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.

The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. We previously characterized 215 dried blood spots (DBS) representing different FMR1 genotypes and ages with a Luminex-based immunoassay (qFMRP). We found variable FMRP levels in the normal samples and identified affected males by the drastic reduction of FMRP. Here, to establish the variability of expression of FMRP in a larger random population we quantified FMRP in 2,000 anonymous fresh newborn DBS. We also evaluated the effect of long term storage on qFMRP by retrospectively assaying 74 aged newborn DBS that had been stored for 7-84 months that included normal and full mutation individuals. These analyses were performed on 3 mm DBS disks. To identify the alleles associated with the lowest FMRP levels in the fresh DBS, we analyzed the DNA in the samples that were more than two standard deviations below the mean. Analysis of the fresh newborn DBS revealed a broad distribution of FMRP with a mean approximately 7-fold higher than that we previously reported for fresh DBS in normal adults and no samples whose FMRP level indicated FXS. DNA analysis of the lowest FMRP DBS showed that this was the low extreme of the normal range and included a female carrying a 165 CGG repeat premutation. In the retrospective study of aged newborn DBS, the FMRP mean of the normal samples was less than 30% of the mean of the fresh DBS. Despite the degraded signal from these aged DBS, qFMRP identified the FXS individuals. The assay showed that newborn DBS contain high levels of FMRP that will allow identification of males and potentially females, affected by FXS. The assay is also an effective screening tool for aged DBS stored for up to four years.

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1—normally containing 6–54 repeats—to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55–200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns.

Background Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. Gene therapy may offer an efficient method to ameliorate the symptoms of this disorder. Methods An AAVphp.eb-hSyn-mFMR1IOS7 vector and an empty control were injected into the tail vein of adult Fmr1 knockout (KO) mice and wildtype (WT) controls. The KO mice were injected with 2 × 10¹³ vg/kg of the construct. The control KO and WT mice were injected with an empty vector. Four weeks following treatment, the animals underwent a battery of tests: open field, marble burying, rotarod, and fear conditioning. The mouse brains were studied for levels of the Fmr1 product FMRP. Results: No significant levels of FMRP were found outside the CNS in the treated animals. The gene delivery was highly efficient, and it exceeded the control FMRP levels in all tested brain regions. There was also improved performance in the rotarod test and partial improvements in the other tests in the treated KO animals. Conclusion: These experiments demonstrate efficient, brain-specific delivery of Fmr1 via peripheral administration in adult mice. The gene delivery led to partial alleviation of the Fmr1 KO phenotypical behaviors. FMRP oversupply may explain why not all behaviors were significantly affected. Since AAV.php vectors are less efficient in humans than in the mice used in the current experiment, studies to determine the optimal dose using human-suitable vectors will be necessary to further demonstrate feasibility.

Autonomic dysfunction is implicated in a range of psychological conditions, including depression and anxiety. The fragile X mental retardation-1 (FMR1) premutation is a common genetic mutation that affects ~1:150 women and is associated with psychological vulnerability. This study examined cardiac indicators of autonomic function among women with the FMR1 premutation and control women as potential biomarkers for psychological risk that may be linked to FMR1. Baseline inter-beat interval and respiratory sinus arrhythmia (a measure of parasympathetic vagal tone) were measured in 35 women with the FMR1 premutation and 28 controls. The women completed anxiety and depression questionnaires. FMR1 genetic indices (i.e., CGG repeat, quantitative FMRP, FMR1 mRNA, activation ratio) were obtained for the premutation group. Respiratory sinus arrhythmia was reduced in the FMR1 premutation group relative to controls. While depression symptoms were associated with reduced respiratory sinus arrhythmia among control women, these variables were unrelated in the FMR1 premutation. Elevated FMR1 mRNA was associated with higher respiratory sinus arrhythmia. Women with the FMR1 premutation demonstrated autonomic dysregulation characterized by reduced vagal tone. Unlike patterns observed in the general population and in study controls, vagal activity and depression symptoms were decoupled in women with the FMR1 premutation, suggesting independence between autonomic regulation and psychopathological symptoms that is atypical and potentially specific to the FMR1 premutation. The association between vagal tone and mRNA suggests that molecular variation associated with FMR1 plays a role in autonomic regulation.

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.