Trinucleotide Repeat Instability Research Articles

DNA Base Damage Repair Crosstalks with Chromatin Structures to Contract Expanded GAA Repeats in Friedreich's Ataxia.

Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington's disease and Friedreich's ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol β, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol β on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases.

Loss of TDP-43 promotes somatic CAG repeat expansion in Huntington’s disease knock-in mice

TAR binding protein 43 (TDP-43) is normally present in the nucleus but mislocalized in the cytoplasm in a number of neurodegenerative diseases including Huntington’s disease (HD). The nuclear loss of TDP-43 impairs gene transcription and regulation. However, it remains to be investigated whether loss of TDP-43 influences trinucleotide CAG repeat expansion in the HD gene, a genetic cause for HD. Here we report that CRISPR/Cas9 mediated-knock down of endogenous TDP-43 in the striatum of HD knock-in mice promoted CAG repeat expansion, accompanied by the increased expression of the DNA mismatch repair genes, Msh3 and Mlh1, which have been reported to increase trinucleotide repeat instability. Furthermore, suppressing Msh3 and Mlh1 by CRISPR/Cas9 targeting diminished the CAG repeat expansion. These findings suggest that nuclear TDP-43 deficiency may dysregulate the expression of DNA mismatch repair genes, leading to CAG repeat expansion and contributing to the pathogenesis of CAG repeat diseases.

Inhibitory Effects of Mismatch Binding Molecules on the Repair Reaction of Uracil-Containing DNA.

The stable R-loop formed during transcription induces enzyme-mediated deamination of cytosine, and the uracil in the DNA produced activates the base excision repair (BER) pathway. DNA cleavage involved in the BER pathway is thought to be one of the possible causes of trinucleotide repeat instability. Here, we performed an in vitro assay to investigate the effect of a DNA-binding small molecule, naphthyridine carbamate dimer (NCD), on BER enzyme reactions. The gel electrophoretic mobility shift assay (EMSA) and thermal melting analysis revealed the binding of NCD to a 5'-XGG-3'/5'-XGG-3' triad (X = C or U or apurinic/apyrimidinic site), which is a mimic of a BER enzyme substrate. Polyacrylamide gel electrophoresis (PAGE) of the reaction products of these substrates with hSMUG1 and APE1 enzymes in the presence of NCD showed that NCD interfered with the repair reaction in the 5'-XGG-3'/5'-XGG-3' triad. These findings would broaden the potential of small molecules in modulating trinucleotide repeat instability.

Suppression of trinucleotide repeat expansion in spermatogenic cells in Huntington’s disease

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington’s disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10 nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, only FAN1, OGG1, and PCNA showed significant suppression. When the methylation profile of HD cells was analyzed, FAN1 and OGG1 showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

Replication dependent and independent mechanisms of GAA repeat instability

Trinucleotide repeat instability is a driver of human disease. Large expansions of (GAA)n repeats in the first intron of the FXN gene are the cause Friedreich’s ataxia (FRDA), a progressive degenerative disorder which cannot yet be prevented or treated. (GAA)n repeat instability arises during both replication-dependent processes, such as cell division and intergenerational transmission, as well as in terminally differentiated somatic tissues. Here, we provide a brief historical overview on the discovery of (GAA)n repeat expansions and their association to FRDA, followed by recent advances in the identification of triplex H-DNA formation and replication fork stalling. The main body of this review focuses on the last decade of progress in understanding the mechanism of (GAA)n repeat instability during DNA replication and/or DNA repair. We propose that the discovery of additional mechanisms of (GAA)n repeat instability can be achieved via both comparative approaches to other repeat expansion diseases and genome-wide association studies. Finally, we discuss the advances towards FRDA prevention or amelioration that specifically target (GAA)n repeat expansions.

CAG Repeat Instability in the Peripheral and Central Nervous System of Transgenic Huntington's Disease Monkeys.

Huntington’s Disease (HD) is an autosomal dominant disease that results in severe neurodegeneration with no cure. HD is caused by the expanded CAG trinucleotide repeat (TNR) on the Huntingtin gene (HTT). Although the somatic and germline expansion of the CAG repeats has been well-documented, the underlying mechanisms had not been fully delineated. Increased CAG repeat length is associated with a more severe phenotype, greater TNR instability, and earlier age of onset. The direct relationship between CAG repeat length and molecular pathogenesis makes TNR instability a useful measure of symptom severity and tissue susceptibility. Thus, we examined the tissue-specific TNR instability of transgenic nonhuman primate models of Huntington’s disease. Our data show a similar profile of CAG repeat expansion in both rHD1 and rHD7, where high instability was observed in testis, liver, caudate, and putamen. CAG repeat expansion was observed in all tissue samples, and tissue- and CAG repeat size-dependent expansion was observed. Correlation analysis of CAG repeat expansion and the gene expression profile of four genes in different tissues, clusterin (CLU), transferrin (TF), ribosomal protein lateral stalk subunit P1 (RPLP1), and ribosomal protein L13a (RPL13A), showed a strong correlation with CAG repeat instability. Overall, our data, along with previously published studies, can be used for studying the biology of CAG repeat instability and identifying new therapeutic targets.

MYOTONIC DYSTROPHY: EP.237 A systematic review and meta-analysis of cancer risk in myotonic dystrophy: consistent with a general increased risk of cancer

Dystrophia myotonica type 1 (DM; Steinert's disease) is an autosomal dominant, multi-system disease caused by CTG trinucleotide repeats in the DMPK gene. Case reports of pilomatricoma, a rare benign skin cancer, in DM patients have raised questions of excess cancer incidence in these patients. To investigate whether DM patients have an increased risk of developing cancer, we systematically searched three electronic databases from database inception through to 20/08/2019. A random effects meta-analysis was used to produce a pooled risk estimate for cancer risk in DM patients. Nine studies reporting on 3,915 DM patients met the eligibility criteria for inclusion. Four studies reported a risk estimate for all cause cancer. Pooled risk estimate showed DM patients had a statistically significant increased odds of developing cancer (pooled OR 1.72 (95% CI 1.45-1.99)). Elevated odds were observed for 10 cancer sites including endometrial (OR 7.92 (95% CI 4.82-13.05)), melanoma (OR 2.49 (1.29-4.83)), brain (OR 5.66 (3.89-8.23)), bladder (OR 5.60 (2.84-11.01)), thyroid (OR 9.75 (4.73-20.11)), ovarian (OR 5.60 (2.84-11.02)), testicular (OR 6.09 (1.96-18.95)), colon/colorectal (OR 2.45 (1.52-3.95)), non-Hodgkin's lymphoma (OR 2.77 (1.20-6.34)) and pancreatic cancer (OR 3.15 (1.12-8.88)). This data supports that the pathogenic DM1 trinucleotide expansion confers a general increase in cancer risk and raises questions about cancer surveillance in DM patients and trinucleotide repeat instability as a potentially carcinogenic phenomenon.

CAG repeat instability in embryonic stem cells and derivative spermatogenic cells of transgenic Huntington’s disease monkey

The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington's disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type-specific and size-dependent. Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

The Role of The DNA Damage Checkpoint in The Relocalization of CAG Trinucleotide Repeats to The Nuclear Pore Complex During S‐phase

The instability of CAG trinucleotide repeats has been known to cause neurodegenerative diseases including Huntington’s disease and myotonic muscular dystrophy. Moreover, CAG trinucleotide repeats are capable of forming lesion inducing hairpin structures during the replication of DNA during S‐phase. These lesions must be repaired in order for DNA to be faithfully replicated and properly segregated during mitotic division. Failure to correct these lesions may result in deleterious mutations at these sequences. The DNA damage checkpoint elicits the first response to damage including double stranded breaks and collapsed replication forks resulting from CAG hairpin stalling. Notably, these collapsed forks are relocalized from the center of the nucleus to the nuclear pore complex (NPC) for repair. It is hypothesized that the DNA damage checkpoint plays a role in signaling relocalization of these lesions to the NPC during S‐phase.To test this hypothesis, a GFP based zoning assay system was designed to assess CAG repeat relocalization to the NPC in several gene backgrounds defective in the DNA damage checkpoint response. These zoning assays have indicated that the DNA damage checkpoint does indeed play a role in the relocalization of CAG repeats during S‐phase. Several mutants tested also displayed abnormal nuclear arrangement. This research demonstrates that the DNA damage checkpoint is necessary for the relocalization of CAG tracts to the NPC which is known to be necessary for repair of persistent DNA lesions. Further studies will determine the mechanisms by which the checkpoint is involved in this relocalization and why abnormal nuclear arrangement is sometimes seen in mutants that are unable to elicit a proper checkpoint response.Support or Funding InformationAll support and funding is provided through the Tufts University Biology Department and specifically the Freudenreich Lab.

The role of D‐loop synthesis and progression in trinucleotide repeat stability via a break induced replication (BIR) model.

Homologous recombination (HR) is a complex, multi‐step process, that has been linked to increasing instability of trinucleotide repeats in Saccharomyces cerevisiae. One step of HR, formation and progression of the D‐loop, involves the invasion of ssDNA into a homologous sequence in the genome. Due to the involvement of a variety of polymerases and accessory factors during D‐loop synthesis and progression, it is possible to hypothesize that there is a high risk of slippage and stalling which could lead to changes in length (instability) of a CAG repeat. Break induced replication (BIR) is a genomic repair process that involves the progression of a D‐loop over long tracts of DNA, and thus is a good model to analyze this structure. In this project an assay system, involving a galactose inducible HO double strand break, results in the formation of a D‐loop due to homology to the URA3 gene on both the left and right arms of chromosome V. Repair through this tract of DNA on the right arm of chromosome V has been characterized through measures of viability, PCR of trinucleotide repeat tract, as well as Southern and Western blots. The characterization of this repair currently leads to the ultimate goal of discerning the impact of different DNA repair mutants on tract length changes within the assay system.Support or Funding InformationTufts University, Freudenreich Lab

Methods to Study Trinucleotide Repeat Instability Induced by DNA Damage and Repair.

Trinucleotide repeat (TNR) instability (expansion and deletion) is associated with more than 42 human neurodegenerative diseases and cancer and mediated by DNA replication, repair, recombination, and gene transcription. Somatic TNR instability is involved in the progression of TNR expansion diseases and can be modulated by DNA damage repair and gene transcription. Recent studies from our group and others have shown that DNA base damage and its repair play an active role in modulating TNR instability and are responsible for somatic age-dependent CAG repeat expansion in neurons of Huntington's disease mice induced by oxidative DNA damage. However, it remains to be elucidated how DNA damage, non-B form DNA structures, and DNA repair enzymes and cofactors can coordinate to regulate somatic TNR instability. Understanding the molecular mechanisms underlying DNA damage and repair-mediated somatic TNR instability is critically important for identification of new therapeutic targets for treatment and prevention of TNR-related diseases. Here we describe the methods to study the locations and distribution of DNA base lesions and their effects on TNR instability through DNA base excision repair in in vitro reconstituted human systems.

The deoxyribose phosphate lyase of DNA polymerase β suppresses a processive DNA synthesis to prevent trinucleotide repeat instability.

Trinucleotide repeat (TNR) instability is associated with over 42 neurodegenerative diseases and cancer, for which the molecular mechanisms remain to be elucidated. We have shown that the DNA base excision repair (BER) pathway and its central component, DNA polymerase β (pol β), in particular, its polymerase activity plays an active role in regulating somatic TNR instability. Herein, we revealed a unique role of the pol β dRP lyase in preventing somatic TNR instability. We found that deficiency of pol β deoxyribose phosphate (dRP) lyase activity locked the pol β dRP lyase domain to a dRP group, and this ‘tethered’ pol β to its template forcing the polymerase to perform a processive DNA synthesis. This subsequently promoted DNA strand slippage allowing pol β to skip over a template loop and causing TNR deletion. We showed that the effects were eliminated by complementation of the dRP lyase deficiency with wild-type pol β protein. The results indicate that pol β dRP lyase activity restrained the pol β-dRP interaction to suppress a pol β processive DNA synthesis, thereby preventing TNR deletion. This further implicates a potential of pol β dRP lyase inhibition as a novel treatment of TNR-expansion diseases.

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy.

Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.

The flash-small-pool PCR: how to transform blotting and numerous hybridization steps into a simple denatured PCR.

Numerous human diseases are associated with abnormal expansion of unstable trinucleotide repeats (TNRs). TNR instability mechanisms are complex, and remain only partially understood. Small-pool-PCR (SP-PCR) is the reference method to assess TNR instability. SP-PCR amplifies a low number of DNA molecules and is followed by Southern blot. However, SP-PCR remains expensive and time consuming. Here, we describe an optimized SP-PCR that can be done in a day, which reduces cost and experimental biases: the flash-small-pool PCR (FSP-PCR). This method consists of a fluorescent PCR on a few DNA molecules, followed by an alkaline gel electrophoresis revealed with a near infra-red detector system. With reduced experimental steps, cost, and time consumption, microsatellite analysis will become more accessible due to FSP-PCR.

Genetic testing for clinically suspected spinocerebellar ataxias: report from a tertiary referral centre in India

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative syndromes, characterized by a wide range of muscular weakness and motor deficits, caused due to cerebellar degeneration. The prevalence of the syndromes of SCA varies across the world and is known to be linked to the instability of trinucleotide repeats within the high-end normal alleles, along with susceptible haplotype. We estimated sizes of the CAG or GAA repeat expansions at the SCA1, SCA2, SCA3, SCA12 and frataxin loci among 864 referrals of subjects to genetic counselling and testing (GCAT) clinic, National Institute of Mental Health and Neurosciences, Bengaluru, India, with suspected SCA. The most frequent mutations detected were SCA1 (n = 100 (11.6%)) and SCA2 (n = 98 (11.3%)) followed by SCA3 (n = 40 (4.6%)), FRDA (n = 20 (2.3%)) and SCA12 (n = 8 (0.9%)).

An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract

Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5’-(2-phosphoryl-1,4-dioxobutane)(DOB) in CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase β and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair (MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components, particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.

Modulation of trinucleotide repeat instability by DNA polymerase β polymorphic variant R137Q.

Trinucleotide repeat (TNR) instability is associated with human neurodegenerative diseases and cancer. Recent studies have pointed out that DNA base excision repair (BER) mediated by DNA polymerase β (pol β) plays a crucial role in governing somatic TNR instability in a damage-location dependent manner. It has been shown that the activities and function of BER enzymes and cofactors can be modulated by their polymorphic variations. This could alter the function of BER in regulating TNR instability. However, the roles of BER polymorphism in modulating TNR instability remain to be elucidated. A previous study has shown that a pol β polymorphic variant, polβR137Q is associated with cancer due to its impaired polymerase activity and its deficiency in interacting with a BER cofactor, proliferating cell nuclear antigen (PCNA). In this study, we have studied the effect of the pol βR137Q variant on TNR instability. We showed that pol βR137Q exhibited weak DNA synthesis activity to cause TNR deletion during BER. We demonstrated that similar to wild-type pol β, the weak DNA synthesis activity of pol βR137Q allowed it to skip over a small loop formed on the template strand, thereby facilitating TNR deletion during BER. Our results further suggest that carriers with pol βR137Q polymorphic variant may not exhibit an elevated risk of developing human diseases that are associated with TNR instability.

Crosstalk between MSH2-MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair.

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.