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

Abstract

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.

Cerebral Expression of Metabotropic Glutamate Receptor Subtype 5 in Idiopathic Autism Spectrum Disorder and Fragile X Syndrome: A Pilot Study

“…We confirmed our earlier finding that mGluR 5 expression is reduced in all brain regions in men with FXS [ 17 ] on a sample of men with FXS compared to participants of both sexes with IASD [ 14 , 23 ] and TD [ 14 , 17 , 25 , 26 ]. In men with FXS, reduced mGluR 5 expression in (A) cortical regions provides a basis for ID and (B) limbic regions provides a basis for the neurobehavioral symptoms [ 10 , 17 ].…”

“…We confirmed our earlier finding of reduced cerebral mGluR 5 expression [ 17 ] in a sample of men with FXS in contrast to participants with IASD and TD of both sexes. The significantly reduced mGluR 5 expression in all brain regions of men with FXS provides a possible molecular basis for the neurobehavioral phenotype of individuals with FXS [ 10 ]. Reduced cortical mGluR 5 expression may provide a basis for the cognitive deficits (delayed socialization) of individuals with FXS [ 56 ].…”

“…Additionally, FXS and some other subtypes of ASD are also characterized by intellectual disability (ID) [ 6 ]. Dysfunction of protein synthesis mediated by abnormal pathways including metabotropic glutamate receptors (mGluR) plays a role in the pathometabolism of IASD [ 7 , 8 ] and FXS [ 9 , 10 , 11 , 12 ]. Despite the evidence for dysfunction of mGluR 5 expression in IASD, conflicting findings include the decreased expression in the dorsolateral prefrontal cortex [ 13 ] and increased expression in the post-central gyrus and the cerebellum [ 14 ].…”

“…The deficits of FMRP in fmr1 KO mouse models result in dysfunction of crucial group 1 metabotropic glutamatergic pathways leading to dysregulated downstream signaling cascades including the mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) pathways [ 17 ]. The correction of mGluR-LTD and behavioral symptoms in fmr1 KO mouse models suggests that the a biomarker to measure mGluR 5 expression in the living human brain represents a means to apply precision molecular medicine to ameliorate behavioral symptoms of FXS and possibly other subtypes of ASD [ 9 , 10 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ].…”

A Bibliometric Insight of Genetic Factors in ASD: Emerging Trends and New Developments

“…Hundreds of candidate genes of ASDs have been identified, such as, KMT5B , FMR1 , NAA15 , and CHD8 . [ 16 , 17 , 18 ].…”