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

Zhao, Xiao-Nan; Gazy, Inbal; Hayward, Bruce E.; Pintado, Elizabeth; Hwang, Ye Hyun; Tassone, Flora; Usdin, Karen

doi:10.3390/brainsci9030052

Cited by 20 publications

(26 citation statements)

References 77 publications

(170 reference statements)

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“…Here we report the establishment of FX KI mESC lines that show a high frequency of progressive CGG repeat expansions in tissue culture. These expansions resemble those seen in the blood and saliva of human PM carriers in that all the repeat PCR profiles are consistent with a high frequency of expansions most of which involve the addition of 1-2 repeats [Figures 1-3 and (Zhao et al, 2019)]. Expansions in the mESCs also show a dependence on MSH2 (Figure 3) consistent with data from GWA studies that implicate MMR in the REDs (Bettencourt et al, 2016;Morales et al, 2016;Moss et al, 2017;Flower et al, 2019).…”

Section: Discussionsupporting

confidence: 76%

“…The failure to see expansion in the 130 repeat line is consistent with observations from mice and likely reflects a much slower expansion rate than is seen in the cell lines with larger repeat tracts. The repeat PCR profile for both cell lines containing larger repeat tracts is similar to what is seen in somatic cells from human PM carriers (Zhao et al, 2019) and in iPSCs from individuals with myotonic dystrophy type 1, another RED that shows large, maternally transmitted intergenerational expansions (Du et al, 2013). Computer simulations that are consistent with this sort of expansion profile require a high frequency of expansion events that add only 1-2 repeats with each event (Mollersen et al, 2010).…”

Section: Results Mescs With An Expanded Cgg Tract In the Fmr1 Gene Shsupporting

confidence: 59%

“…Repeat PCR analysis of the smallest cell line tested, one having 130 repeats, showed an initial PCR profile that had a left-skew, a characteristic we have previously shown to be associated with alleles that do not expand in vivo [ (Figure 1A; Zhao et al, 2019)]. These cell lines show no significant increase in repeat number after 52 days in culture and no change in the skewness of the PCR profile.…”

Section: Results Mescs With An Expanded Cgg Tract In the Fmr1 Gene Shmentioning

confidence: 71%

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

Gazy

Miller

Kim

et al. 2020

Front. Cell Dev. Biol.

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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.

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Section: Discussionsupporting

confidence: 76%

Section: Results Mescs With An Expanded Cgg Tract In the Fmr1 Gene Shsupporting

confidence: 59%

Section: Results Mescs With An Expanded Cgg Tract In the Fmr1 Gene Shmentioning

confidence: 71%

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

Gazy

Miller

Kim

et al. 2020

Front. Cell Dev. Biol.

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“…This is consistent with earlier similar results in female premutation carriers [36,38,76], which have been interpreted as resulting from small sample sizes impacting adversely an analytic power, particularly in the setting of the potentially weaker and more complex genetic effect on the phenotype of women with X-linked mutations. In addition, it is known that there is a brain-blood difference in FMR1 mRNA expression, and potentially of the CGG expansion [14,[77][78][79]. Similar concerns apply to the activation ratio (AR), which did not show any relation to any of the cognitive or other phenotypic test scores in the recent comprehensive study [37].…”

Section: Discussionmentioning

confidence: 97%

Relationships between motor scores and cognitive functioning in FMR1 female premutation X carriers indicate early involvement of cerebello-cerebral pathways.

Storey

Bui

Stimpson

et al. 2020

Preprint

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Background Smaller expansions of CGG trinucleotide repeats in the FMR1 X-linked gene termed ‘premutation’ lead to a neurodegenerative disorder: Fragile X Associated Tremor/Ataxia Syndrome (FXTAS) in nearly half of aged carrier males, and 8-16% females. Core features include intention tremor, ataxia, and cognitive decline, and white matter lesions especially in cerebellar and periventricular locations. A ‘toxic’ role of elevated and expanded FMR1 mRNA has been linked to the pathogenesis of this disorder. The emerging issue concerns the trajectory of the neurodegenerative changes: is the pathogenetic effect confined to overt clinical manifestations? Here we explore the relationships between motor and cognitive scale scores in a sample of 57 asymptomatic adult female premutation carriers of broad age range. Methods Three motor scale scores (ICARS-for tremor/ataxia, UPDRS-for parkinsonism, and the Clinical Tremor) were related to 11 cognitive tests and two psychiatric pathology scores - DASS and SCL-90 - using Spearman’s rank correlations. Robust regression, applied in relationships between all phenotypic measures, and genetic molecular and demographic data, identified age and educational levels as common correlates of these measures, which were incorporated as confounders in correlation analysis. Results Cognitive tests demonstrating significant correlations with motor scores were those assessing psychomotor speed/visual attention (SDMT; TMT-A) and those dependent on various aspects of executive functioning for their execution: sequencing and alternation (TMTB-A); working memory (DS backwards); and non-verbal reasoning (MR). The largest number of motor x cognitive correlations involved ICARS and Tremor scales, which reflect the type of motor dysfunctions seen in FXTAS. Conclusions Subtle motor impairments correlating with cognitive deficits may occur in female premutation carriers not meeting diagnostic criteria for FXTAS. This pattern of cognitive deficits is consistent with those seen in other cerebellar disorders. Our results provide evidence that more than one category of clinical manifestation reflecting cerebellar changes – motor and cognitive - may be simultaneously affected by premutation carriage across a broad age range in asymptomatic carriers. Future longitudinal studies should determine whether cognitive dysfunction tracking motor impairments is driven by the premutation status common to all carriers, or only to a subset of carriers who will develop FXTAS in older age.

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“…Confidence in target selection is derived from a range In other words, target identification is being informed by human data, which increases the confidence that such drug targets are valid. We view the relationship between DDR and the bona fide cause of the disease (the trinucleotide repeat sequence) as highly compelling especially as these variant associations are not limited to HD and have been observed in the context of other trinucleotide repeat expansion diseases [35][36][37][38][39]. The observation that genetic inactivation of mismatch repair (MMR) pathway genes eliminates the somatic instability of trinucleotide repeats in disease models is in contrast to the increased di-and tetranucleotide instability typically associated with these genes although it could be argued that we are lacking a full characterisation of microsatellites in the human genome [40][41][42].…”

mentioning

confidence: 99%

Drugging DNA Damage Repair Pathways for Trinucleotide Repeat Expansion Diseases

Benn¹,

Gibson²,

Reynolds³

2021

JHD

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DNA damage repair (DDR) mechanisms have been implicated in a number of neurodegenerative diseases (both genetically determined and sporadic). Consistent with this, recent genome-wide association studies in Huntington’s disease (HD) and other trinucleotide repeat expansion diseases have highlighted genes involved in DDR mechanisms as modifiers for age of onset, rate of progression and somatic instability. At least some clinical genetic modifiers have been shown to have a role in modulating trinucleotide repeat expansion biology and could therefore provide new disease-modifying therapeutic targets. In this review, we focus on key considerations with respect to drug discovery and development using DDR mechanisms as a target for trinucleotide repeat expansion diseases. Six areas are covered with specific reference to DDR and HD: 1) Target identification and validation; 2) Candidate selection including therapeutic modality and delivery; 3) Target drug exposure with particular focus on blood-brain barrier penetration, engagement and expression of pharmacology; 4) Safety; 5) Preclinical models as predictors of therapeutic efficacy; 6) Clinical outcome measures including biomarkers.

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

Cited by 20 publications

References 77 publications

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

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

Relationships between motor scores and cognitive functioning in FMR1 female premutation X carriers indicate early involvement of cerebello-cerebral pathways.

Drugging DNA Damage Repair Pathways for Trinucleotide Repeat Expansion Diseases

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