Genetic Modifiers of CAG.CTG Repeat Instability in Huntington's Disease Mouse Models (original) (raw)
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PLoS Genetics, 2013
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 strainspecific 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 null 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 MutLc (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 MutSb (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.
Neurobiology of Disease, 2009
Modifying the length of the Huntington's disease (HD) CAG repeat, the major determinant of age of disease onset, is an attractive therapeutic approach. To explore this we are investigating mechanisms of intergenerational and somatic HD CAG repeat instability. Here, we have crossed HD CAG knockin mice onto backgrounds deficient in mismatch repair genes, Msh3 and Msh6, to discern the effects on CAG repeat size and disease pathogenesis. We find that different mechanisms predominate in inherited and somatic instability, with Msh6 protecting against intergenerational contractions and Msh3 required both for increasing CAG length and for enhancing an early disease phenotype in striatum. Therefore, attempts to decrease inherited repeat size may entail a full understanding of Msh6 complexes, while attempts to block the age-dependent increases in CAG size in striatal neurons and to slow the disease process will require a full elucidation of Msh3 complexes and their function in CAG repeat instability.
Genetics, 2016
Huntington's disease is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in Huntington's disease families, partly underlying the genetic anticipation seen in this disorder. Huntington's disease CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest Huntington's disease knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identi...
MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice
PLoS Genetics, 2013
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.
Parent-of-origin differences of mutant HTT CAG repeat instability in Huntington’s disease
European Journal of Medical Genetics, 2011
Background: Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). The CAG domain of mutant HTT is unstable upon intergenerational transmission, however, little is known about the underlying mechanisms. Methods: From the HD archives of the Leiden University Medical Centre DNA samples from all parentoffspring pairs involving 36 CAG repeats or more were selected. To minimize procedural variability, CAG repeat lengths in both mutant and normal HTT were reassessed using the same standardized protocol, which resulted in the identification of 337 parent-offspring transmissions. The effects of both parental (mutant and normal CAG repeat size, age and gender) and offspring (gender and season of conception) characteristics on CAG repeat instability were assessed. Results: Paternal transmissions were often associated with CAG repeat expansion, whereas maternal transmissions mainly resulted in CAG repeat contraction (mean change: þ1.76 vs. À0.07, p < 0.001). Only in paternal transmissions larger mutant CAG repeat size was associated with a greater degree of CAG repeat expansion (b ¼ 0.73; p < 0.001). Conversely, only in maternal transmissions larger CAG repeat size of the normal allele was associated with a greater degree of CAG repeat contraction (b ¼ À0.07; p ¼ 0.029). Parental age, offspring gender and season of conception were not related to CAG repeat instability. Conclusion: Our findings suggest a slight maternal contraction bias as opposed to a paternal expansion bias of the mutant HTT CAG repeat during intergenerational transmission, which only in the maternal line is associated with normal HTT CAG repeat size.
Putting the Brakes on Huntington Disease in a Mouse Experimental Model
PLoS genetics, 2015
Huntington disease (HD) is a hereditary neurodegenerative disorder that causes a progressively debilitating impact on movement, cognition, speech, and mood. It most commonly develops during adulthood and worsens over a 10-15-year period. The genetic basis of HD is an expansion of the (CAG) n trinucleotide repeat in the first exon of the HTT gene [1,2]. Although the function of the normal HTT protein is not well established, in-frame repeat expansion results in the accumulation of an abnormally long polyglutamine tract, which is believed to contribute to mutant protein toxicity and neural degeneration [3]. Consequently, CAG repeat length is inversely correlated with age of onset and severity of disease. Disease-size CAG repeats are prone to further lengthening, which leads to two distinct aspects of their instability: expansions during intergenerational transmissions and somatic expansions occurring throughout the lifetime of an individual (Fig 1A). An outstanding question in the field is whether somatic expansions contribute to disease. The problem is that somatic expansions occur in the context of expressing an already toxic protein, making it difficult to address this question. Two commonly discussed ideas are that (i) disease arises with time because of expression of the toxic protein or RNA or that (ii) the onset of disease depends on gradual accumulation of somatic expansions in patients' tissues throughout life. To distinguish between these scenarios, it is critical to assess to what extent, if any, somatic expansions accelerate disease progression. Since 1993 when the cause of HD was first reported, substantial effort has been made to understand the molecular basis of repeat expansions and the mechanisms of disease pathophysiology. While these studies were conducted in various model systems [4,5], mouse models appeared to be particularly well suited for unraveling disease pathophysiology. Despite a few caveats, such as the much longer CAG repeat lengths (i.e., >100 repeats) needed to recapitulate disease symptoms and the relatively small-scale of expansions, mouse models of HD have led to principal contributions in the field. First, they exhibit both intergenerational and somatic repeat expansions. Second, candidate gene analysis uncovered a surprising role of the mismatch repair (MMR) complex MutSβ in promoting rather than protecting against CAG repeat expansions (Fig 1) [6,7]. A breakthrough in distinguishing between the molecular mechanisms of intergenerational and somatic expansions came with the discovery that loss of 8-oxoguanine glycosylase (OGG1) specifically decreased CAG expansions in somatic cells, but it had no effect on intergenerational transmissions [8]. The main function of OGG1 is to remove 8-oxoguanine (8-oxoG), a mutagenic base by-product accumulating in DNA after its exposure to reactive oxygen species (ROS). It was hypothesized, therefore, that aberrant repair of DNA oxidative damage specifically elevates repeat instability in long-lived differentiated cells, such as neurons. OGG1 excises 8-oxoG, generating a nick in the damaged DNA strand, which is further processed to permit
Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities
Nature Reviews Neurology, 2020
Huntington disease (HD) is a neurodegenerative disease caused by CAG repeat expansion in the HTT gene and involves a complex web of pathogenic mechanisms. Mutant HTT disrupts transcription, interferes with immune and mitochondrial function, and is aberrantly modified post-translationally. Evidence suggests that the mHTT RNA is toxic, and at the DNA level, somatic CAG repeat expansion in vulnerable cells influences disease course. Genome-wide association studies have identified DNA repair pathways as modifiers of somatic instability and disease course in HD and other repeat expansion diseases. In animal models of HD, nucleocytoplasmic transport is disrupted and its restoration is neuroprotective. Novel cerebrospinal fluid (CSF) and plasma biomarkers are amongst the earliest detectable changes in individuals with premanifest HD, and have the sensitivity to detect therapeutic benefit. Therapeutically, the first human trial of a HTT-lowering antisense oligonucleotide successfully, and safely, reduced CSF concentration of mHTT in individuals with HD. A larger trial, powered to detect clinical efficacy, is underway, along with trials of other HTT-lowering approaches. In this Review, we discuss new insights into the molecular pathogenesis of HD and future therapeutic strategies, including the modulation of DNA repair and targeting the DNA mutation itself. direction-dependence might be because CAG and CTG repeats have different propensities to form slipped structures, or are processed differently by repair machinery. Excitingly, most of the HD-modifying variants and pathways converge on specific DNA repair mechanisms, particularly mismatch repair, and influence somatic instability 98,110,112,117,122. These observations suggest that downregulation of MSH3, MutLα, MutLγ and LIG1, the inhibition of interactions between them, or the upregulation of FAN1 and PMS1, could reduce somatic CAG expansion and improve the course of HD (Acknowledgements S.J.T.
Journal of Huntington's Disease
The discovery in the early 1990s of the expansion of unstable simple sequence repeats as the causative mutation for a number of inherited human disorders, including Huntington’s disease (HD), opened up a new era of human genetics and provided explanations for some old problems. In particular, an inverse association between the number of repeats inherited and age at onset, and unprecedented levels of germline instability, biased toward further expansion, provided an explanation for the wide symptomatic variability and anticipation observed in HD and many of these disorders. The repeats were also revealed to be somatically unstable in a process that is expansion-biased, age-dependent and tissue-specific, features that are now increasingly recognised as contributory to the age-dependence, progressive nature and tissue specificity of the symptoms of HD, and at least some related disorders. With much of the data deriving from affected individuals, and model systems, somatic expansions ha...
Plos Genetics, 2009
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age-and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 59-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 59-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLb was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLb strand displacement activity during LP-BER promotes the formation of stable 59-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion.