FMR1 Research Articles

Stress and genetics influence hair cortisol in FMR1 premutation carrier mothers of children with fragile X syndrome

To investigate genetic and environmental influences on cortisol levels, mothers of children with fragile X syndrome (FXS) were studied four times over a 7.5-year period. All participants (n = 84) were carriers of the FMR1 “premutation”, a genetic condition associated with impaired HPA axis functioning. Genetic variation was indicated by expansions in the number of CGG (cytosine-guanine-guanine) repeats in the FMR1 gene (67–138 repeats in the present sample). The environmental factor was cumulative exposure to adverse life events during the study period. Cortisol was measured at the beginning of the study via saliva samples and at the end of the study via hair samples; hormone values from these two specimen types were significantly correlated. The interactions between CGG repeat number and adverse life events significantly predicted hair cortisol concentration, including after accounting for the initial salivary cortisol level. For those with fewer CGG repeats, stress exposure was associated with elevated cortisol, the expected response to stress, although women with a higher number of CGGs had a reduced cortisol response to adverse events, which might be related to HPA dysfunction. These results indicate that both exogenous and endogenous factors affect HPA functioning in this population of women.

Therapeutic Development for CGG Repeat Expansion-Associated Neurodegeneration.

Non-coding repeat expansions, such as CGG, GGC, CUG, CCUG, and GGGGCC, have been shown to be involved in many human diseases, particularly neurological disorders. Of the diverse pathogenic mechanisms proposed in these neurodegenerative diseases, dysregulated RNA metabolism has emerged as an important contributor. Expanded repeat RNAs that form particular structures aggregate to form RNA foci, sequestering various RNA binding proteins and consequently altering RNA splicing, transport, and other downstream biological processes. One of these repeat expansion-associated diseases, fragile X-associated tremor/ataxia syndrome (FXTAS), is caused by a CGG repeat expansion in the 5’UTR region of the fragile X mental retardation 1 (FMR1) gene. Moreover, recent studies have revealed abnormal GGC repeat expansion within the 5’UTR region of the NOTCH2NLC gene in both essential tremor (ET) and neuronal intranuclear inclusion disease (NIID). These CGG repeat expansion-associated diseases share genetic, pathological, and clinical features. Identification of the similarities at the molecular level could lead to a better understanding of the disease mechanisms as well as developing novel therapeutic strategies. Here, we highlight our current understanding of the molecular pathogenesis of CGG repeat expansion-associated diseases and discuss potential therapeutic interventions for these neurological disorders.

Significance and case analysis of FMR1 mutation screening during early and middle pregnancy

To screen for mutations of fragile X mental retardation 1 (FMR1) gene during early and middle pregnancy and provide prenatal diagnosis for those carrying high-risk CGG trinucleotide expansions. Peripheral blood samples of 2316 pregnant women at 12 to 21(+6) gestational weeks were collected for the extraction of genomic DNA. CGG repeats of the FMR1 gene were detected by fluorescence PCR and capillary electrophoresis. Genetic counseling and prenatal diagnosis were provided for 3 women carrying the premutations. The carrier rate of CGG repeats of the FMR1 gene was 1 in 178 for the intermediate type and 1 in 772 for the premutation types. The highest frequency allele of CGG was 29 repeats, which accounted for 49.29%, followed by 30 repeats (28.56%) and 36 repeats (8.83%). In case 1, the fetus had a karyotype of 45,X, in addition with premutation type of CGG expansion of the FMR1 gene. Following genetic counseling, the couple chose to terminate the pregnancy through induced labor. The numbers of CGG repeats were respectively 70/- and 29/30 for the husband and wife. In case 2, amniocentesis was performed at 20 weeks of gestation. The number of CGG repeats of the FMR1 gene was 29/-. No abnormality was found in the fetal karyotype and chromosomal copy number variations. The couple chose to continue with the pregnancy. Case 3 refused prenatal diagnosis after genetic counseling and gave birth to a girl at full term, who had a birth weight of 2440 g and no obvious abnormality found during follow-up. Pregnant women should be screened for FMR1 gene mutations during early and middle pregnancy, and those with high-risk CGG expansions should undergo prenatal diagnosis, genetic counseling and family study.

Analysis and prenatal diagnosis of FMR1 gene mutations among patients with unexplained mental retardation

To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation. For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEaseTM PCR. Prenatal diagnosis was provided to carriers of pre- and full-mutations. The pattern of X chromosome inactivation (XCI) was determined for women with mental retardation and full mutations. For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele. FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.

054 Characterization of Sleep Phenotypes and Sleep-Dependent Memory Consolidation in a Mouse Model of Fragile X Syndrome

Abstract Introduction Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by disruption of Fmr1 gene function, leading to intellectual disability. FXS individuals report increased incidence of sleep disruptions such as loss of NREM sleep, irregular sleep/wake cycles, and circadian rhythm disturbances that warrant pharmacological intervention. Since sleep has critical roles in the promotion of memory consolidation, it is unknown whether disrupted cognitive function in FXS is exacerbated by abnormal sleep. We characterized the link between sleep loss phenotypes and cognition in FXS mice (Fmr1 KO). We hypothesized that normalizing sleep in Fmr1 KO mice could improve sleep-dependent cognitive function. Because direct activation of G-protein inward rectifying potassium (GIRK) channels by ML297 has been found to promote NREM sleep, we tested how ML297 affected sleep and memory consolidation phenotypes in Fmr1 KO mice. Methods Wild type (WT) and Fmr1 KO were implanted with electrodes for electroencephalogram/electromyogram (EEG/EMG) recording of wakefulness, NREM and REM sleep. Sleep-dependent memory consolidation was measured using single-trial contextual fear conditioning (CFC). ML297 or vehicle was administered after CFC training to measure the effects on sleep and fear memory consolidation. Results Fmr1 KO mice showed reduced sleep in the hours following CFC learning compared to wild type littermates, and reduced contextual fear memory consolidation. Post-CFC sleep deprivation disrupted memory consolidation in wild type littermates, but not Fmr1 KO mice. Both NREM sleep time and NREM bout length were reduced in Fmr1 KO mice, and preliminary data suggest reduced NREM delta (0.5–4 Hz) power in the prefrontal cortex. These deficits were present at baseline and also following CFC. Post-CFC training administration of ML297 rescued NREM sleep and contextual fear memory deficits in Fmr1 KO mice. Conclusion Our study showed a strong link between NREM sleep loss and cognitive deficits in Fmr1 KO mice. Critically, normalization of NREM sleep through direct activation of GIRK channels rescues cognitive deficits seen in Fmr1 KO mice, suggesting a new therapeutic approach to treating cognitive deficits associated with FXS. Support (if any) This work was supported by a Rackham Merit Fellowship to JDM.

Emergence of Developmental Delay in Infants and Toddlers With an FMR1 Mutation.

Children with FMR1 gene expansions are known to experience a range of developmental challenges, including fragile X syndrome. However, little is known about early development and symptom onset, information that is critical to guide earlier identification, more accurate prognoses, and improved treatment options. Data from 8 unique studies that used the Mullen Scales of Early Learning to assess children with an FMR1 gene expansion were combined to create a data set of 1178 observations of >500 young children. Linear mixed modeling was used to explore developmental trajectories, symptom onset, and unique developmental profiles of children <5 years of age. Boys with an FMR1 gene full mutation showed delays in early learning, motor skills, and language development as young as 6 months of age, and both sexes with a full mutation were delayed on all developmental domains by their second birthday. Boys with a full mutation continued to gain skills over early childhood at around half the rate of their typically developing peers; girls with a full mutation showed growth at around three-quarters of the rate of their typically developing peers. Although children with a premutation were mostly typical in their developmental profiles and trajectories, mild but significant delays in fine motor skills by 18 months were detected. Children with the FMR1 gene full mutation demonstrate significant developmental challenges within the first 2 years of life, suggesting that earlier identification is needed to facilitate earlier implementation of interventions and therapeutics to maximize effectiveness.

Laboratory testing for fragile X, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Pathogenic variants in the FMR1 gene are associated with fragile X syndrome, fragile X–associated tremor ataxia syndrome (FXTAS), and fragile X–associated primary ovarian insufficiency (FXPOI). This document provides updated information regarding FMR1 pathogenic variants, including prevalence, genotype–phenotype correlations, and variant nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction (PCR) amplification of FMR1, including triplet repeat–primed and methylation-specific PCR.The American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document ACMG Technical Standards for Clinical Genetics Laboratories, which is now maintained online (http://www.acmg.net). This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document is the fourth update to the original standards and guidelines for fragile X testing that were published in 2001, with revisions in 2005 and 2013, respectively.This versionClarifies the clinical features associated with different FMRI variants (Section 2.3)Discusses important reporting considerations (Section 3.3.1.3)Provides updates on technology (Section 4.1)

Evaluation of Serotonin 5‐HT 1A , 5‐HT 2A , and 5‐HT 2C Receptors and the Serotonin Transporter in an Fmr1 Knockout Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD). Diminished serotonin (5-hydroxytryptamine, 5-HT) synthesis has been reported in ASD. The 5-HT transporter (SERT) and receptors (5-HTRs) have been targeted to manage ASD and FXS symptoms. Serotonergic neurons project to glutamate and GABA neurons expressing 5-HT1ARs, 5-HT2ARs, and 5-HT2CRs that modulate cellular excitation-inhibition which is altered in ASD and FXS. Here, we assessed the expression and function of these 5-HTRs and SERT in adult (P60-180) Fmr1 knockout (KO) mice, a model of FXS, compared to wild type (WT) mice. We performed autoradiography on brain sections using [3H]5-CT for 5-HT1ARs, [3H]Ketanserin for 5-HT2ARs, [3H]Mesulergine for 5-HT2CRs, [3H]Citalopram for SERT, and appropriate non-specific and off-target blocking ligands. Brain regions of interest were chosen and analyzed using ImageJ. Mean pixel intensity from the images was normalized to the mean of background intensity, and non-specific binding was subtracted from total binding to obtain normalized specific binding. Saturation binding assays, using whole brain membrane homogenates, were performed for 5-HT1ARs with [3H]5-CT and SB-269970 to block 5-HT7Rs; WAY-100635 was used to isolate 5-HT1AR specific binding. Behavioral effects of (R)-8-OH-DPAT, (±)-DOI, and lorcaserin were used to probe in vivo functional activity of 5-HT1A, 5-HT2A, and 5-HT2CRs, respectively. Responses to (R)-8-OH-DPAT (1 and 2 mg/kg), including flat body posture, head weaving, straub tail, hind limb abduction, piloerection, tremor, rearing, grooming, and head twitch responses (HTRs), were recorded by two independent observers. Locomotor effects of lorcaserin (1, 3, and 10 mg/kg) were recorded using Noldus Ethovision XT software, and HTRs elicited by (±)-DOI (1 mg/kg) were recorded using a hand-held tally counter. Sex effects were assessed in all tests. Preliminary results suggest that compared to WT males, Fmr1 KO males (N=3 per group) have higher 5-HT1AR expression in lateral septum and frontal cortex, lower 5-HT2AR expression in isocortex layer V, and lower 5-HT2CR expression in anterior olfactory nucleus and nucleus accumbens. Increased 5-HT1AR expression was confirmed by saturation binding analyses (N=6 per group). Male mice of both genotypes had lower 5-HT2CR expression in choroid plexus and caudate putamen (N=3 per group). Behavioral effects of (R)-8-OH-DPAT were significantly more pronounced in both male and female (N=5-7 per group) Fmr1 KO mice, relative to WT mice. A minor decrease in DOI-elicited HTRs was observed in Fmr1 KO males compared to WT males (14 vs. 17, N=8-9 per group). No genotype differences in locomotor activity were observed after lorcaserin administration (N=6 per group). Ongoing studies are assessing SERT expression and in vivo function via autoradiography and the tail suspension test, respectively. This study provides the first observations of perturbations in key 5-HTRs in Fmr1 KO mice, suggesting dysfunctions in the central 5-HT system in FXS.

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.

Evaluation of Perineuronal Nets and Their Regulation by Serotonin 5‐HT 7 Receptors in a Juvenile Fmr1 Knockout Mouse Model of Fragile X Syndrome

Fragile X Syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. Individuals with FXS also often experience several other neuropsychiatric symptoms, including seizures. FXS is caused by silencing of the FMR1 gene, preventing expression of FMRP, an RNA-binding protein that regulates protein synthesis. A widespread theory is that the lack of FMRP causes an imbalance in excitatory—inhibitory neurotransmission, leading to excessive excitatory tone, which may explain the relatively high prevalence of seizures in individuals diagnosed with FXS and autism. The central serotonin system can modulate symptoms of FXS, and certain serotonin receptors (5-HTRs) are functionally linked with proteins known to be altered in FXS, suggesting targeting them may be therapeutic for FXS. For example, reports show that activation of 5-HT7Rs increases activity of matrix metalloproteinase 9 (MMP-9), which is upregulated in the absence of FMRP; MMP-9 degrades perineuronal nets (PNNs) that form around parvalbumin-positive GABAergic interneurons, suggesting blockade of 5-HT7Rs may restore MMP-9 homeostasis, leading to enhanced inhibitory tone in FXS. The formation of PNNs coincides with the closing of juvenile plasticity. Previous studies show that PNN density in the auditory cortex is reduced in juvenile Fmr1 knockout mice, and coincident hyperexcitability of the auditory cortex may contribute to the audiogenic seizure (AGS) phenotype that is prominent in juvenile Fmr1 knockout mice. Recently, we reported that a novel serotonin receptor modulator, FPT, that has potent 5-HT7R competitive antagonist activity, completely blocks AGSs in juvenile Fmr1 knockout mice. Here we report that the selective 5-HT7R antagonist, SB-269970, reduced AGS lethality in juvenile Fmr1 knockout mice compared to vehicle, but effects were not statistically significant (P=0.21, N=12 each). Preliminary results support previous observations of reduced density of PNNs in the auditory cortex of juvenile Fmr1 knockout mice (N=8) compared to WT mice (N=8). Ongoing studies are investigating PNN density following semi-chronic administration of SB-269970 or FPT to determine the efficacy of 5-HT7R blockade or other pharmacological mechanisms of FPT to normalize PNN density in juvenile Fmr1 knockout mice to wild-type mice levels.

A Pivotal Study of ZYN002 Cannabidiol (CBD) Transdermal Gel in Children and Adolescents With Fragile X Syndrome [CONNECT-FX (ZYN2-CL-016)

ZYN002 is a pharmaceutically manufactured transdermal cannabidiol gel. CONNECT-FX was a randomized, double-blind, placebo-controlled study to evaluate efficacy and safety of ZYN002 in patients 3 to 17 years with full FMR1 gene mutation.

Hyperexcitability and Loss of Feedforward Inhibition Contribute to Aberrant Plasticity in the Fmr1KO Amygdala.

Fragile X syndrome (FXS) is a neurodevelopmental disorder (NDD) characterized by intellectual disability, autism spectrum disorders (ASDs), and anxiety disorders. The disruption in the function of the FMR1 gene results in a range of alterations in cellular and synaptic function. Previous studies have identified dynamic alterations in inhibitory neurotransmission in early postnatal development in the amygdala of the mouse model of FXS. However, little is known about how these changes alter microcircuit development and plasticity in the lateral amygdala (LA). Using whole-cell patch clamp electrophysiology, we demonstrate that principal neurons (PNs) in the LA exhibit hyperexcitability with a concomitant increase in the synaptic strength of excitatory synapses in the BLA. Further, reduced feed-forward inhibition appears to enhance synaptic plasticity in the FXS amygdala. These results demonstrate that plasticity is enhanced in the amygdala of the juvenile Fmr1 knock-out (KO) mouse and that E/I imbalance may underpin anxiety disorders commonly seen in FXS and ASDs.

Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

CGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.

SUMMARYTranscriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.

Adult-onset neuronal intranuclear inclusion disease, with both stroke-like onset and encephalitic attacks: a case report

BackgroundNeuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease, the clinical manifestations of which are complex and easily misdiagnosed. NIID clinical characteristics are varied, affecting the central and peripheral nervous systems and autonomic nerves. In this study, we present an NIID case with both stroke-like onset and encephalitic attacks, which is a rare case report.Case presentationA 68-year-old Chinese female presented with sudden aphasia and limb hemiplegia as the first symptoms, as well as fever, cognitive impairment and mental irritability from encephalitic attacks. During hospitalization, a brain magnetic resonance imaging (MRI) examination detected high signal intensity from diffusion-weighted imaging (DWI) of the bilateral frontal grey matter-white matter junction. Electrophysiological tests revealed the main site of injury was at the myelin sheath in the motor nerves. A skin biopsy revealed eosinophilic spherical inclusion bodies in the nuclei of small sweat gland cells, fibroblasts and fat cells, whilst immunohistochemistry revealed that p62 and ubiquitin antibodies were positive. From genetic analyses, the patient was not a carrier of the fragile X mental retardation 1 (FMR1) permutation, but repeated GGC sequences in the NOTCH2NLC gene confirmed an NIID diagnosis. Through antipsychotic and nutritional support therapy, the patient’s symptoms were completely relieved within 3 weeks.ConclusionsThis report of an NIID case with both stroke-like onset and encephalitic attacks provides new information for NIID diagnoses, and a comprehensive classification of clinical characteristics.

SENP1 in the Retrosplenial Agranular Cortex Regulates Core Autistic-Like Symptoms in Mice

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder, in which core symptoms are defects of social interaction and evidently repetitive behaviors. Although around 50-70 % of ASD patients have comorbidity of intellectual disabilities (ID) or developmental delay (DD), there are some ASD patients who exhibit only core symptoms but without ID/DD, raising the question whether there are genetic components and neural circuits specific for core symptoms of ASD. Here, by focusing on ASD patients who do not show compound ID or DD, we identified a de novo heterozygous gene-truncating mutation of the Sentrin-specific peptidase1 (SENP1) gene, coding the small ubiquitin-like modifiers (SUMO) deconjugating enzyme, as a potentially new candidate gene for ASD. We found that Senp1 haploinsufficient mice exhibited core symptoms of autism such as deficits in social interaction and repetitive behaviors, but normal learning and memory ability. Moreover, we found that the inhibitory and excitatory synaptic functions were severely affected in the retrosplenial agranular (RSA) cortex of Senp1 haploinsufficient mice. Lack of Senp1 led to over SUMOylation and degradation of fragile X mental retardation protein (FMRP) proteins, which is coded by the FMR1 gene, also implicated in syndromic ASD. Importantly, re-introducing SENP1 or FMRP specifically in RSA fully rescued the defects of synaptic functions and core autistic-like symptoms of Senp1 haploinsufficient mice. Together, these results demonstrated that disruption of the SENP1-FMRP regulatory axis in the RSA may cause core autistic symptoms, which provide a candidate brain region of ASD for potential therapeutic intervene by neural modulation approaches.

Fragile X premutation rCGG repeats impair synaptic growth and synaptic transmission at Drosophila larval neuromuscular junction.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell-developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. Here, we used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. We show that presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by riboCGG (rCGG) repeats mediates presynaptically during larval neuromuscular junction development. We also suggest that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats.

Cerebral Microbleeds in Fragile X-Associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome is a neurodegenerative disease of late onset developed by carriers of the premutation in the fragile x mental retardation 1 (FMR1) gene. Pathological features of neurodegeneration in fragile X-associated tremor/ataxia syndrome include toxic levels of FMR1 mRNA, ubiquitin-positive intranuclear inclusions, white matter disease, iron accumulation, and a proinflammatory state. The objective of this study was to analyze the presence of cerebral microbleeds in the brains of patients with fragile X-associated tremor/ataxia syndrome and investigate plausible causes for cerebral microbleeds in fragile X-associated tremor/ataxia syndrome. We collected cerebral and cerebellar tissue from 15 fragile X-associated tremor/ataxia syndrome cases and 15 control cases carrying FMR1 normal alleles. We performed hematoxylin and eosin, Perls and Congo red stains, ubiquitin, and amyloid β protein immunostaining. We quantified the number of cerebral microbleeds, amount of iron, presence of amyloid β within the capillaries, and number of endothelial cells containing intranuclear inclusions. We evaluated the relationships between pathological findings using correlation analysis. We found intranuclear inclusions in the endothelial cells of capillaries and an increased number of cerebral microbleeds in the brains of those with fragile X-associated tremor/ataxia syndrome, both of which are indicators of cerebrovascular dysfunction. We also found a suggestive association between the amount of capillaries that contain amyloid β in the cerebral cortex and the rate of disease progression. We propose microangiopathy as a pathologic feature of fragile X-associated tremor/ataxia syndrome. © 2021 International Parkinson and Movement Disorder Society.

'Essential Tremor' Phenotype in FMR1 Premutation/Gray Zone Sibling Series: Exploring Possible Genetic Modifiers.

Fragile X-associated tremor/ataxia syndrome (FXTAS) occurs in carriers of fragile X mental retardation 1 (FMR1) X-linked small CGG expansion (gray zone [GZ] and premutation [PM]) alleles, containing 41-200 repeats. Major features comprise kinetic tremor, gait ataxia, cognitive decline and cerebellar peduncular white matter lesions, but atypical/incomplete FXTAS may occur. We explored the possibility of polygenic effects modifying the FXTAS spectrum phenotypes. We used three motor scales and selected cognitive tests in a series of three males and three females from a single sibship carrying PM or GZ alleles (44 to 75 repeats). The molecular profiles from these siblings were determined by genomewide association study with single-nucleotide polymorphism (SNP) genotyping by Illumina Global Screening Array. Nonparametric linkage analysis was applied and Parkinson's disease (PD) polygenic risk scores (PRSs) were calculated for all the siblings, based on 107 known risk variants. All male and female siblings manifested similar kinetic tremor phenotypes. In contrast to FXTAS, they showed negligible gait ataxia, and few white matter lesions on MRI. Cognitive functioning was unaffected. Suggestive evidence of linkage to a broad region of the short arm of chromosome 10 was obtained, and median PD PRS for the sibship fell within the top 30% of a sample of over 500,000 UK and Australian controls. The genomewide study results are suggestive of modifying effects of genetic risk loci linked to PD, on the neurological phenotype of FMR1-CGG small expansion carriers, resulting in an oligosymptomatic kinetic tremor seen in FXTAS spectrum, but also consistent with essential tremor.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.