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

2021 ◽  
Author(s):  
Anna E Boggs ◽  
Lauren M Schmitt ◽  
Richard D McLane ◽  
Tatyana Adayev ◽  
Giuseppe LaFauci ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Clinical Relevance ◽  
Fragile X ◽  
Dried Blood Spots ◽  
Full Mutation ◽  
X Protein ◽  
Blood Spots ◽  
Fragile X Mental Retardation ◽  
Iq Scores

Abstract 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.

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

2021 ◽  
Author(s):  
Anna Boggs ◽  
Lauren Schmitt ◽  
Richard McLane ◽  
Tatyana Adayev ◽  
Giuseppe LaFauci ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Clinical Relevance ◽  
Fragile X ◽  
Dried Blood Spots ◽  
Full Mutation ◽  
X Protein ◽  
Blood Spots ◽  
Fragile X Mental Retardation ◽  
Iq Scores

Abstract 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.

scholarly journals A Single Common Assay for Robust and Rapid Fragile X Mental Retardation Syndrome Screening From Dried Blood Spots

2018 ◽  
Vol 9 ◽  
Author(s):  
Vivienne J. Tan ◽  
Mulias Lian ◽  
Sultana M.H. Faradz ◽  
Tri I. Winarni ◽  
Samuel S. Chong
Keyword(s):  
Mental Retardation ◽  
Fragile X ◽  
Dried Blood Spots ◽  
Blood Spots ◽  
Fragile X Mental Retardation

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 Comparative Behavioral Phenotypes of Fmr1 KO, Fxr2 Het, and Fmr1 KO/Fxr2 Het Mice

2019 ◽  
Vol 9 (1) ◽  
pp. 13 ◽  
Author(s):  
Rachel Saré ◽  
Christopher Figueroa ◽  
Abigail Lemons ◽  
Inna Loutaev ◽  
Carolyn Beebe Smith
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Fragile X ◽  
Protein Product ◽  
Behavioral Phenotypes ◽  
Memory Impairments ◽  
Fragile X Mental Retardation ◽  
Behavioral Abnormalities ◽  
Mental Retardation Protein ◽  
Functional Consequences

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene leading to loss of the protein product fragile X mental retardation protein (FMRP). FXS is the most common monogenic cause of intellectual disability. There are two known mammalian paralogs of FMRP, FXR1P, and FXR2P. The functions of FXR1P and FXR2P and their possible roles in producing or modulating the phenotype observed in FXS are yet to be identified. Previous studies have revealed that mice lacking Fxr2 display similar behavioral abnormalities as Fmr1 knockout (KO) mice. In this study, we expand upon the behavioral phenotypes of Fmr1 KO and Fxr2+/− (Het) mice and compare them with Fmr1 KO/Fxr2 Het mice. We find that Fmr1 KO and Fmr1 KO/Fxr2 Het mice are similarly hyperactive compared to WT and Fxr2 Het mice. Fmr1 KO/Fxr2 Het mice have more severe learning and memory impairments than Fmr1 KO mice. Fmr1 KO mice display significantly impaired social behaviors compared to WT mice, which are paradoxically reversed in Fmr1 KO/Fxr2 Het mice. These results highlight the important functional consequences of loss or reduction of FMRP and FXR2P.

scholarly journals New Targeted Treatments for Fragile X Syndrome

2019 ◽  
Vol 15 (4) ◽  
pp. 251-258 ◽  
Author(s):  
Dragana Protic ◽  
Maria J. Salcedo-Arellano ◽  
Jeanne Barbara Dy ◽  
Laura A. Potter ◽  
Randi J. Hagerman
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Fragile X Syndrome ◽  
Behavioral Problems ◽  
Rna Binding ◽  
Fragile X ◽  
Symptomatic Treatment ◽  
Fmr1 Gene ◽  
Fragile X Mental Retardation ◽  
Cgg Repeats

Fragile X Syndrome (FXS) is the most common cause of inherited intellectual disability with prevalence rates estimated to be 1:5,000 in males and 1:8,000 in females. The increase of >200 Cytosine Guanine Guanine (CGG) repeats in the 5’ untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene results in transcriptional silencing on the FMR1 gene with a subsequent reduction or absence of fragile X mental retardation protein (FMRP), an RNA binding protein involved in the maturation and elimination of synapses. In addition to intellectual disability, common features of FXS are behavioral problems, autism, language deficits and atypical physical features. There are still no currently approved curative therapies for FXS, and clinical management continues to focus on symptomatic treatment of comorbid behaviors and psychiatric problems. Here we discuss several treatments that target the neurobiological pathway abnormal in FXS. These medications are clinically available at present and the data suggest that these medications can be helpful for those with FXS.

Mosaicism in Fragile X syndrome: A family case series

2021 ◽  
pp. 174462952199534
Author(s):  
Wilmar Saldarriaga ◽  
Laura Yuriko González-Teshima ◽  
Jose Vicente Forero-Forero ◽  
Hiu-Tung Tang ◽  
Flora Tassone
Keyword(s):  
Mental Retardation ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Fmr1 Gene ◽  
Molecular Testing ◽  
Full Mutation ◽  
Mild Phenotype ◽  
Variable Expression ◽  
Fragile X Mental Retardation ◽  
Classic Phenotype

Fragile X syndrome (FXS) has a classic phenotype, however its expression can be variable among full mutation males. This is secondary to variable methylation mosaicisms and the number of CGG triplet repeats in the non-coding region of the Fragile X Mental Retardation 1 ( FMR1) gene, producing a variable expression of the Fragile X Mental Retardation Protein (FMRP). Here we report a family with several individuals affected by FXS: a boy with a hypermethylated FMR1 mutation and a classic phenotype; a man with an FMR1 gene mosaicism in the range of premutation (PM) and full mutation (FM), who has a mild phenotype due to which FXS was initially disregarded; and the cases of four women with a FM and mosaicism. This report highlights the importance of DNA molecular testing for the diagnosis of FXS in patients with developmental delay, intellectual disability and/or autism due to the variable phenotype that occurs in individuals with FMR1 mosaicisms.

scholarly journals Fragile X mental retardation protein interactions with a G quadruplex structure in the 3′-untranslated region of NR2B mRNA

2015 ◽  
Vol 11 (12) ◽  
pp. 3222-3230 ◽  
Author(s):  
Snezana Stefanovic ◽  
Brett A. DeMarco ◽  
Ayana Underwood ◽  
Kathryn R. Williams ◽  
Gary J. Bassell ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Protein Interactions ◽  
Untranslated Region ◽  
Fragile X ◽  
Common Cause ◽  
Fragile X Mental Retardation ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Mental Retardation Protein

Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP).

scholarly journals Fragile X mental retardation protein is a size-dependent translational activator

2018 ◽  
Author(s):  
Ethan J. Greenblatt ◽  
Allan C. Spradling
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Fragile X ◽  
Ovarian Follicle ◽  
Autism Spectrum ◽  
Ribosome Profiling ◽  
Large Proteins ◽  
Fragile X Mental Retardation ◽  
Stored Mrna ◽  
Neural Dysfunction

SummaryFMR1 enhances translation of large neural/oocyte proteinsMutations in the highly conserved Fragile X mental retardation gene (Fmr1) cause the most common inherited human intellectual disability/autism spectrum disorder. Fmr1 is also needed for ovarian follicle development, and lesions are the largest genetic cause of premature ovarian failure (POF). FMR1 associates with ribosomes and is thought to repress translation, but identifying functional targets has been difficult. We analyzed FMR1’s role in quiescent Drosophila oocytes stored prior to ovulation, cells that depend entirely on translation of stored mRNA. Ribosome profiling revealed that in quiescent oocytes FMR1 stimulates the translation of large proteins, including at least twelve proteins whose human homologs are associated with dominant intellectual disability disorders, and 25 others associated with neural dysfunction. Knockdown of Fmr1 in unstored oocytes did not affect embryo development, but more than 50% of embryos derived from stored oocytes lacking FMR1 developed severe neural defects. Fmr1’s previously unappreciated role promoting the translation of large proteins from stored mRNAs in oocytes and neurons may underlie POF as well as multiple aspects of neural dysfunction.

scholarly journals Fragile X Syndrome

2014 ◽  
pp. 190-198 ◽  
Author(s):  
Wilmar Saldarriaga ◽  
Flora Tassone ◽  
Laura Yuriko González-Teshima ◽  
Jose Vicente Forero-Forero ◽  
Sebastián Ayala-Zapata ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Intellectual Disability ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Gene Promoter ◽  
Molecular Techniques ◽  
Pharmaceutical Management ◽  
Developmental Problems ◽  
Fragile X Mental Retardation ◽  
Cgg Repeats

Fragile X Syndrome (FXS) is a genetic disease due to a CGG trinucleotide expansion, named full mutation (greater than 200 CGG repeats), in the fragile X mental retardation 1 gene locus Xq27.3; which leads to an hypermethylated region in the gene promoter therefore silencing it and lowering the expression levels of the fragile X mental retardation 1, a protein involved in synaptic plasticity and maturation. Individuals with FXS present with intellectual disability, autism, hyperactivity, long face, large or prominent ears and macroorchidism at puberty and thereafter. Most of the young children with FXS will present with language delay, sensory hyper arousal and anxiety. Girls are less affected than boys, only 25% have intellectual disability. Given the genomic features of the syndrome, there are patients with a number of triplet repeats between 55 and 200, known as premutation carriers. Most carriers have a normal IQ but some have developmental problems. The diagnosis of FXS has evolved from karyotype with special culture medium, to molecular techniques that are more sensitive and specific including PCR and Southern Blot. During the last decade, the advances in the knowledge of FXS, has led to the development of investigations on pharmaceutical management or targeted treatments for FXS. Minocycline and sertraline have shown efficacy in children.

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