scholarly journals Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

2018 ◽  
Author(s):  
Jill M. Haenfler ◽  
Geena Skariah ◽  
Caitlin M. Rodriguez ◽  
Andre Monteiro da Rocha ◽  
Jack M. Parent ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Embryonic Stem ◽  
Transcriptional Silencing ◽  
Hesc Line ◽  
Human Patient ◽  
Cgg Repeat ◽  
Heterochromatin Formation ◽  
Fmr1 Mrna

ABSTRACTFragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5’ untranslated region of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing, and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the CRISPR/dCAS9 system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCAS9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 mRNA expression despite promoter and repeat methylation. These studies demonstrate that FMR1 mRNA expression can be selectively reactivated in human patient cells, creating a pathway forward for therapeutic development in Fragile X Syndrome.

scholarly journals Autism Profiles of Males With Fragile X Syndrome

2008 ◽  
Vol 113 (6) ◽  
pp. 427-438 ◽  
Author(s):  
Susan W. Harris ◽  
David Hessl ◽  
Beth Goodlin-Jones ◽  
Jessica Ferranti ◽  
Susan Bacalman ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Fragile X ◽  
Autism Diagnosis ◽  
Cgg Repeat ◽  
Molecular Features ◽  
Fmr1 Mrna ◽  
Two Measures ◽  
Dsm Iv ◽  
Relationship Of ◽  
The Relationship

Abstract Autism, which is common in individuals with fragile X syndrome, is often difficult to diagnose. We compared the diagnostic classifications of two measures for autism diagnosis, the ADOS and the ADI-R, in addition to the DSM-IV-TR in 63 males with this syndrome. Overall, 30% of the subjects met criteria for autistic disorder and 30% met criteria for PDD-NOS. The classifications on the ADOS and DSM-IV-TR were most similar, whereas the ADI-R classified subjects as autistic much more frequently. We further investigated the relationship of both FMRP and FMR1 mRNA to symptoms of autism in this cohort and found no significant relationship between the measures of autism and molecular features, including FMRP, FMR1 mRNA, and CGG repeat number.

scholarly journals The Contribution of Pluripotent Stem Cell (PSC)-Based Models to the Study of Fragile X Syndrome (FXS)

2019 ◽  
Vol 9 (2) ◽  
pp. 42 ◽  
Author(s):  
Manar Abu Diab ◽  
Rachel Eiges
Keyword(s):  
Stem Cells ◽  
Gene Silencing ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Embryonic Stem ◽  
Cgg Repeat ◽  
Molecular Features ◽  
Epigenetic Gene Silencing ◽  
Mental Retardation Protein ◽  
Induced Pluripotent

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a deficiency in the fragile X mental retardation protein (FMRP) due to a CGG repeat expansion in the 5′-UTR of the X-linked FMR1 gene. When CGGs expand beyond 200 copies, they lead to epigenetic gene silencing of the gene. In addition, the greater the allele size, the more likely it will become unstable and exhibit mosaicism for expansion size between and within tissues in affected individuals. The timing and mechanisms of FMR1 epigenetic gene silencing and repeat instability are far from being understood given the lack of appropriate cellular and animal models that can fully recapitulate the molecular features characteristic of the disease pathogenesis in humans. This review summarizes the data collected to date from mutant human embryonic stem cells, induced pluripotent stem cells, and hybrid fusions, and discusses their contribution to the investigation of FXS, their key limitations, and future prospects.

scholarly journals Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 356 ◽  
Author(s):  
Daman Kumari ◽  
Nicholas Sciascia ◽  
Karen Usdin
Keyword(s):  
Dna Methylation ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Fmr1 Gene ◽  
Mrna Levels ◽  
H3k9 Methylation ◽  
Methyl Transferase ◽  
Cgg Repeat ◽  
Heterochromatin Formation ◽  
Gene Reactivation

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.

scholarly journals The Feasibility and Utility of Hair Follicle Sampling To Measure FMRP and FMR1 mRNA in Children With or Without Fragile X Syndrome

2021 ◽  
Author(s):  
Isha Jalnapurkar ◽  
Jean A. Frazier ◽  
Mark Roth ◽  
David M. Cochran ◽  
Ann Foley ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Hair Follicle ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Hair Follicles ◽  
Buccal Swab ◽  
Typically Developing ◽  
Cgg Repeat ◽  
Fragile X Mental Retardation ◽  
Fmr1 Mrna

Abstract Background: Fragile X syndrome (FXS) is the most common cause inherited cause of intellectual disability in males and the most common single gene cause of autism. This X-linked disorder is caused by an expansion of a trinucleotide CGG repeat (>200 base pairs) on the promotor region of the fragile X mental retardation 1 gene (FMR1). This leads to the deficiency or absence of the encoded protein, Fragile X mental retardation protein (FMRP). FMRP has a central role in the translation of mRNAs involved in synaptic connections and plasticity. Recent studies have demonstrated the benefit of therapeutics focused on reactivation of the FMR1 locus towards improving key clinical phenotypes via restoration of FMRP and ultimately disease modification. A key step in future studies directed towards this effort is the establishment of proof of concept (POC) for FMRP reactivation in individuals with FXS. For this it is key to determine the feasibility of repeated collection of tissues or fluids to measure FMR1 and FMRP. Methods: Individuals, ages 3 to 22 years of age, with FXS and those who were typically developing participated in this single-site pilot clinical biomarker study. The repeated collection of hair follicles was compared with the collection of blood and buccal swabs for detection of FMR1 mRNA and FMRP and related molecules. Results: There were n = 15 participants, of whom 10 had a diagnosis of FXS (7.0 ± 3.56 years) and 5 were typically developing (8.2 ± 2.77 years). Absolute levels of FMRP and FMR1 mRNA were substantially higher in healthy participants compared to full mutation and mosaic FXS participants, and lowest in the FXS boys. Measurement of FMR1 and FMRP levels by any method did not show any notable variation by collection location at home versus office across the various sample collection methodologies of hair follicle, blood sample, and buccal swab. Conclusion: Findings demonstrated that repeated sampling of hair follicles in individuals with FXS, in both, home and office settings, is feasible, repeatable, and can be used for measurement of FMR1 and FMRP in longitudinal studies.

Learning-disabled Males With a Fragile X CGG Expansion in the Upper Premutation Size Range

PEDIATRICS ◽  
1996 ◽  
Vol 97 (1) ◽  
pp. 122-126
Author(s):  
Randi J. Hagerman ◽  
Louise W. Staley ◽  
Rebecca O'Conner ◽  
Kellie Lugenbeel ◽  
David Nelson ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Fragile X Syndrome ◽  
Size Range ◽  
Cpg Island ◽  
Fragile X ◽  
Learning Disabled ◽  
Full Mutation ◽  
Cgg Repeat ◽  
Responsible Gene ◽  
Repeat Segment

There is a broad spectrum of clinical involvement in both boys and girls affected by fragile X syndrome. Although this disorder is best known as the most common inherited cause of mental retardation, it also can manifest as learning disabilities in individuals with IQs in the broad range of normal. Boys are usually retarded, and girls are usually learning disabled with fragile X syndrome.1 The responsible gene, fragile X mental retardation 1 (FMR1), was isolated in 1991, and the mutation was found to involve expansion of a trinucleotide (CGG) repeat segment. Individuals with fragile X syndrome have a CGG expansion of more than 200 repeats associated with hypermethylation of both the expansion and an adjacent CpG island (full mutation).2,3

scholarly journals Integrative analysis identifies key molecular signatures underlying neurodevelopmental deficits in fragile X syndrome

2019 ◽  
Author(s):  
Kagistia Hana Utami ◽  
Niels H. Skotte ◽  
Ana R. Colaço ◽  
Nur Amirah Binte Mohammad Yusof ◽  
Bernice Sim ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Fragile X ◽  
Neurodevelopmental Disorder ◽  
Embryonic Stem ◽  
Neural Progenitor Cell ◽  
Integrative Analysis ◽  
Network Activity ◽  
Neural Cells ◽  
Molecular Signatures ◽  
Neurodevelopmental Abnormalities

AbstractFragile X syndrome (FXS) is an incurable neurodevelopmental disorder with no effective treatment. FXS is caused by epigenetic silencing ofFMR1and loss of FMRP expression. To investigate the consequences of FMRP deficiency in the context of human physiology, we established isogenicFMR1knockout (FMR1KO) human embryonic stem cells (hESCs). Integrative analysis of the transcriptomic and proteomic profiles of hESC-derived FMRP-deficient neurons revealed several dysregulated pathways important for brain development including processes related to axon development, neurotransmission, and the cell cycle. We functionally validated alterations in a number of these pathways, showing abnormal neural rosette formation and increased neural progenitor cell proliferation inFMR1KO cells. We further demonstrated neurite outgrowth and branching deficits along with impaired electrophysiological network activity in FMRP-deficient neurons. Using isogenicFMR1KO hESC-derived neurons, we reveal key molecular signatures and neurodevelopmental abnormalities arising from loss of FMRP. We anticipate that theFMR1KO hESCs and the neuronal transcriptome and proteome datasets will provide a platform to delineate the pathophysiology of FXS in human neural cells.

scholarly journals FMRP Levels in Human Peripheral Blood Leukocytes Correlates with Intellectual Disability

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1780
Author(s):  
Mark Roth ◽  
Lucienne Ronco ◽  
Diego Cadavid ◽  
Blythe Durbin-Johnson ◽  
Randi J. Hagerman ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Peripheral Blood ◽  
Fragile X ◽  
Repeat Expansion ◽  
Repeat Number ◽  
Cgg Repeat ◽  
Fragile X Mental Retardation ◽  
Cgg Repeats ◽  
Fmr1 Mrna

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. FXS is an X-linked, neurodevelopmental disorder caused by a CGG trinucleotide repeat expansion in the 5′ untranslated region (UTR) of the Fragile X Mental Retardation gene, FMR1. Greater than 200 CGG repeats results in epigenetic silencing of the gene leading to the deficiency or absence of Fragile X mental retardation protein (FMRP). The loss of FMRP is considered the root cause of FXS. The relationship between neurological function and FMRP expression in peripheral blood mononuclear cells (PBMCs) has not been well established. Assays to detect and measure FMR1 and FMRP have been described; however, none are sufficiently sensitive, precise, or quantitative to properly characterize the relationships between cognitive ability and CGG repeat number, FMR1 mRNA expression, or FMRP expression measured in PBMCs. To address these limitations, two novel immunoassays were developed and optimized, an electro-chemiluminescence immunoassay and a multiparameter flow cytometry assay. Both assays were performed on PMBCs isolated from 27 study participants with FMR1 CGG repeats ranging from normal to full mutation. After correcting for methylation, a significant positive correlation between CGG repeat number and FMR1 mRNA expression levels and a significant negative correlation between FMRP levels and CGG repeat expansion was observed. Importantly, a high positive correlation was observed between intellectual quotient (IQ) and FMRP expression measured in PBMCs.

scholarly journals Cascade Testing for Fragile X Syndrome in a Rural Setting in Cameroon (Sub-Saharan Africa)

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 136
Author(s):  
Karen Kengne Kamga ◽  
Séraphin Nguefack ◽  
Khuthala Minka ◽  
Edmond Wonkam Tingang ◽  
Alina Esterhuizen ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Fragile X ◽  
Autism Spectrum ◽  
Sub Saharan Africa ◽  
Rural Setting ◽  
Premature Menopause ◽  
Molecular Testing ◽  
Full Mutation ◽  
Cgg Repeat ◽  
Cgg Repeats

Fragile X Syndrome (FXS), an X-linked dominant monogenic condition, is the main genetic cause of intellectual disability (ID) and autism spectrum disorder (ASD). FXS is associated with an expansion of CGG repeat sequence in the Fragile X Mental Retardation gene 1 (FMR1) on chromosome X. Following a neuropediatric assessment of two male siblings who presented with signs of FXS that was confirmed with molecular testing, we provided cascade counselling and testing to the extended family. A total of 46 individuals were tested for FXS; among them, 58.70% (n = 27) were females. The mean age was 9.4 (±5) years for children and 45.9 (±15.9) years for adults. Pedigree analysis suggested that the founder of these families was likely a normal transmitting male. Four out of 19 males with clinical ID were confirmed to have a full mutation for FXS, while 14/27 females had a pathologic CGG expansion (>56 CGG repeats) on one of their X chromosomes. Two women with premature menopause were confirmed of being carriers of premutation (91 and 101 CGG repeats). We also identified maternal alleles (91 and 126 CGG repeats) which expanded to a full mutation in their offspring (>200 CGG repeats). This study is a rare report on FXS from Africa and illustrates the case scenario of implementing genetic medicine for a neurogenetic condition in a rural setting.

scholarly journals Fragile X syndrome carrier screening in pregnant women in Chinese Han population

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chia-Cheng Hung ◽  
Chien-Nan Lee ◽  
Yu-Chu Wang ◽  
Chih-Ling Chen ◽  
Tze-Kang Lin ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Fragile X Syndrome ◽  
Chinese Women ◽  
Fragile X ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Carrier Screening ◽  
Chinese Han ◽  
Cgg Repeat ◽  
Taiwanese Women

Abstract Fragile X syndrome (FXS) is the most frequent genetic cause of intellectual disability (ID). It was previously believed that the FXS prevalence was low in Chinese population, and the cost-efficiency of FXS carrier screening was questioned. This retrospective observational study was conducted between September 2014 and May 2017 to determine the prevalence of FXS carriers in a large Chinese cohort of pregnant women. The FMR1 CGG repeat status was determined in 20,188 pregnant Taiwanese women and we identified 26 women with premutation (PM). The PM allele was transmitted to the fetus in 17 pregnancies (56.6%), and six of 17 expanded to full mutation (FM). One asymptomatic woman had a FM allele with 280 CGG repeats. Prenatal genetic diagnosis of her first fetus revealed a male carrying a FMR1 gene deletion of 5′ UTR and exon 1. Her second fetus was a female carrying a FM allele as well. This is so far the largest study of the FXS carrier screening in Chinese women. The prevalence of premutation allele for FXS in normal asymptomatic Taiwanese women was found to be as high as 0.13% (1 in 777) in this study. The empirical evidence suggests that reproductive FXS carrier screening in Taiwan might be cost-effective.

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