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

Abstract

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.

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

“…Previous methods of quantifying FXP have had low signal to noise ratios that increase at the lower limit of quantification and therefore mask potential low level protein expression (19). Although these previous methods have shown initial clinical relevance by documenting relationships with general cognitive ability, due to the restricted range of FXP expression captured by these methods, the full scope of its clinical utility is limited (19).…”

“…Previous methods of quantifying FXP have had low signal to noise ratios that increase at the lower limit of quantification and therefore mask potential low level protein expression (19). Although these previous methods have shown initial clinical relevance by documenting relationships with general cognitive ability, due to the restricted range of FXP expression captured by these methods, the full scope of its clinical utility is limited (19). In the current study, using several key assay optimization techniques, including standard curve and reagent optimization, we have developed a reliable and reproducible means to quantify FXP in blood using blood spot cards.…”

“…50 µL of blood was pipetted onto ID Bloodstain Cards (Whatman Bloodstain Cards, WB100014) producing at least two cards with 13 spots each from one sample collection. Cards were dried and stored with desiccant packs in lowgas-permeable bags (VWR, 89027-022) within 4-24 hours after spotting to ensure DBS stability, in accordance with dried blood spot (DBS) guidelines and published protocols (18,19) (Figure 1).…”

“…Moreover, this assay can be used with eluates from dried blood spots, which facilitates the potential future application of this assay outside the research laboratory and into clinical settings. The FXP Luminex assay was used in a recent study to measure FXP levels in 42 samples of individuals with FXS and demonstrated that males with severe ID had lower FXP than males with mild or moderate ID (19). However, the lower limit of FXP detection was above zero indicating difficulty differentiating "true zero" FXP expression from potential low or trace level FXP levels, thus also limiting its capacity to be more clinically relevant among males with full mutation.…”

Response to Placebo in Fragile X Syndrome Clinical Trials: An Initial Analysis

“…These tests show good test–retest reproducibility in ID populations, including FXS, covering a broad range of function; tests such as expressive language sampling (ELS) [ 19 ] and NIH Toolbox [ 20 ], that are expected to be sensitive to short-term change, are in development for trials in FXS [ 8 , 9 ]. Recent advances in molecular-phenotype relationships underscore links between FMR1 expansion, gene methylation, FMRP deficit and overall severity of neurobehavioral phenotypes [ 21 , 22 ]. Despite these concerns, the extent to which the placebo effect has impacted FXS trial outcomes in general or specific outcome measures is unknown [ 9 ].…”