Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5'UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

Preliminary Findings on CTG Expansion Determination in Different Tissues from Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is characterized by a high genetic and clinical variability. Determination of the genetic variability in DM1 might help to determine whether there is an association between CTG (Cytosine-Thymine-Guanine) expansion and the clinical manifestations of this condition. We studied the variability of the CTG expansion (progenitor, mode, and longest allele, respectively, and genetic instability) in three tissues (blood, muscle, and tissue) from eight patients with DM1. We also studied the association of genetic data with the patients' clinical characteristics. Although genetic instability was confirmed in all the tissues that we studied, our results suggest that CTG expansion is larger in muscle and skin cells compared with peripheral blood leukocytes. While keeping in mind that more research is needed in larger cohorts, we have provided preliminary evidence suggesting that the estimated progenitor CTG size in muscle could be potentially used as an indicator of age of disease onset and muscle function impairment.

Modeling Polyglutamine Expansion Diseases with Induced Pluripotent Stem Cells.

Polyglutamine expansion disorders, which include Huntington's disease, have expanded CAG repeats that result in polyglutamine expansions in affected proteins. How this specific feature leads to distinct neuropathies in 11 different diseases is a fascinating area of investigation. Most proteins affected by polyglutamine expansions are ubiquitously expressed, yet their mechanisms of selective neurotoxicity are unknown. Induced pluripotent stem cells have emerged as a valuable tool to model diseases, understand molecular mechanisms, and generate relevant human neural and glia subtypes, cocultures, and organoids. Ideally, this tool will generate specific neuronal populations that faithfully recapitulate specific polyglutamine expansion disorder phenotypes and mimic the selective vulnerability of a given disease. Here, we review how induced pluripotent technology is used to understand the effects of the disease-causing polyglutamine protein on cell function, identify new therapeutic targets, and determine how polyglutamine expansion affects human neurodevelopment and disease. We will discuss ongoing challenges and limitations in our use of induced pluripotent stem cells to model polyglutamine expansion diseases.

Molecular Mechanisms and Therapeutics for SCA17.

Spinocerebellar ataxia type 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP), which functions as a general transcription factor. Like other polyQ expansion-mediated diseases, SCA17 is characterized by late-onset and selective neurodegeneration, despite the disease protein being ubiquitously expressed in the body. To date, the pathogenesis of polyQ diseases is not fully understood, and there are no effective treatments for these devastating disorders. The well-characterized function of TBP and typical neurodegeneration in SCA17 give us opportunities to understand how polyQ expansion causes selective neurodegeneration and to develop effective therapeutics. In this review, we discuss the molecular mechanisms behind SCA17, focusing on transcriptional dysregulation as its major cause. Mounting evidence suggests that reversing transcriptional alterations induced by mutant TBP and reducing the expression of mutant TBP are promising strategies to treat SCA17.

Molecular Mechanisms and Therapeutics for the GAA·TTC Expansion Disease Friedreich Ataxia.

Friedreich ataxia (FRDA), the most common inherited ataxia, is caused by transcriptional silencing of the nuclear FXN gene, encoding the essential mitochondrial protein frataxin. Currently, there is no approved therapy for this fatal disorder. Gene silencing in FRDA is due to hyperexpansion of the triplet repeat sequence GAA·TTC in the first intron of the FXN gene, which results in chromatin histone modifications consistent with heterochromatin formation. Frataxin is involved in mitochondrial iron homeostasis and the assembly and transfer of iron-sulfur clusters to various mitochondrial enzymes and components of the electron transport chain. Frataxin insufficiency leads to progressive spinocerebellar neurodegeneration, causing symptoms of gait and limb ataxia, slurred speech, muscle weakness, sensory loss, and cardiomyopathy in many patients, resulting in death in early adulthood. Numerous approaches are being taken to find a treatment for FRDA, including excision or correction of the repeats by genome engineering methods, gene activation with small molecules or artificial transcription factors, delivery of frataxin to affected cells by protein replacement therapy, gene therapy, or small molecules to increase frataxin protein levels, and therapies aimed at countering the cellular consequences of reduced frataxin. This review will summarize the mechanisms involved in repeat-mediated gene silencing and recent efforts aimed at development of therapeutics.

Molecular genetics of congenital myotonic dystrophy.

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease showing strong genetic anticipation, and is caused by the expansion of a CTG repeat tract in the 3'-UTR of the DMPK gene. Congenital Myotonic Dystrophy (CDM1) represents the most severe form of the disease, with prenatal onset, symptoms distinct from adult onset DM1, and a high rate of perinatal mortality. CDM1 is usually associated with very large CTG expansions, but this correlation is not absolute and cannot explain the distinct clinical features and the strong bias for maternal transmission. This review focuses upon the molecular and epigenetic factors that modulate disease severity and might be responsible for CDM1. Changes in the epigenetic status of the DM1 locus and in gene expression have recently been observed. Increasing evidence supports a role of a CTCF binding motif as a cis-element, upstream of the DMPK CTG tract, whereby CpG methylation of this site regulates the interaction of the insulator protein CTCF as a modulating trans-factor responsible for the inheritance and expression of CDM1.

Emerging differences between Huntington's disease-like 2 and Huntington's disease: A comparison using MRI brain volumetry.

Huntington's Disease-Like 2 (HDL2), caused by a CTG/CAG expansion in JPH3 on chromosome 16q24, is the most common Huntington's Disease (HD) phenocopy in populations with African ancestry. Qualitatively, brain MRIs of HDL2 patients have been indistinguishable from HD. To determine brain regions most affected in HDL2 a cross-sectional study using MRI brain volumetry was undertaken to compare the brains of nine HDL2, 11 HD and nine age matched control participants. Participants were ascertained from the region in South Africa with the world's highest HDL2 incidence. The HDL2 and HD patient groups showed no significant differences with respect to mean age at MRI, disease duration, abnormal triplet repeat length, or age at disease onset. Overall, intracerebral volumes were smaller in both affected groups compared to the control group. Comparing the HDL2 and HD groups across multiple covariates, cortical and subcortical volumes were similar with the exception that the HDL2 thalamic volumes were smaller. Consistent with other similarities between the two diseases, these results indicate a pattern of neurodegeneration in HDL2 that is remarkably similar to HD. However smaller thalamic volumes in HDL2 raises intriguing questions into the pathogenesis of both disorders, and how these volumetric differences relate to their respective phenotypes.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

Repeat-associated non-AUG (RAN) translation and other molecular mechanisms in Fragile X Tremor Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset inherited neurodegenerative disorder characterized by progressive intention tremor, gait ataxia and dementia associated with mild brain atrophy. The cause of FXTAS is a premutation expansion, of 55 to 200 CGG repeats localized within the 5'UTR of FMR1. These repeats are transcribed in the sense and antisense directions into mutants RNAs, which have increased expression in FXTAS. Furthermore, CGG sense and CCG antisense expanded repeats are translated into novel proteins despite their localization in putatively non-coding regions of the transcript. Here we focus on two proposed disease mechanisms for FXTAS: 1) RNA gain-of-function, whereby the mutant RNAs bind specific proteins and preclude their normal functions, and 2) repeat-associated non-AUG (RAN) translation, whereby translation through the CGG or CCG repeats leads to the production of toxic homopolypeptides, which in turn interfere with a variety of cellular functions. Here, we analyze the data generated to date on both of these potential molecular mechanisms and lay out a path forward for determining which factors drive FXTAS pathogenicity.

MutSß abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions.

CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSß (Msh2-Msh3 heterodimer). To evaluate properties of MutSß that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSß, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSß. Msh3-/- cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3-/- cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSß on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSß complex, indicating that MutSß abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3-/- cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSß and rely heavily on Msh3 ATPase function.

Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca2+ channel CaV2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of CaV2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in CaV2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability.

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA)n Repeats Associated with Friedreich's Ataxia.

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA)n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach.

A Ligand That Targets CUG Trinucleotide Repeats.

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9-diaminoalkyl-substituted 1,10-phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (Tm ) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time-of-flight (ESI-TOF) mass spectrometry showed that DAP bound to r(CUG)9 but not to r(CAG)9 and r(CGG)9 . The dual luciferase assay clearly indicated DAP bound to the r(CUG)n repeat by affecting the translation in vitro.

The expanding biology of the C9orf72 nucleotide repeat expansion in neurodegenerative disease.

A nucleotide repeat expansion (NRE) within the chromosome 9 open reading frame 72 (C9orf72) gene was the first of this type of mutation to be linked to multiple neurological conditions, including amyotrophic lateral sclerosis and frontotemporal dementia. The pathogenic mechanisms through which the C9orf72 NRE contributes to these disorders include loss of C9orf72 function and gain-of-function mechanisms of C9orf72 driven by toxic RNA and protein species encoded by the NRE. These mechanisms have been linked to several cellular defects - including nucleocytoplasmic trafficking deficits and nuclear stress - that have been observed in both patients and animal models.

Expansión del triplete citosina-guanina-guanina: 3 casos de síndrome OMIM en una misma familia / Triplet expansion cytosine-guanine-guanine: Three cases of OMIM syndrome in the same family

Introducción: El aumento del número de repeticiones del triplete citosina-guanina-guanina (CGG), en el gen FMR1 es responsable de 3 síndromes OMIM con fenotipo clínico bien diferenciado: síndrome de X frágil (SXF) y 2 enfermedades en adultos portadores de la premutación (55-200 repeticiones CGG): insuficiencia ovárica primaria (FXPOI) y síndrome de temblor-ataxia (FXTAS) asociado al SXF. Observación clínica o métodos: Se estudió la mutación dinámica CGG del gen FMR1 en muestras de ADN de sangre periférica del caso índice y familiares de primer, segundo y tercer grado mediante TP-PCR, así como el porcentaje de metilación. Resultados: Se confirmó el diagnóstico del SXF en 3 pacientes (21,4%), 8 pacientes (57,1%) se encontraban en el rango de premutación, un paciente varón con mosaicismo premutación-mutación completa (7,1%) y 2 pacientes (14,3%) con estudio normal. De los 8 pacientes premutados, 3 presentaron FXPOI y un paciente varón FXTAS. Discusión: Nuestro estudio muestra la importancia de realizar un diagnóstico precoz del SXF y su consecuente estudio familiar y consejo genético, que permita identificar nuevos pacientes afectos o pacientes premutados con síndromes relacionados con el gen FMR1 (FXTAS, FXPOI) (AU)

Introduction: The dynamic increase in the number of triplet repeats of cytosine-guanine-guanine (CGG) in the FMR1gene mutation is responsible for three OMIM syndromes with a distinct clinical phenotype: Fragile X syndrome (FXS) and two pathologies in adult carriers of the premutation (55-200 CGG repeats): Primary ovarian insufficiency (FXPOI) and tremor-ataxia syndrome (FXTAS) associated with FXS. Clinical observation and methods: CGG mutation dynamics of the FMR1 gene were studied in DNA samples from peripheral blood from the index case and other relatives of first, second and third degree by TP-PCR, and the percentage methylation. Results: Diagnosis of FXS was confirmed in three patients (21.4%), eight patients (57.1%) were confirmed in the premutation range transmitters, one male patient with full mutation/permutation mosaicism (7.1%) and two patients (14.3%) with normal study. Of the eight permutated patients, three had FXPOI and one male patient had FXTAS. Discussion: Our study suggests the importance of making an early diagnosis of SXF in order to carry out a family study and genetic counselling, which allow the identification of new cases or premutated patients with FMR1 gene- associated syndromes (FXTAS, FXPOI) (AU)

Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat.

Spinocerebellar ataxia type 2 (SCA2) is a progressive autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 disease phenotype is characterized by cerebellar atrophy, gait ataxia, and slow saccades. ATXN2 mutation causes gains of toxic and normal functions of the ATXN2 gene product, ataxin-2, and abnormally slow Purkinje cell firing frequency. Previously we investigated features of ATXN2 controlling expression and noted expression differences for ATXN2 constructs with varying CAG lengths, suggestive of repeat associated non-AUG translation (RAN translation). To determine whether RAN translation occurs for ATXN2 we assembled various ATXN2 constructs with ATXN2 tagged by luciferase, HA or FLAG tags, driven by the CMV promoter or the ATXN2 promoter. Luciferase expression from ATXN2-luciferase constructs lacking the ATXN2 start codon was weak vs AUG translation, regardless of promoter type, and did not increase with longer CAG repeat lengths. RAN translation was detected on western blots by the anti-polyglutamine antibody 1C2 for constructs driven by the CMV promoter but not the ATXN2 promoter, and was weaker than AUG translation. Strong RAN translation was also observed when driving the ATXN2 sequence with the CMV promoter with ATXN2 sequence downstream of the CAG repeat truncated to 18 bp in the polyglutamine frame but not in the polyserine or polyalanine frames. Our data demonstrate that ATXN2 RAN translation is weak compared to AUG translation and is dependent on ATXN2 sequences flanking the CAG repeat.

Lack of C9ORF72 coding mutations supports a gain of function for repeat expansions in amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in C9ORF72 are a common cause of familial and apparently sporadic amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The mechanism by which expansions cause neurodegeneration is unknown, but current evidence supports both loss-of-function and gain-of-function mechanisms. We used pooled next-generation sequencing of the C9ORF72 gene in 389 ALS patients to look for traditional loss-of-function mutations. Although rare variants were identified, none were likely to be pathogenic, suggesting that mutations other than the repeat expansion are not a common cause of ALS, and providing supportive evidence for a gain-of-function mechanism. We also show by repeat-primed PCR genotyping that the C9ORF72 expansion frequency varies by geographical region within the United States, with an unexpectedly high frequency in the Mid-West. Finally we also show evidence of somatic instability of the expansion size by Southern blot, with the largest expansions occurring in brain tissue.

Predictors of atrio-ventricular conduction disease, long-term outcomes in patients with myotonic dystrophy types I and II.

BACKGROUND: Patients with myotonic dystrophy (DM) have an annual mortality of approximately 3.5%, one-third of which is sudden cardiac death. The predictors of cardiac conduction disease in these patients are incompletely defined. METHODS: A single-center cohort study included 211 patients with DM type 1 (DM1) and 25 DM type 2 (DM2). A severe electrocardiogram (ECG) abnormality was defined as a PR interval of ≥240 ms or QRS duration of ≥120 ms. RESULTS: A severe ECG abnormality was found in 24% of DM1 patients and 17% of DM2 patients. Among DM1 patients, those with a severe ECG abnormality were older (41.6 ± 14.6 vs 35.4 ± 12.6 years) and more likely to have hypertension (13.2% vs 4.2%, P = 0.038), heart failure (4.4% vs 0%, P = 0.056), atrial arrhythmias (6.6% vs 0.7%, P < 0.001), a higher number of trinucleotide repeats (689 ± 451 vs 474 ± 322, P = 0.01), and a family history of sudden cardiac death (26.7% vs 5.6%, P < 0.001) or pacemaker implantation (20% vs 0.7%, P < 0.001). Pacemakers or defibrillators were implanted in 14% of all patients, including 65% of patients with severe ECG abnormalities. During 57 ± 46 months, 13 patients died (1.16% per year), including three patients who died suddenly, two of whom had normally functioning pacemakers. CONCLUSION: In DM1, atrio-ventricular conduction disease is associated with increasing age, concomitant cardiovascular disease, nucleotide repeat length, and family history. The systematic identification of conduction disease and aggressive use of prophylactic pacemakers is associated with low rate of sudden cardiac death.

Neuronal aggregates are associated with phenotypic onset in the R6/2 Huntington's disease transgenic mouse.

Huntington's disease (HD) is caused by the expansion of the polyglutamine tract expressed in the huntingtin protein. Data from patients show a strong negative correlation between CAG repeat size and age of disease onset. Recent studies in mixed background C57×CBA R6/2 mice suggest the inverse correlation observed in the human disease may not be replicated in some animal models of HD. To further clarify the relationship between repeat length and age of onset, congenic C57BL6/J R6/2 transgenic mice expressing 110, 260 or 310 CAG were tested in a comprehensive behavioral battery at multiple ages. Data confirmed the findings of earlier studies and indicate that on a pure C57BL6/J genetic background, R6/2 mice with larger repeats exhibit a delay in phenotypic onset with increasing polyglutamine size (6 weeks in 110 CAG and 17 weeks in 310 CAG mice). Further analysis confirmed a decrease in transgene transcript expression in 310 CAG mice as well as differential aggregated protein localization in association with repeat length. Mice expressing 110 CAG developed aggregates that localized almost exclusively to the nucleus of neuronal cells in the striatum and cortex. In contrast, tissue from 310 CAG mice exhibited predominantly extranuclear inclusions. Novel mutant protein analysis obtained using time-resolved fluorescence resonance energy transfer (FRET) revealed that soluble protein levels decreased with disease onset in R6/2 mice while aggregated protein levels increased. We believe that these data suggest a role for aggregation and inclusion localization in HD pathogenesis and propose a mechanism for the age of onset delay observed in R6/2 mice.

The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions.