mlh3 genetic backgrounds. Mlh3+/+, CAG103; Mlh3+/−, CAG101; Mlh3−/−, CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3. Mlh3+/+, CAG103.3±1.5SD, n = 3; Mlh3+/−, CAG101.3±0.5SD, n = 4; Mlh3−/−, CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.
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Related In: Results  -  Collection

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

pgen-1003930-g006: Mlh3 is required for striatal and liver HTT CAG repeat instability in B6.HdhQ111/+ mice.(A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ mice on different Mlh3 genetic backgrounds. Mlh3+/+, CAG103; Mlh3+/−, CAG101; Mlh3−/−, CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3. Mlh3+/+, CAG103.3±1.5SD, n = 3; Mlh3+/−, CAG101.3±0.5SD, n = 4; Mlh3−/−, CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p<0.05; ***, p<0.001; ****, p<0.0001.
Mentions: Given the critical role of MLH1 in somatic HTT CAG expansion we were interested in investigating further this MLH1-mediated pathway. It is known that MLH1 is an obligate subunit of three MutL complexes: MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1) and MutLγ (MLH1–MLH3) (reviewed in [37], [38]). These MutL heterodimers are essential downstream factors in MMR and are recruited to the MMR reaction following the binding of mismatched DNA by MutSα (MSH2–MSH6) or MutSβ (MSH2–MSH3). Outside of its role in meiotic recombination [39], MutLγ appears to function predominantly with MutSβ both in somatic and germ cells [40], [41]. Given the specific requirement for MutSβ in somatic CAG expansion in HdhQ111 mice [19] and other mouse models of CAG/CTG disease [22], [25], [26], we hypothesized that MLH3 may also play a major role in somatic expansion. A role for MLH3 had also been suggested from findings in a mouse model of myotonic dystrophy type 1 in which knockout of Pms2, encoding MLH1's major binding partner, reduced the rate of somatic CTG expansion by ∼50%, but did not eliminate somatic expansions [24]. We therefore crossed B6.HdhQ111 with Mlh3 mice (B6) [39] and quantified HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6.HdhQ111/+ animals on Mlh3+/+, Mlh3+/− and Mlh3−/− genetic backgrounds (Figure 6). Slightly reduced striatum- and liver-specific CAG instability was observed in Mlh3+/− mice when compared to Mlh3+/+ animals (2-tailed unpaired t-tests: striatum, p = 0.06; liver, p = 0.03). Interestingly, no instability was present in Mlh3−/− striatum or liver (2 tailed unpaired t-tests: p<0.0001 for both tissues compared to Mlh3+/+), demonstrating, as for MLH1, that MLH3 is absolutely required for somatic HTT CAG instability in B6.HdhQ111 mice, and implying that MutLγ dimers act in this process. The slight reduction of instability in Mlh3+/− mice (Figure 6), not apparent in Mlh1+/− mice (Figure 4) suggests that Mlh3 may be a limiting factor in somatic expansion, as previously reported for Msh3[19], [26]. The relatively strong impacts of heterozygous loss of Mlh3 and Msh3 compared to heterozygous loss of the Mlh1 and Msh2 genes encoding their respective binding partners may be explained in part by the lower levels of MSH3 compared to MSH2 and of MLH3 compared to MLH1 [42], [43].

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