Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

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 HdhQ111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.HdhQ111) than on a 129 background (129.HdhQ111). Linkage mapping in (B6x129).HdhQ111 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.HdhQ111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. HdhQ111 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.

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

“…More recently, it was shown that short CAG/CTG slip-out structures were efficiently repaired by human MutL and MutS complexes [27,28], and to a lesser extent by MutS [27]. In mice in which Msh2, Msh3, Pms2, Mlh1 or Mlh3 were reduced or abolished, CAG/CTG repeat expansions were decreased [29][30][31][32][33][34][35][36][37]. This is also true when Msh2 was partially or totally depleted in a mouse model for fragile X premutation [38].…”

Genetic Modifiers of CAG.CTG Repeat Instability in Huntington's Disease Mouse Models

“…A second MutL homolog has been shown to act as an enhancer of CAG repeat expansions. Indeed, expansion-biased somatic mosaicism is reduced in Mlh3 heterozygous knockout mice and totally abolished in Mlh3 homozygous knockout mice suggesting that MLH3 is a limiting factor on the process of expansion as reported for MSH3 protein [69].…”

“…It has been reported that Msh2 alleles delay the accumulation of mutant protein and destruction of mutant huntingtin in striatum and in specific neuron type from knock-in HdhQ111 mice [58,67]. Moreover, MLH1 also contributes to nuclear huntingtin and HD inclusion phenotypes [69]. Both data suggest that MSH2 and MLH1 may enhance the HD pathogenic process by modulating the somatic mosaicism in cooperation with MSH3 and MLH3 via the mismatch repair pathway.…”

“…To identify other genetic modifiers of CAG repeat instability, linkage analyses have been performed in different HdhQ111 strains that showed CAG repeat instability variation [69]. A single quantitative trait locus on chromosome 9 and particularly in MutL homolog Mlh1 gene has been identified and associated with CAG repeat instability.…”

Trinucleotide Repeat Instability is Modulated by DNA Base Lesions and DNA Base Excision Repair

“…The loss of other MMR proteins such as PMS2 and MLHrelated endonucleases have also been found to suppress TNR expansions (195). MLH1, which is involved in all 3 mammalian MutL complexes, and MLH3 were found to be involved in somatic TNR expansions in HD mouse models (195,196). The protein PCNA acts as a BER cofactor to stimulate FEN1 cleavage and further interacts with all core BER proteins.…”