Fragile X-related Disorders Research Articles

Intersection of the Fragile X-related disorders and the DNA damage response

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.

PMS2 has both pro-mutagenic and anti-mutagenic effects on repeat instability in the Repeat Expansion Diseases.

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.

Mitochondrial Dysfunction Causes Cell Death in Patients Affected by Fragile-X-Associated Disorders.

Mitochondria are involved in multiple aspects of neurodevelopmental processes and play a major role in the pathogenetic mechanisms leading to neuro-degenerative diseases. Fragile-X-related disorders (FXDs) are genetic conditions that occur due to the dynamic expansion of CGG repeats of the FMR1 gene encoding for the RNA-binding protein FMRP, particularly expressed in the brain. This gene expansion can lead to premutation (PM, 56-200 CGGs), full mutation (FM, >200 CGGs), or unmethylated FM (UFM), resulting in neurodegeneration, neurodevelopmental disorders, or no apparent intellectual disability, respectively. To investigate the mitochondrial mechanisms that are involved in the FXD patients, we analyzed mitochondrial morphology and bioenergetics in fibroblasts derived from patients. Donut-shaped mitochondrial morphology and excessive synthesis of critical mitochondrial proteins were detected in FM, PM, and UFM cells. Analysis of mitochondrial oxidative phosphorylation in situ reveals lower respiration in PM fibroblasts. Importantly, mitochondrial permeability transition-dependent apoptosis is sensitized to reactive oxygen species in FM, PM, and UFM models. This study elucidated the mitochondrial mechanisms that are involved in the FXD phenotypes, and indicated altered mitochondrial function and morphology. Importantly, a sensitization to permeability transition and apoptosis was revealed in FXD cells. Overall, our data suggest that mitochondria are novel drug targets to relieve the FXD symptoms.

Non-canonical DNA/RNA structures associated with the pathogenesis of Fragile X-associated tremor/ataxia syndrome and Fragile X syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X syndrome (FXS) are primary examples of fragile X-related disorders (FXDs) caused by abnormal expansion of CGG repeats above a certain threshold in the 5′-untranslated region of the fragile X mental retardation (FMR1) gene. Both diseases have distinct clinical manifestations and molecular pathogenesis. FXTAS is a late-adult-onset neurodegenerative disorder caused by a premutation (PM) allele (CGG expansion of 55–200 repeats), resulting in FMR1 gene hyperexpression. On the other hand, FXS is a neurodevelopmental disorder that results from a full mutation (FM) allele (CGG expansions of ≥200 repeats) leading to heterochromatization and transcriptional silencing of the FMR1 gene. The main challenge is to determine how CGG repeat expansion affects the fundamentally distinct nature of FMR1 expression in FM and PM ranges. Abnormal CGG repeat expansions form a variety of non-canonical DNA and RNA structures that can disrupt various cellular processes and cause distinct effects in PM and FM alleles. Here, we review these structures and how they are related to underlying mutations and disease pathology in FXS and FXTAS. Finally, as new CGG expansions within the genome have been identified, it will be interesting to determine their implications in disease pathology and treatment.

Mechanisms of the FMR1 Repeat Instability: How Does the CGG Sequence Expand?

A dynamic mutation in exon 1 of the FMR1 gene causes Fragile X-related Disorders (FXDs), due to the expansion of an unstable CGG repeat sequence. Based on the CGG sequence size, two types of FMR1 alleles are possible: “premutation” (PM, with 56-200 CGGs) and “full mutation” (FM, with >200 triplets). Premutated females are at risk of transmitting a FM allele that, when methylated, epigenetically silences FMR1 and causes Fragile X syndrome (FXS), a very common form of inherited intellectual disability (ID). Expansions events of the CGG sequence are predominant over contractions and are responsible for meiotic and mitotic instability. The CGG repeat usually includes one or more AGG interspersed triplets that influence allele stability and the risk of transmitting FM to children through maternal meiosis. A unique mechanism responsible for repeat instability has not been identified, but several processes are under investigations using cellular and animal models. The formation of unusual secondary DNA structures at the expanded repeats are likely to occur and contribute to the CGG expansion. This review will focus on the current knowledge about CGG repeat instability addressing the CGG sequence expands.

Stool is a sensitive and noninvasive source of DNA for monitoring expansion in repeat expansion disease mouse models.

ABSTRACTRepeat 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.

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

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.

Mechanisms of Genome Instability in the Fragile X-Related Disorders.

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.

(Dys)function Follows Form: Nucleic Acid Structure, Repeat Expansion, and Disease Pathology in FMR1 Disorders.

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.

Modifiers of Somatic Repeat Instability in Mouse Models of Friedreich Ataxia and the Fragile X-Related Disorders: Implications for the Mechanism of Somatic Expansion in Huntington's Disease.

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.

A point mutation in the nuclease domain of MLH3 eliminates repeat expansions in a mouse stem cell model of the Fragile X-related disorders.

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.

CGG Repeat Expansion, and Elevated Fmr1 Transcription and Mitochondrial Copy Number in a New Fragile X PM Mouse Embryonic Stem Cell Model.

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 O2-sensitive. Thus, this new stem cell model should facilitate studies of both repeat expansion and the consequences of expansion during early embryonic development.

All three mammalian MutL complexes are required for repeat expansion in a mouse cell model of the Fragile X-related disorders.

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.

Altered mitochondrial function in cells carrying a premutation or unmethylated full mutation of the FMR1 gene

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.

Isolation and Analysis of the CGG-Repeat Size in Male and Female Gametes from a Fragile X Mouse Model.

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.

Repeat Instability in the Fragile X-Related Disorders: Lessons from a Mouse Model.

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.

Assays for Determining Repeat Number, Methylation Status, and AGG Interruptions in the Fragile X-Related Disorders.

Knowledge of the CGG•CCG-repeat number, AGG interruption status, and the extent of DNA methylation of the FMR1 geneare vital for both diagnosis of thefragile 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.

Double-strand break repair plays a role in repeat instability in a fragile X mouse model

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.

MutLγ promotes repeat expansion in a Fragile X mouse model while EXO1 is protective.

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5’ end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSβ) and MSH2/MSH6 (MutSα), together with Polβ, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSβ in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans.

Timing of Expansion of Fragile X Premutation Alleles During Intergenerational Transmission in a Mouse Model of the Fragile X-Related Disorders.

Fragile X syndrome (FXS) is caused by the maternal expansion of an unstable CGG-repeat tract located in the first exon of the FMR1 gene. Further changes in repeat number occur during embryogenesis resulting in individuals sometimes being highly mosaic. Here we show in a mouse model that, in males, expansions are already present in primary spermatocytes with no additional expansions occurring in later stages of gametogenesis. We also show that, in females, expansion occurs in the post-natal oocyte. Additional expansions and a high frequency of large contractions are seen in two-cell stage embryos. Expansion in oocytes, which are non-dividing, would be consistent with a mechanism involving aberrant DNA repair or recombination rather than a problem with chromosomal replication. Given the difficulty of replicating large CGG-repeat tracts, we speculate that very large expanded alleles may be prone to contract in the mitotically proliferating spermatagonial stem cells in men. However, expanded alleles may not be under such pressure in the non-dividing oocyte. The high degree of both expansions and contractions seen in early embryos may contribute to the high frequency of somatic mosaicism that is observed in humans. Our data thus suggest an explanation for the fact that FXS is exclusively maternally transmitted and lend support to models for repeat expansion that are based on problems arising during DNA repair.