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We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated pa­tients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably re­sulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing. [ABSTRACT FROM AUTHOR]

Background: Trinucleotide repeat expansions are an emerging class of genetic variants associated with various movement disorders. Unbiased genome-wide analyses can reveal novel genotype-phenotype associations and provide a diagnosis for patients and families., Objective: The aim was to identify the genetic cause of a severe progressive movement disorder phenotype in 2 affected brothers., Methods: A family of 2 affected brothers and unaffected parents had extensive phenotyping since birth. Whole-genome and long-read sequencing methods characterized genetic variants and methylation status., Results: Two male siblings with a CGG repeat expansion in the 5'-untranslated region (UTR) of disco-interacting protein 2 homolog B (DIP2B) presented with a novel DIP2B phenotype, including neurodevelopmental disability, dysmorphic traits, and a severe progressive movement disorder (chorea, dystonia, and ataxia)., Conclusions: This is the first report of a severe progressive movement disorder phenotype associated with a CGG repeat expansion in the DIP2B 5'-UTR. © 2025 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2025 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Fragile X syndrome (FXS) is a genetic condition caused by the inheritance of alleles with >200 CGG repeats in the 5' UTR of the fragile X messenger ribonucleoprotein 1 ( FMR1 ) gene. These full mutation (FM) alleles are associated with DNA methylation and gene silencing, which result in intellectual disabilities, developmental delays, and social and behavioral issues. Mosaicism for both the size of the CGG repeat tract and the extent of its methylation is commonly observed in individuals with the FM. Mosaicism has also been reported in carriers of premutation (PM) alleles, which have 55-200 CGG repeats. PM alleles confer risk for the fragile X premutation-associated conditions (FXPAC), including FXTAS, FXPOI, and FXAND, conditions thought to be due to the toxic consequences of transcripts containing large CGG-tracts. Unmethylated FM (UFM) alleles are transcriptionally and translationally active. Thus, they produce transcripts with toxic effects. These transcripts do produce some FMRP, the encoded product of the FMR1 gene, albeit with reduced translational efficiency. As a result, mosaicism can result in a complex clinical presentation. Here, we review the concept of mosaicism in both FXS and in PM carriers, including its potential clinical significance.

The Repeat Expansion Diseases (REDs) are a large group of human genetic disorders that result from an increase in the number of repeats in a disease-specific tandem repeat or microsatellite. Emerging evidence suggests that the repeats trigger an error-prone form of DNA repair that causes the expansion mutation by exploiting a limitation in normal mismatch repair. Furthermore, while much remains to be understood about how the mutation causes pathology in different diseases in this group, there is evidence to suggest that some of the downstream consequences of repeat expansion trigger the DNA damage response in ways that contribute to disease pathology. This review will discuss these subjects in the context of the Fragile X-related disorders (aka the FMR1 disorders) that provide a particularly interesting example of the intersection between the repeats and the DNA damage response that may also be relevant for many other diseases in this group., Competing Interests: Declaration of Competing Interest All of the authors have nothing to declare., (Published by Elsevier B.V.)

Background and Objectives: The number of GAA repeats in the FXN gene is a major but not sole determinant of the clinical presentation of Friedreich ataxia (FRDA). The objective of this study was to establish whether the length of the GAA repeat tract in the FGF14 gene, which is associated with another neurodegenerative disorder (SCA27B), affects the clinical presentation (age at onset, mFARS score) of patients with FRDA., Methods: The number of GAA repeats in the FXN and FGF14 genes was determined using PCR in a cohort of 221 patients with FRDA. Next, we compared absolute lengths of the FGF14 GAAs with FXN GAAs, followed by correlative analyses to determine potential effects of FGF14 GAA length on age at onset and clinical presentation (mFARS) of FRDA., Results: We found no significant correlation between the size of the GAA repeats in FXN and FGF14 loci in our FRDA cohort. Moreover, the number of GAAs in FGF14 did not affect the clinical presentation of FRDA even in a small number of cases where a long FGF14 allele was present., Discussion: Despite both molecular and clinical similarities between FRDA and SCA27B, the length of the GAA repeats in the FGF14 gene, including potentially pathogenic alleles, did not influence the clinical presentation of FRDA., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Microsatellite instability is responsible for the human Repeat Expansion Disorders. The mutation responsible differs from classical cancer-associated microsatellite instability (MSI) in that it requires the mismatch repair proteins that normally protect against MSI. LIG4, an enzyme essential for non-homologous end-joining (NHEJ), the major pathway for double-strand break repair (DSBR) in mammalian cells, protects against expansion in mouse models. Thus, NHEJ may compete with the expansion pathway for access to a common intermediate. This raises the possibility that expansion involves an NHEJ-independent form of DSBR. Pol θ, a polymerase involved in the theta-mediated end joining (TMEJ) DSBR pathway, has been proposed to play a role in repeat expansion. Here we examine the effect of the loss of Pol θ on expansion in FXD mouse embryonic stem cells (mESCs), along with the effects of mutations in Rad52 , Rad54l and Rad54b, genes important for multiple DSBR pathways. None of these mutations significantly affected repeat expansion. These observations put major constraints on what pathways are likely to drive expansion. Together with our previous demonstration of the protective effect of nucleases like EXO1 and FAN1, and the importance of Pol β, they suggest a plausible model for late steps in the expansion process., Competing Interests: Competing interest statement The authors have no competing interests Conflict of Interest: The authors have no conflict of interest

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14. Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14. We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure cerebellar involvement in SCA27B., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Genome Wide Association studies (GWAS) have implicated PMS2 as a modifier of somatic expansion in Huntington's disease (HD), one of >45 known Repeat Expansion Diseases (REDs). PMS2 is a subunit of the MutLα complex, a major component of the mismatch repair (MMR) system, a repair pathway that is involved in the generation of expansions in many different REDs. However, while MLH3, a subunit of a second MutL complex, MutLγ, is required for all expansions, PMS2 has been shown to protect against expansion in some model systems but to drive expansion in others. To better understand PMS2's behavior, we have compared the effect of the loss of PMS2 in different tissues of an HD mouse model (CAG/CTG repeats) and a mouse model for the Fragile X-related disorders (FXDs), disorders that result from a CGG/CCG repeat expansion. Mice heterozygous for Pms2 show increased expansions in most expansion-prone tissues in both disease models. However, in Pms2 null mice expansions of both repeats increased in some tissues but decreased in others. Thus, the previously reported differences in the effects of PMS2 in different model systems do not reflect fundamentally different roles played by PMS2 in different REDs, but rather the paradoxical effects of PMS2 in different cellular contexts. These findings have important implications not only for the mechanism of expansion and the development of therapeutic approaches to reduce the pathology generated by repeat expansion, but also for our understanding of normal MMR., Competing Interests: Conflict of interest statement The authors declare no competing or financial interests.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~ 120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLβ, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

A long CGG-repeat tract in the FMR1 gene induces the epigenetic silencing that causes fragile X syndrome (FXS). Epigenetic changes include H4K20 trimethylation, a heterochromatic modification frequently implicated in transcriptional silencing. Here, we report that treatment with A-196, an inhibitor of SUV420H1/H2, the enzymes responsible for H4K20 di-/trimethylation, does not affect FMR1 transcription, but does result in increased chromosomal duplications. Increased duplications were also seen in FXS cells treated with SCR7, an inhibitor of Lig4, a ligase essential for NHEJ. Our study suggests that the fragile X (FX) locus is prone to spontaneous DNA damage that is normally repaired by NHEJ. We suggest that heterochromatinization of the FX allele may be triggered, at least in part, in response to this DNA damage., Competing Interests: The authors declare no competing interests.

Carriers of the FMR1 premutation (PM) allele are at risk of one or more clinical conditions referred to as FX premutation-associated conditions (FXPAC). Since the FMR1 gene is on the X chromosome, the activation ratio (AR) may impact the risk, age of onset, progression, and severity of these conditions. The aim of this study was to evaluate the reliability of AR measured using different approaches and to investigate potential correlations with clinical outcomes. Molecular and clinical assessments were obtained for 30 PM female participants, and AR was assessed using both Southern blot analysis (AR-Sb) and methylation PCR (AR-mPCR). Higher ARs were associated with lower FMR1 transcript levels for any given repeat length. The higher AR-Sb was significantly associated with performance, verbal, and full-scale IQ scores, confirming previous reports. However, the AR-mPCR was not significantly associated ( p > 0.05) with these measures. Similarly, the odds of depression and the number of medical conditions were correlated with higher AR-Sb but not correlated with a higher AR-mPCR. This study suggests that AR-Sb may be a more reliable measure of the AR in female carriers of PM alleles. However, further studies are warranted in a larger sample size to fully evaluate the methylation status in these participants and how it may affect the clinical phenotype.

Carriers of a premutation allele (PM) in the FMR1 gene are at risk of developing a number of Fragile X premutation asssociated disorders (FXPAC), including Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-associated neuropsychiatric disorders (FXAND). We have recently reported somatic CGG allele expansion in female PM; however, its clinical significance remains unclear. The aim of this study was to examine the potential clinical association between somatic FMR1 allele instability and PM associated disorders. Participants comprised of 424 female PM carriers age 0.3- 90 years. FMR1 molecular measures and clinical information on the presence of medical conditions, were determined for all subjects for primary analysis. Two sub-groups of participants (age ≥ 25, N = 377 and age ≥ 50, N = 134) were used in the analysis related to presence of FXPOI and FXTAS, respectively. Among all participants (N = 424), the degree of instability (expansion) was significantly higher (median 2.5 vs 2.0, P = 0.026) in participants with a diagnosis of attention deficit hyperactivity disorder (ADHD) compared to those without. FMR1 mRNA expression was significantly higher in subjects with any psychiatric disorder diagnosis (P = 0.0017); specifically, in those with ADHD (P = 0.009), and with depression (P = 0.025). Somatic FMR1 expansion was associated with the presence of ADHD in female PM and FMR1 mRNA levels were associated with the presence of mental health disorders. The findings of our research are innovative as they suggest a potential role of the CGG expansion in the clinical phenotype of PM and may potentially guide clinical prognosis and management., (© 2023. The Author(s).)

Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~3000 genomes and ~1500 exomes with optical genome mapping and long-read sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping and long-read sequencing)-to effectively flag complex repeat expansions using short-read datasets and thus facilitate diagnosis of ultra-rare disorders., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA) n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA) n repeats can be disrupted by treatment with a (GAA) n -binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Roughly 3% of the human genome consists of microsatellites or tracts of short tandem repeats (STRs). These STRs are often unstable, undergoing high-frequency expansions (increases) or contractions (decreases) in the number of repeat units. Some microsatellite instability (MSI) is seen at multiple STRs within a single cell and is associated with certain types of cancer. A second form of MSI is characterised by expansion of a single gene-specific STR and such expansions are responsible for a group of 40+ human genetic disorders known as the repeat expansion diseases (REDs). While the mismatch repair (MMR) pathway prevents genome-wide MSI, emerging evidence suggests that some MMR factors are directly involved in generating expansions in the REDs. Thus, MMR suppresses some forms of expansion while some MMR factors promote expansion in other contexts. This review will cover what is known about the paradoxical effect of MMR on microsatellite expansion in mammalian cells.

The fragile X mental retardation (FMR1) gene contains an expansion-prone CGG repeat within its 5' UTR. Alleles with 55-200 repeats are known as premutation (PM) alleles and confer risk for one or more of the FMR1 premutation (PM) disorders that include Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-Associated Neuropsychiatric Disorders (FXAND). PM alleles expand on intergenerational transmission, with the children of PM mothers being at risk of inheriting alleles with > 200 CGG repeats (full mutation FM) alleles) and thus developing Fragile X Syndrome (FXS). PM alleles can be somatically unstable. This can lead to individuals being mosaic for multiple size alleles. Here, we describe a detailed evaluation of somatic mosaicism in a large cohort of female PM carriers and show that 94% display some evidence of somatic instability with the presence of a series of expanded alleles that differ from the next allele by a single repeat unit. Using two different metrics for instability that we have developed, we show that, as with intergenerational instability, there is a direct relationship between the extent of somatic expansion and the number of CGG repeats in the originally inherited allele and an inverse relationship with the number of AGG interruptions. Expansions are progressive as evidenced by a positive correlation with age and by examination of blood samples from the same individual taken at different time points. Our data also suggests the existence of other genetic or environmental factors that affect the extent of somatic expansion. Importantly, the analysis of candidate single nucleotide polymorphisms (SNPs) suggests that two DNA repair factors, FAN1 and MSH3, may be modifiers of somatic expansion risk in the PM population as observed in other repeat expansion disorders., (© 2022. The Author(s).)

Repeat expansion diseases are a large group of human genetic disorders caused by expansion of a specific short tandem repeat tract. Expansion in somatic cells affects age of onset and disease severity in some of these disorders. However, alleles in DNA derived from blood, a commonly used source of DNA, usually show much less expansion than disease-relevant cells in the central nervous system in both humans and mouse models. Here we examined the extent of expansion in different DNA sources from mouse models of the fragile X-related disorders, Huntington's disease, spinocerebellar ataxia type 1 and spinocerebellar ataxia type 2. We found that DNA isolated from stool is a much better indicator of somatic expansion than DNA from blood. As stool is a sensitive and noninvasive source of DNA, it can be useful for studies of factors affecting the risk of expansion, or the monitoring of treatments aimed at reducing expansion in preclinical trials, as it would allow expansions to be examined longitudinally in the same animal and allow significant changes in expansion to be observed much earlier than is possible with other DNA sources., Competing Interests: Competing interests J.B.C. is on the scientific advisory board, and has shares in Triplet Therapeutics, which is a company focused on modulating somatic instability in HD and other diseases. Other authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5' UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54-200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lokanga, Kumari and Usdin.)

Fragile X-related disorders (FXDs), also known as FMR1 disorders, are examples of repeat expansion diseases (REDs), clinical conditions that arise from an increase in the number of repeats in a disease-specific microsatellite. In the case of FXDs, the repeat unit is CGG/CCG and the repeat tract is located in the 5' UTR of the X-linked FMR1 gene. Expansion can result in neurodegeneration, ovarian dysfunction, or intellectual disability depending on the number of repeats in the expanded allele. A growing body of evidence suggests that the mutational mechanisms responsible for many REDs share several common features. It is also increasingly apparent that in some of these diseases the pathologic consequences of expansion may arise in similar ways. It has long been known that many of the disease-associated repeats form unusual DNA and RNA structures. This review will focus on what is known about these structures, the proteins with which they interact, and how they may be related to the causative mutation and disease pathology in the FMR1 disorders.

Huntington's disease (HD) is one of a large group of human disorders that are caused by expanded DNA repeats. These repeat expansion disorders can have repeat units of different size and sequence that can be located in any part of the gene and, while the pathological consequences of the expansion can differ widely, there is evidence to suggest that the underlying mutational mechanism may be similar. In the case of HD, the expanded repeat unit is a CAG trinucleotide located in exon 1 of the huntingtin (HTT) gene, resulting in an expanded polyglutamine tract in the huntingtin protein. Expansion results in neuronal cell death, particularly in the striatum. Emerging evidence suggests that somatic CAG expansion, specifically expansion occurring in the brain during the lifetime of an individual, contributes to an earlier disease onset and increased severity. In this review we will discuss mouse models of two non-CAG repeat expansion diseases, specifically the Fragile X-related disorders (FXDs) and Friedreich ataxia (FRDA). We will compare and contrast these models with mouse and patient-derived cell models of various other repeat expansion disorders and the relevance of these findings for somatic expansion in HD. We will also describe additional genetic factors and pathways that modify somatic expansion in the FXD mouse model for which no comparable data yet exists in HD mice or humans. These additional factors expand the potential druggable space for diseases like HD where somatic expansion is a significant contributor to disease impact.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1-4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases, genetic disorders that result from the expansion of a disease-specific microsatellite. In those Repeat Expansion Disease models where it has been examined, expansion is dependent on functional mismatch repair (MMR) factors, including MutLγ, a heterodimer of MLH1/MLH3, one of the three MutL complexes found in mammals and a minor player in MMR. In contrast, MutLα, a much more abundant MutL complex that is the major contributor to MMR, is either not required for expansion or plays a limited role in expansion in many model systems. How MutLγ acts to generate expansions is unclear given its normal role in protecting against microsatellite instability and while MLH3 does have an associated endonuclease activity, whether that contributes to repeat expansion is uncertain. We show here, using a gene-editing approach, that a point mutation that eliminates the endonuclease activity of MLH3 eliminates expansions in an FXD mouse embryonic stem cell model. This restricts the number of possible models for repeat expansion and supports the idea that MutLγ may be a useful druggable target to reduce somatic expansion in those disorders where it contributes to disease pathology., (Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that result from expansion of a CGG-repeat tract located at the 5' end of the FMR1 gene. While expansion affects transmission risk and can also affect disease risk and severity, the underlying molecular mechanism responsible is unknown. Despite the fact that expanded alleles can be seen both in humans and mouse models in vivo , existing patient-derived cells do not show significant repeat expansions even after extended periods in culture. In order to develop a good tissue culture model for studying expansions we tested whether mouse embryonic stem cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene are permissive for expansion. We show here that these mESCs have a very high frequency of expansion that allows changes in the repeat number to be seen within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that this may be due in part to the fact that non-homologous end-joining (NHEJ), which is able to protect against expansions in some cell types, is not effective in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are MSH2-dependent, consistent with those seen in vivo . While comparable human Genome Wide Association (GWA) studies are not available for the FXDs, such studies have implicated MSH2 in expansion in other REDs. The shared unusual requirement for MSH2 for this type of microsatellite instability suggests that this new cell-based system is relevant for understanding the mechanism responsible for this peculiar type of mutation in humans. The high frequency of expansions and the ease of gene editing these cells should expedite the identification of factors that affect expansion risk. Additionally, we found that, as with cells from human premutation (PM) carriers, these cell lines have elevated mitochondrial copy numbers and Fmr1 hyperexpression, that we show here is O 2 -sensitive. Thus, this new stem cell model should facilitate studies of both repeat expansion and the consequences of expansion during early embryonic development., (Copyright © 2020 Gazy, Miller, Kim and Usdin.)

Expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLβ, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLβ is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLβ may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier., Competing Interests: The authors have declared that no competing interests exist.

In fragile X syndrome (FXS), expansion of a CGG repeat tract in the 5'-untranslated region of the FMR1 gene to >200 repeats causes transcriptional silencing by inducing heterochromatin formation. Understanding the mechanism of FMR1 silencing is important as gene reactivation is a potential treatment approach for FXS. To date, only the DNA demethylating drug 5-azadeoxycytidine (AZA) has proved effective at gene reactivation; however, this drug is toxic. The repressive H3K9 methylation mark is enriched on the FMR1 gene in FXS patient cells and is thus a potential druggable target. However, its contribution to the silencing process is unclear. Here, we studied the effect of small molecule inhibitors of H3K9 methylation on FMR1 expression in FXS patient cells. Chaetocin showed a small effect on FMR1 gene reactivation and a synergistic effect on FMR1 mRNA levels when used in combination with AZA. Additionally, chaetocin, BIX01294 and 3-Deazaneplanocin A (DZNep) were able to significantly delay the re-silencing of AZA-reactivated FMR1 alleles. These data are consistent with the idea that H3K9 methylation precedes DNA methylation and that removal of DNA methylation is necessary to see the optimal effect of histone methyl-transferase (HMT) inhibitors on FMR1 gene expression. Nonetheless, our data also show that drugs targeting repressive H3K9 methylation marks are able to produce sustained reactivation of the FMR1 gene after a single dose of AZA.

Analysis of individual gametes has a number of applications in the study of the mechanism of repeat expansion in mouse models of the fragile X-related disorders, as well as in mouse models of other repeat expansion diseases. This chapter describes the techniques required to isolate oocytes and male gametes of different stages of maturity, along with the techniques required to accurately determine the repeat number in these gametes.

Most cases of fragile X syndrome (FXS) result from aberrant methylation of the FMR1 gene. Methylation occurs when the number of tandemly arranged cytosine guanine guanine (CGG)-repeats in the 5' end of the transcriptional unit of FMR1 exceeds a certain critical threshold, thought to be between 200 and 400 repeats. Such alleles are referred to as full mutation (FM) alleles. Premutation (PM) alleles, alleles with 55-200 repeats, are generally not aberrantly methylated and in fact may have hyperexpression of the FMR1 mRNA. We describe here a male who meets the diagnostic criteria for FXS, who is highly mosaic with a mixture of multiple PM and FM alleles and 50% methylation. However, the methylated alleles are limited to two alleles in the PM range, ~165 and ~175 repeats respectively, with the FM alleles being unmethylated. This finding has implications for FXS diagnosis as well as for efforts to delete the repeat in individuals with FXS using a CRISPR-Cas9 approach., (© 2019 Wiley Periodicals, Inc.)

The fragile X-related disorders (FXDs) are a group of clinical conditions that result primarily from an unusual mutation, the expansion of a CGG-repeat tract in exon 1 of the FMR1 gene. Mouse models are proving useful for understanding many aspects of disease pathology in these disorders. There is also reason to think that such models may be useful for understanding the molecular basis of the unusual mutation responsible for these disorders. This review will discuss what has been learnt to date about mechanisms of repeat instability from a knock-in FXD mouse model and what the implications of these findings may be for humans carrying expansion-prone FMR1 alleles.

More than ~200 CGG repeats in the 5' untranslated region of the FMR1 gene results in transcriptional silencing and the absence of the FMR1 encoded protein, FMRP. FMRP is an RNA-binding protein that regulates the transport and translation of a variety of brain mRNAs in an activity-dependent manner. The loss of FMRP causes dysregulation of many neuronal pathways and results in an intellectual disability disorder, fragile X syndrome (FXS). Currently, there is no effective treatment for FXS. In this review, we discuss reactivation of the FMR1 gene as a potential approach for FXS treatment with an emphasis on the use of small molecules to inhibit the pathways important for gene silencing.

Expansion of a CGG-repeat tract in the 5' UTR of FMR1 is responsible for the Fragile X-related disorders (FXDs), FXTAS, FXPOI and FXS. Previous work in a mouse model of these disorders has implicated proteins in the base excision and the mismatch repair (MMR) pathways in the expansion mechanism. However, the precise role of these factors in this process is not well understood. The essential role of MutLγ, a complex that plays a minor role in MMR but that is essential for resolving Holliday junctions during meiosis, raises the possibility that expansions proceed via a Holliday junction-like intermediate that is processed to generate a double-strand break (DSB). We show here in an FXD mouse model that LIG4, a ligase essential for non-homologous end-joining (NHEJ), a form of DSB repair (DSBR), protects against expansions. However, a mutation in MRE11, a nuclease that is important for several other DSBR pathways including homologous recombination (HR), has no effect on the extent of expansion. Our results suggest that the expansion pathway competes with NHEJ for the processing of a DSB intermediate. Thus, expansion likely proceeds via an NHEJ-independent DSBR pathway that may also be HR-independent., (Published by Elsevier B.V.)

Knowledge of the CGG•CCG-repeat number, AGG interruption status, and the extent of DNA methylation of the FMR1 gene are vital for both diagnosis of the fragile X-related disorders and for basic research into disease mechanisms. We describe here assays that we use in our laboratory to assess these parameters. Our assays are PCR-based and include one for repeat size that can also be used to assess the extent of methylation and a related assay that allows the AGG interruption pattern to be reliably determined even in women. A second more quantitative methylation assay is also described. We also describe our method for cloning of repeats to generate the reference standards necessary for the accurate determination of repeat number and AGG interruption status.