Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers

Gustin, Stephanie; Wang, Guangwen; Baker, Valerie M; Latham, Gary J.; Sebastiano, Vittorio

doi:10.1007/s10815-018-1237-y

Cited by 2 publications

(13 citation statements)

References 41 publications

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“…Each sample was run at two input concentrations of 31 ng/ul and 6.25 ng/ul, respectively. FMR1 mRNA expression was quanti ed relative to expression of the control housekeeping gene USP33 [42].…”

Section: Southern Blot Analysismentioning

confidence: 99%

“…Quantitative FMRP analysis FMRP levels were determined using a quantitative FMRP (qFMRP) assay developed by Lafauci et al [43], per the method described by Gustin et al [42]. This assay utilizes a recombinant FMRP peptide (GST-SR7) as a standard for quanti cation of FMRP level.…”

Section: Southern Blot Analysismentioning

confidence: 99%

“…Recently, a series of FMR1-based molecular methods quantifying genetic and epigenetic features of the mutation with greater sensitivity, speci city, and breadth have been reported [20,21,[39][40][41]. These FMR1 assays can be applied across different specimen types, such as blood and buccal epithelium, and combined with FMRP measurements [42,43]. Speci cally, advanced PCR-based methods can reliably amplify small and large FMR1 repeat expansions, detect low-level repeat size mosaicism, determine the number and sequence context of sequences that interrupt the repeat tract, quantify X-inactivation, and measure skewed FMR1 gene silencing that was masked using less sensitive techniques such as Southern blotting [11,17,20,21,39,[44][45][46].…”

Section: Introductionmentioning

confidence: 99%

“…Speci cally, advanced PCR-based methods can reliably amplify small and large FMR1 repeat expansions, detect low-level repeat size mosaicism, determine the number and sequence context of sequences that interrupt the repeat tract, quantify X-inactivation, and measure skewed FMR1 gene silencing that was masked using less sensitive techniques such as Southern blotting [11,17,20,21,39,[44][45][46]. In addition, more precise FMRP assays allow a better delineation of the range of FMR1 protein levels and, consequently, more accurate FMR1-FMRP correlations [42,43,47]. This integrated approach was recently used to explore repeat instability in a reprogrammed stem cell model of fragile X-associated primary ovarian insu ciency (FXPOI) patients [42].…”

Section: Introductionmentioning

confidence: 99%

“…In addition, more precise FMRP assays allow a better delineation of the range of FMR1 protein levels and, consequently, more accurate FMR1-FMRP correlations [42,43,47]. This integrated approach was recently used to explore repeat instability in a reprogrammed stem cell model of fragile X-associated primary ovarian insu ciency (FXPOI) patients [42].…”

Section: Introductionmentioning

confidence: 99%

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A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients With Neurobehavioral Assessments

Budimirović

Schlageter

Sadic-Filipovic

et al. 2020

Preprint

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Background. Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying FMR1 silencing, CGG repeat expansion, is well characterized; however, delineation of the pathway from DNA to RNA to protein using biosamples from well characterized patients with FXS is limited. Since FXS is a common and prototypical genetic disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD), a comprehensive assessment of the FMR1 DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority.Methods. We applied nine sensitive and quantitative assays evaluating FMR1 DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of FMR1 expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different FMR1 expansions, with emphasis on those with FXS (N = 42 total, N = 31 with FMRP measurements). The group with FMRP data was also evaluated comprehensively in terms of its neurobehavioral profile, which allowed molecular-neurobehavioral correlations. Results. FMR1 CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with previous FMR1 genomic and protein studies. They also demonstrated a high level of agreement between blood and buccal specimens. These assays further corroborated previous reports of the relatively high prevalence of methylation mosaicism. Molecular-neurobehavioral correlations confirmed the inverse relationship between overall severity of the FXS phenotype and decrease in FMRP levels. Other intriguing findings included a potential relationship between diagnosis of FXS with ASD and very low levels of FMRP, compared to FXS without ASD. Conclusions. The results underscore the link between FMR1 expansion, gene methylation, and FMRP deficit. The association between FMRP deficiency and overall severity of the neurobehavioral phenotype invites follow up studies in larger patient cohorts. They would be valuable to confirm and potentially extend our initial findings of a relationship between ASD and other neurobehavioral features and the magnitude of FMRP deficit. Molecular profiling of individuals with FXS may have important implications in research and clinical practice.

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Section: Southern Blot Analysismentioning

confidence: 99%

Section: Southern Blot Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients With Neurobehavioral Assessments

Budimirović

Schlageter

Sadic-Filipovic

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients with Neurobehavioral Assessments

et al. 2020

View full text Add to dashboard Cite

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying FMR1 silencing, CGG repeat expansion, is well characterized; however, delineation of the pathway from DNA to RNA to protein using biosamples from well characterized patients with FXS is limited. Since FXS is a common and prototypical genetic disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD), a comprehensive assessment of the FMR1 DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority. We applied nine sensitive and quantitative assays evaluating FMR1 DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of FMR1 expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different FMR1 expansions, with emphasis on those with FXS (N = 42 total, N = 31 with FMRP measurements). The group with FMRP data was also evaluated comprehensively in terms of its neurobehavioral profile, which allowed molecular–neurobehavioral correlations. FMR1 CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with findings of previous FMR1 genomic and protein studies. They also demonstrated a high level of agreement between blood and buccal specimens. These assays further corroborated previous reports of the relatively high prevalence of methylation mosaicism (slightly over 50% of the samples). Molecular-neurobehavioral correlations confirmed the inverse relationship between overall severity of the FXS phenotype and decrease in FMRP levels (N = 26 males, mean 4.2 ± 3.3 pg FMRP/ng genomic DNA). Other intriguing findings included a significant relationship between the diagnosis of FXS with ASD and two-fold lower levels of FMRP (mean 2.8 ± 1.3 pg FMRP/ng genomic DNA, p = 0.04), in particular observed in younger age- and IQ-adjusted males (mean age 6.9 ± 0.9 years with mean 3.2 ± 1.2 pg FMRP/ng genomic DNA, 57% with severe ASD), compared to FXS without ASD. Those with severe ID had even lower FMRP levels independent of ASD status in the male-only subset. The results underscore the link between FMR1 expansion, gene methylation, and FMRP deficit. The association between FMRP deficiency and overall severity of the neurobehavioral phenotype invites follow up studies in larger patient cohorts. They would be valuable to confirm and potentially extend our initial findings of the relationship between ASD and other neurobehavioral features and the magnitude of FMRP deficit. Molecular profiling of individuals with FXS may have important implications in research and clinical practice.

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Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers

Abstract: This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.

Cited by 2 publications

References 41 publications

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients With Neurobehavioral Assessments

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients With Neurobehavioral Assessments

A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients with Neurobehavioral Assessments

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