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

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.

Experiences of the Molecular Diagnosis of Fragile X Syndrome in Ecuador

Fragile X syndrome (FXS) is the most common cause of hereditary intellectual disability and the second most common cause of intellectual disability of genetic etiology. This complex neurodevelopmental disorder is caused by an alteration in the CGG trinucleotide expansion in fragile X mental retardation gene 1 (FMR1) leading to gene silencing and the subsequent loss of its product: fragile X mental retardation protein 1 (FMRP). Molecular diagnosis is based on polymerase chain reaction (PCR) screening followed by Southern blotting (SB) or Triplet primer-PCR (TP-PCR) to determine the number of CGG repeats in the FMR1 gene. We performed, for the first time, screening in 247 Ecuadorian male individuals with clinical criteria to discard FXS. Analysis was carried out by the Genetics Service of the Hospital de Especialidades No. 1 de las Fuerzas Armadas (HE-1), Ecuador. The analysis was performed using endpoint PCR for CGG fragment expansion analysis of the FMR1 gene. Twenty-two affected males were identified as potentially carrying the full mutation in FMR1 and thus diagnosed with FXS that is 8.1% of the sample studied. The average age at diagnosis of the positive cases was 13 years of age, with most cases from the geographical area of Pichincha (63.63%). We confirmed the familial nature of the disease in four cases. The range of CGG variation in the population was 12–43 and followed a modal distribution of 27 repeats. Our results were similar to those reported in the literature; however, since it was not possible to differentiate between premutation and mutation cases, we can only establish a molecular screening approach to identify an expanded CGG repeat, which makes it necessary to generate national strategies to optimize molecular tests and establish proper protocols for the diagnosis, management, and follow-up of patients, families, and communities at risk of presenting FXS.

Altered expression of fragile X mental retardation-1 (FMR1) in the thymus in autoimmune myasthenia gravis

Predisposition to autoimmunity and inflammatory disorders is observed in patients with fragile X-associated syndromes. These patients have increased numbers of CGG triplets in the 5’ UTR region of FMR1 (Fragile X Mental Retardation 1) gene, that affects its expression. FMR1 is decreased in the thymus of myasthenia gravis (MG) patients, a prototypical autoimmune disease. We thus analyzed the number of CGG triplets in FMR1 in MG, and explored the regulatory mechanisms affecting thymic FMR1 expression. We measured the number of CGGs using thymic DNA from MG and controls, but no abnormalities in CGGs were found in MG that could explain thymic decrease of FMR1. We next analyzed by RT-PCR the expression of FMR1 and its transcription factors in thymic samples, and in thymic epithelial cell cultures in response to inflammatory stimuli. In control thymuses, FMR1 expression was higher in males than females, and correlated with CTCF (CCCTC-binding factor) expression. In MG thymuses, decreased expression of FMR1 was correlated with both CTCF and MAX (Myc-associated factor X) expression. Changes in FMR1 expression were supported by western blot analyses for FMRP. In addition, we demonstrated that FMR1, CTCF and MAX expression in thymic epithelial cells was also sensitive to inflammatory signals. Our results suggest that FMR1 could play a central role in the thymus and autoimmunity. First, in relation with the higher susceptibility of females to autoimmune diseases. Second, due to the modulation of its expression by inflammatory signals that are known to be altered in MG thymuses.

FMRP Levels in Human Peripheral Blood Leukocytes Correlates with Intellectual Disability

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.

Development of a Quantitative FMRP Assay for Mouse Tissue Applications

Fragile X syndrome results from the absence of the FMR1 gene product—Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Optimization, Validation and Initial Clinical Implications of a Luminex-based Immunoassay for the Quantification of Fragile X Protein from Dried Blood Spots

Background: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability affecting 1 in 4,000 males and 1 in 6-8,000 females. FXS is caused by a trinucleotide expansion in the 5’UTR of the Fragile X Mental Retardation (FMR1) gene which in full mutation carriers (>200 repeats) leads to hypermethylation and transcriptional silencing of the gene and lack of expression of Fragile X Protein (FXP, formerly known as Fragile X Mental Retardation Protein, FMRP). Phenotypic presentation of FXS is highly variable, and molecular markers explaining or predicting this variability are lacking. Recent studies suggest that trace amounts of FXP can be detected even in fully methylated individuals and may have clinical relevance; however, the lack of available reproducible, sensitive assays to detect FXP in peripheral tissue makes evaluation of peripheral FXP as a source of clinical variability challenging. Methods: We optimized a Luminex-based assay to detect FXP in dried blot spots for increased reproducibility and sensitivity by improving reagent concentrations and buffer conditions. The optimized assay was used to quantify FXP in 187 individuals (101 males, 86 females; 0-78.4 years) including 35 typically developing controls (24 males, 11 females), 103 individuals carrying full mutations (70 males, 33 females), and 49 individuals with premutations (7 males, 42 females). A subset of these individuals showed repeat number or methylation mosaicism. We investigated the clinical relevance of peripheral FXP levels by examining its relationship with general intellectual functioning in a subset of individuals with available IQ scores. Results: We show that the optimized assay is highly reproducible and detects a wide range of FXP levels. Mosaic individuals had, on average, higher FXP levels than fully methylated individuals, and trace amounts of FXP were consistently detectable in a subset of individuals with full mutation FXS. IQ scores were positively correlated with peripheral FXP levels in male and female individuals with full mutation FXS. Conclusions: We demonstrate that our optimized Luminex-based assay to detect FXP is reproducible, highly sensitive, and related to the core intellectual disability phenotype. Further, our data suggest that trace amounts of FXP detectable in dried blood spots of individuals with FXS could be clinically relevant and may be used to stratify individuals with FXS for optimized treatment. Future studies are needed with larger sample sizes, evaluating FXP across development and expanded analysis of the relevance of FXP levels for behavioral and electrophysiological phenotypes in FXS.

Optimization, validation and initial clinical implications of a Luminex-based immunoassay for the quantification of Fragile X Protein from Dried Blood Spots

BackgroundFragile X syndrome (FXS) is the most common inherited form of intellectual disability affecting 1 in 4,000 males and 1 in 6-8,000 females. FXS is caused by a trinucleotide expansion in the 5’UTR of the Fragile X Mental Retardation (FMR1) gene which in full mutation carriers (>200 repeats) leads to hypermethylation and transcriptional silencing of the gene and lack of expression of Fragile X Protein (FXP, formerly known as Fragile X Mental Retardation Protein, FMRP). Phenotypic presentation of FXS is highly variable, and molecular markers explaining or predicting this variability are lacking. Recent studies suggest that trace amounts of FXP can be detected even in fully methylated individuals and may have clinical relevance; however, the lack of available reproducible, sensitive assays to detect FXP in peripheral tissue makes evaluation of peripheral FXP as a source of clinical variability challenging. MethodsWe optimized a Luminex-based assay to detect FXP in dried blot spots for increased reproducibility and sensitivity by improving reagent concentrations and buffer conditions. The optimized assay was used to quantify FXP in 187 individuals (101 males, 86 females; 0-78.4 years) including 35 typically developing controls (24 males, 11 females), 103 individuals carrying full mutations (70 males, 33 females), and 49 individuals with premutations (7 males, 42 females). A subset of these individuals showed repeat number or methylation mosaicism. We investigated the clinical relevance of peripheral FXP levels by examining its relationship with general intellectual functioning in a subset of individuals with available IQ scores. ResultsWe show that the optimized assay is highly reproducible and detects a wide range of FXP levels. Mosaic individuals had, on average, higher FXP levels than fully methylated individuals, and trace amounts of FXP were consistently detectable in a subset of individuals with full mutation FXS. IQ scores were positively correlated with peripheral FXP levels in male and female individuals with full mutation FXS. ConclusionsWe demonstrate that our optimized Luminex-based assay to detect FXP is reproducible, highly sensitive, and related to the core intellectual disability phenotype. Further, our data suggest that trace amounts of FXP detectable in dried blood spots of individuals with FXS could be clinically relevant and may be used to stratify individuals with FXS for optimized treatment. Future studies are needed with larger sample sizes, evaluating FXP across development and expanded analysis of the relevance of FXP levels for behavioral and electrophysiological phenotypes in FXS.

In vivo imaging of mGlu5 receptor expression in humans with Fragile X Syndrome towards development of a potential biomarker

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by silencing of the Fragile X Mental Retardation (FMR1) gene. The resulting loss of Fragile X Mental Retardation Protein (FMRP) leads to excessive glutamate signaling via metabotropic glutamate subtype 5 receptors (mGluR5) which has been implicated in the pathogenesis of the disorder. In the present study we used the radioligand 3-[18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB) in simultaneous PET-MR imaging of males with FXS and age- and gender-matched controls to assess the availability of mGlu5 receptors in relevant brain areas. Patients with FXS showed lower [18F]FPEB binding potential (p < 0.01), reflecting reduced mGluR5 availability, than the healthy controls throughout the brain, with significant group differences in insula, anterior cingulate, parahippocampal, inferior temporal and olfactory cortices, regions associated with deficits in inhibition, memory, and visuospatial processes characteristic of the disorder. The results are among the first to provide in vivo evidence of decreased availability of mGluR5 in the brain in individuals with FXS than in healthy controls. The consistent results across the subjects, despite the tremendous challenges with neuroimaging this population, highlight the robustness of the protocol and support for its use in drug occupancy studies; extending our radiotracer development and application efforts from mice to humans.