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

Abstract

<title>Abstract</title> <bold>Background.</bold> Fragile X syndrome (FXS) is caused by silencing of the <italic>FMR1</italic> gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying <italic>FMR1</italic> 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 <italic>FMR1 </italic>DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority.<bold>Methods. </bold>We applied nine sensitive and quantitative assays evaluating <italic>FMR1</italic> DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of <italic>FMR1 </italic>expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different <italic>FMR1 </italic>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. <bold>Results.</bold> <italic>FMR1</italic> CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with previous <italic>FMR1</italic> 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. <bold>Conclusions. </bold>The results<bold> </bold>underscore the link between <italic>FMR1</italic> expansion, gene methylation, and FMRP<italic> deficit. The </italic>association between FMRP deficiency and overall severity <italic>of the neurobehavioral phenotype invites </italic>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.