Circadian Rhythm and Sleep Analyses in a Fruit Fly Model of Fragile X Syndrome Using a Video-Based Automated Behavioral Research System.

Fragile X syndrome (FXS) is caused by the full mutation in the FMR1 gene on the Xq27.3 chromosome region. It is the most common monogenic cause of autism spectrum disorder (ASD) and inherited intellectual disability (ID). Besides ASD and ID and other symptoms, individuals with FXS may exhibit sleep problems and impairment of circadian rhythm (CR). The Drosophila melanogaster models of FXS, such as dFMR1B55, represent excellent models for research in the FXS field. During this study, sleep patterns and CR in dFMR1B55 mutants were analyzed, using a new platform based on continuous high-resolution videography integrated with a highly-customized version of an open-source software. This methodology provides more sensitive results, which could be crucial for all further research in this model of fruit flies. The study revealed that dFMR1B55 male mutants sleep more and can be considered weak rhythmic flies rather than totally arrhythmic and present a good alternative animal model of genetic disorder, which includes impairment of CR and sleep behavior. The combination of affordable videography and software used in the current study is a significant improvement over previous methods and will enable broader adaptation of such high-resolution behavior monitoring methods.

Sleep problems in fragile X syndrome: Cross-sectional analysis of a large clinic-based cohort.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism spectrum disorder, is associated with multiple neurobehavioral abnormalities including sleep difficulties. Nonetheless, frequency, severity, and consequences of sleep problems are still unclear. The Fragile X Online Registry with Accessible Research Database (FORWARD-version-3), including Clinician Report and Parent Report forms, was analyzed for frequency, severity, relationship with behavioral problems, and impact of sleep difficulties in a mainly pediatric cohort. A focused evaluation of sleep apnea was also conducted. Six surveyed sleep difficulties were moderately frequent (~23%-46%), relatively mild, affected predominantly younger males, and considered a problem for 7%-20% of families. Snoring was more prevalent in older individuals. All sleep difficulties were associated with irritability/aggression and most also to hyperactivity. Only severe snoring was correlated with sleep apnea (loud snoring: 30%; sleep apnea: 2%-3%). Sleep difficulties are prevalent in children with FXS and, although they tend to be mild, they are associated with behavioral problems and negative impact to families. Because of its cross-sectional nature, clinic-origin, use of ad hoc data collection forms, and lack of treatment data, the present study should be considered foundational for future research aiming at better recognition and management of sleep problems in FXS.

Fragile X Syndrome: From Molecular Aspect to Clinical Treatment.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the full mutation as well as highly localized methylation of the fragile X mental retardation 1 (FMR1) gene on the long arm of the X chromosome. Children with FXS are commonly co-diagnosed with Autism Spectrum Disorder, attention and learning problems, anxiety, aggressive behavior and sleep disorder, and early interventions have improved many behavior symptoms associated with FXS. In this review, we performed a literature search of original and review articles data of clinical trials and book chapters using MEDLINE (1990-2021) and ClinicalTrials.gov. While we have reviewed the biological importance of the fragile X mental retardation protein (FMRP), the FXS phenotype, and current diagnosis techniques, the emphasis of this review is on clinical interventions. Early non-pharmacological interventions in combination with pharmacotherapy and targeted treatments aiming to reverse dysregulated brain pathways are the mainstream of treatment in FXS. Overall, early diagnosis and interventions are fundamental to achieve optimal clinical outcomes in FXS.

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

Multiple lines of evidence suggest that dysfunction of the metabotropic glutamate receptor subtype 5 (mGluR5) plays a role in the pathogenesis of autism spectrum disorder (ASD). Yet animal and human investigations of mGluR5 expression provide conflicting findings about the nature of dysregulation of cerebral mGluR5 pathways in subtypes of ASD. The demonstration of reduced mGluR5 expression throughout the living brains of men with fragile X syndrome (FXS), the most common known single-gene cause of ASD, provides a clue to examine mGluR5 expression in ASD. We aimed to (A) compare and contrast mGluR5 expression in idiopathic autism spectrum disorder (IASD), FXS, and typical development (TD) and (B) show the value of positron emission tomography (PET) for the application of precision medicine for the diagnosis and treatment of individuals with IASD, FXS, and related conditions. Two teams of investigators independently administered 3-[18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB), a novel, specific mGluR5 PET ligand to quantitatively measure the density and the distribution of mGluR5s in the brain regions, to participants of both sexes with IASD and TD and men with FXS. In contrast to participants with TD, mGluR5 expression was significantly increased in the cortical regions of participants with IASD and significantly reduced in all regions of men with FXS. These results suggest the feasibility of this protocol as a valuable tool to measure mGluR5 expression in clinical trials of individuals with IASD and FXS and related conditions.

Raising Knowledge and Awareness of Fragile X Syndrome in Serbia, Georgia, and Colombia: A Model for Other Developing Countries?

Fragile X syndrome is the most common monogenetic cause of inherited intellectual disability and syndromic autism spectrum disorder. Fragile X syndrome is caused by an expansion (full mutation ≥200 CGGs repeats, normal 10-45 CGGs) of the fragile X mental retardation 1 (FMR1) gene, epigenetic silencing of the gene, which leads to reduction or lack of the gene's product: the fragile X mental retardation protein. In this cross-sectional study, we assessed general and pharmacotherapy knowledge (GK and PTK) of fragile X syndrome and satisfaction with education in neurodevelopmental disorders (NDDs) among senior medical students in Serbia (N=348), Georgia (N=112), and Colombia (N=58). A self-administered 18-item questionnaire included GK (8/18) and PTK (7/18) components and self-assessment of the participants education in NDDs (3/18). Roughly 1 in 5 respondents had correct answers on half or more facts about fragile X syndrome (GK>PTK), which ranged similarly 5-7 in Serbia, 6-8 in Georgia, and 5-8 in Colombia, respectively. No cohort had an average value greater than 9 (60%) that would represent passing score "cut-off." None of the participants answered all the questions correctly. More than two-thirds of the participants concluded that they gained inadequate knowledge of NDDs during their studies, and that their education in this field should be more intense. In conclusion, there is a major gap in knowledge regarding fragile X syndrome among senior medical students in these three developing countries. The finding could at least in part be generalized to other developing countries aimed toward increasing knowledge and awareness of NDDs and fostering an institutional collaboration between developed and developing countries.

Using a Combination of Novel Research Tools to Understand Social Interaction in the Drosophila melanogaster Model for Fragile X Syndrome.

Fragile X syndrome (FXS), the most common monogenic cause of inherited intellectual disability and autism spectrum disorder, is caused by a full mutation (>200 CGG repeats) in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. Individuals with FXS experience various challenges related to social interaction (SI). Animal models, such as the Drosophila melanogaster model for FXS where the only ortholog of human FMR1 (dFMR1) is mutated, have played a crucial role in the understanding of FXS. The aim of this study was to investigate SI in the dFMR1B55 mutants (the groups of flies of both sexes simultaneously) using the novel Drosophila Shallow Chamber and a Python data processing pipeline based on social network analysis (SNA). In comparison with wild-type flies (w1118), SNA analysis in dFMR1B55 mutants revealed hypoactivity, fewer connections in their networks, longer interaction duration, a lower ability to transmit information efficiently, fewer alternative pathways for information transmission, a higher variability in the number of interactions they achieved, and flies tended to stay near the boundaries of the testing chamber. These observed alterations indicate the presence of characteristic strain-dependent social networks in dFMR1B55 flies, commonly referred to as the group phenotype. Finally, combining novel research tools is a valuable method for SI research in fruit flies.

Role of the endocannabinoid system in fragile X syndrome: potential mechanisms for benefit from cannabidiol treatment.

Multiple lines of evidence suggest a central role for the endocannabinoid system (ECS) in the neuronal development and cognitive function and in the pathogenesis of fragile X syndrome (FXS). This review describes the ECS, its role in the central nervous system, how it is dysregulated in FXS, and the potential role of cannabidiol as a treatment for FXS. FXS is caused by deficiency or absence of the fragile X messenger ribonucleoprotein 1 (FMR1) protein, FMRP, typically due to the presence of >200 cytosine, guanine, guanine sequence repeats leading to methylation of the FMR1 gene promoter. The absence of FMRP, following FMR1 gene-silencing, disrupts ECS signaling, which has been implicated in FXS pathogenesis. The ECS facilitates synaptic homeostasis and plasticity through the cannabinoid receptor 1, CB1, on presynaptic terminals, resulting in feedback inhibition of neuronal signaling. ECS-mediated feedback inhibition and synaptic plasticity are thought to be disrupted in FXS, leading to overstimulation, desensitization, and internalization of presynaptic CB1 receptors. Cannabidiol may help restore synaptic homeostasis by acting as a negative allosteric modulator of CB1, thereby attenuating the receptor overstimulation, desensitization, and internalization. Moreover, cannabidiol affects DNA methylation, serotonin 5HT1A signal transduction, gamma-aminobutyric acid receptor signaling, and dopamine D2 and D3 receptor signaling, which may contribute to beneficial effects in patients with FXS. Consistent with these proposed mechanisms of action of cannabidiol in FXS, in the CONNECT-FX trial the transdermal cannabidiol gel, ZYN002, was associated with improvements in measures of social avoidance, irritability, and social interaction, particularly in patients who are most affected, showing ≥90% methylation of the FMR1 gene.

Insight and Recommendations for Fragile X-Premutation-Associated Conditions from the Fifth International Conference on FMR1 Premutation.

The premutation of the fragile X messenger ribonucleoprotein 1 (FMR1) gene is characterized by an expansion of the CGG trinucleotide repeats (55 to 200 CGGs) in the 5' untranslated region and increased levels of FMR1 mRNA. Molecular mechanisms leading to fragile X-premutation-associated conditions (FXPAC) include cotranscriptional R-loop formations, FMR1 mRNA toxicity through both RNA gelation into nuclear foci and sequestration of various CGG-repeat-binding proteins, and the repeat-associated non-AUG (RAN)-initiated translation of potentially toxic proteins. Such molecular mechanisms contribute to subsequent consequences, including mitochondrial dysfunction and neuronal death. Clinically, premutation carriers may exhibit a wide range of symptoms and phenotypes. Any of the problems associated with the premutation can appropriately be called FXPAC. Fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated neuropsychiatric disorders (FXAND) can fall under FXPAC. Understanding the molecular and clinical aspects of the premutation of the FMR1 gene is crucial for the accurate diagnosis, genetic counseling, and appropriate management of affected individuals and families. This paper summarizes all the known problems associated with the premutation and documents the presentations and discussions that occurred at the International Premutation Conference, which took place in New Zealand in 2023.

Fragile X Syndrome: Recent Research Updates toward Capturing Treatments' Improvement in Clinical Trials.

This Brain Sciences 2020 Special Issue of nine manuscripts contribute novel data on treatment updates in fragile X syndrome (FXS) [...].

Latent Class Analysis Identifies Distinctive Behavioral Subtypes in Children with Fragile X Syndrome.

Fragile X syndrome (FXS) is characterized by variable neurobehavioral abnormalities, which leads to difficulties in developing and evaluating treatments and in determining accurate prognosis. We employed a pediatric cross-sectional sample (1,072 males, 338 females) from FORWARD, a clinic-based natural history study, to identify behavioral subtypes by latent class analysis. Input included co-occurring behavioral conditions, sleep and sensory problems, autistic behavior scales (SCQ, SRS-2), and the Aberrant Behavior Checklist revised for FXS (ABCFX). A 5-class solution yielded the most clinically meaningful, pharmacotherapy independent behavioral groups with distinctive SCQ, SRS-2, and ABCFX profiles, and adequate non-overlap (≥ 71%): "Mild" (31%), "Moderate without Social Impairment" (32%), "Moderate with Social Impairment" (7%), "Moderate with Disruptive Behavior" (20%), and "Severe" (9%). Our findings support FXS subtyping, for improving clinical management and therapeutic development.

Drosophila melanogaster as a Model to Study Fragile X-Associated Disorders.

Fragile X syndrome (FXS) is a global neurodevelopmental disorder caused by the expansion of CGG trinucleotide repeats (≥200) in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. FXS is the hallmark of Fragile X-associated disorders (FXD) and the most common monogenic cause of inherited intellectual disability and autism spectrum disorder. There are several animal models used to study FXS. In the FXS model of Drosophila, the only ortholog of FMR1, dfmr1, is mutated so that its protein is missing. This model has several relevant phenotypes, including defects in the circadian output pathway, sleep problems, memory deficits in the conditioned courtship and olfactory conditioning paradigms, deficits in social interaction, and deficits in neuronal development. In addition to FXS, a model of another FXD, Fragile X-associated tremor/ataxia syndrome (FXTAS), has also been established in Drosophila. This review summarizes many years of research on FXD in Drosophila models.

Height and BMI in fragile X syndrome: A longitudinal assessment.

OBJECTIVE: Previously reported data regarding growth parameters in individuals with fragile X syndrome (FXS) are inconsistent. A longitudinal analysis of height and BMI in a large number of individuals with FXS was conducted. METHODS: Age- and sex-specific z scores for height and BMI of 1,223 individuals with FXS were calculated based on published normative data. Mixed-effect linear regression models were fit separately for males and females, and z scores for height and weight were regressed against age and adjusted for intellectual disability (ID) and psychotropic medication use. RESULTS: Mean height z score for both sexes decreased with age and was lower than normative data. Mean BMI z score was greater than normative data in both sexes, and this disparity increased with age. BMI z score in females was greater for those with moderate or severe ID than those with no or mild ID. Individuals taking antipsychotics had higher BMI z scores than those taking no or other medications; those taking anticonvulsants or stimulants had lower BMI z scores. CONCLUSIONS: Individuals with FXS are at elevated risk for overweight and obesity. The risk is higher in individuals taking antipsychotics and among females with severe ID. These findings warrant increased attention to obesity prevention for all individuals with FXS.

A randomized, controlled trial of ZYN002 cannabidiol transdermal gel in children and adolescents with fragile X syndrome (CONNECT-FX).

BACKGROUND: Fragile X syndrome (FXS) is associated with dysregulated endocannabinoid signaling and may therefore respond to cannabidiol therapy. DESIGN: CONNECT-FX was a double-blind, randomized phase 3 trial assessing efficacy and safety of ZYN002, transdermal cannabidiol gel, for the treatment of behavioral symptoms in children and adolescents with FXS. METHODS: Patients were randomized to 12 weeks of ZYN002 (250 mg or 500 mg daily [weight-based]) or placebo, as add-on to standard of care. The primary endpoint assessed change in social avoidance (SA) measured by the Aberrant Behavior Checklist-Community Edition FXS (ABC-CFXS) SA subscale in a full cohort of patients with a FXS full mutation, regardless of the FMR1 methylation status. Ad hoc analyses assessed efficacy in patients with ≥ 90% and 100% methylation of the promoter region of the FMR1 gene, in whom FMR1 gene silencing is most likely. RESULTS: A total of 212 patients, mean age 9.7 years, 75% males, were enrolled. A total of 169 (79.7%) patients presented with ≥ 90% methylation of the FMR1 promoter and full mutation of FMR1. Although statistical significance for the primary endpoint was not achieved in the full cohort, significant improvement was demonstrated in patients with ≥ 90% methylation of FMR1 (nominal P = 0.020). This group also achieved statistically significant improvements in Caregiver Global Impression-Change in SA and isolation, irritable and disruptive behaviors, and social interactions (nominal P-values: P = 0.038, P = 0.028, and P = 0.002). Similar results were seen in patients with 100% methylation of FMR1. ZYN002 was safe and well tolerated. All treatment-emergent adverse events (TEAEs) were mild or moderate. The most common treatment-related TEAE was application site pain (ZYN002: 6.4%; placebo: 1.0%). CONCLUSIONS: In CONNECT-FX, ZYN002 was well tolerated in patients with FXS and demonstrated evidence of efficacy with a favorable benefit risk relationship in patients with ≥ 90% methylation of the FMR1 gene, in whom gene silencing is most likely, and the impact of FXS is typically most severe. TRIAL REGISTRATION: The CONNECT-FX trial is registered on Clinicaltrials.gov (NCT03614663).

Fragile X Mental Retardation Protein and Cerebral Expression of Metabotropic Glutamate Receptor Subtype 5 in Men with Fragile X Syndrome: A Pilot Study.

Multiple lines of evidence suggest that a deficiency of Fragile X Mental Retardation Protein (FMRP) mediates dysfunction of the metabotropic glutamate receptor subtype 5 (mGluR5) in the pathogenesis of fragile X syndrome (FXS), the most commonly known single-gene cause of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Nevertheless, animal and human studies regarding the link between FMRP and mGluR5 expression provide inconsistent or conflicting findings about the nature of those relationships. Since multiple clinical trials of glutamatergic agents in humans with FXS did not demonstrate the amelioration of the behavioral phenotype observed in animal models of FXS, we sought measure if mGluR5 expression is increased in men with FXS to form the basis for improved clinical trials. Unexpectedly marked reductions in mGluR5 expression were observed in cortical and subcortical regions in men with FXS. Reduced mGluR5 expression throughout the living brains of men with FXS provides a clue to examine FMRP and mGluR5 expression in FXS. In order to develop the findings of our previous study and to strengthen the objective tools for future clinical trials of glutamatergic agents in FXS, we sought to assess the possible value of measuring both FMRP levels and mGluR5 expression in men with FXS. We aimed to show the value of measurement of FMRP levels and mGluR5 expression for the diagnosis and treatment of individuals with FXS and related conditions. We administered 3-[18F]fluoro-5-(2-pyridinylethynyl)benzonitrile ([18F]FPEB), a specific mGluR5 radioligand for quantitative measurements of the density and the distribution of mGluR5s, to six men with the full mutation (FM) of FXS and to one man with allele size mosaicism for FXS (FXS-M). Utilizing the seven cortical and subcortical regions affected in neurodegenerative disorders as indicator variables, adjusted linear regression of mGluR5 expression and FMRP showed that mGluR5 expression was significantly reduced in the occipital cortex and the thalamus relative to baseline (anterior cingulate cortex) if FMRP levels are held constant (F(7,47) = 6.84, p < 0.001).These findings indicate the usefulness of cerebral mGluR5 expression measured by PET with [18F]FPEB and FMRP values in men with FXS and related conditions for assessments in community facilities within a hundred-mile radius of a production center with a cyclotron. These initial results of this pilot study advance our previous study regarding the measurement of mGluR5 expression by combining both FMRP levels and mGluR5 expression as tools for meaningful clinical trials of glutamatergic agents for men with FXS. We confirm the feasibility of this protocol as a valuable tool to measure FMRP levels and mGluR5 expression in clinical trials of individuals with FXS and related conditions and to provide the foundations to apply precision medicine to tailor treatment plans to the specific needs of individuals with FXS and related conditions.

What can we learn about autism from studying fragile X syndrome?

Despite early controversy, it is now accepted that a substantial proportion of children with fragile X syndrome (FXS) meets diagnostic criteria for autism spectrum disorder (ASD). This change has led to an increased interest in studying the association of FXS and ASD because of the clinical consequences of their co-occurrence and the implications for a better understanding of ASD in the general population. Here, we review the current knowledge on the behavioral, neurobiological (i.e., neuroimaging), and molecular features of ASD in FXS, as well as the insight into ASD gained from mouse models of FXS. This review covers critical issues such as the selectivity of ASD in disorders associated with intellectual disability, differences between autistic features and ASD diagnosis, and the relationship between ASD and anxiety in FXS patients and animal models. While solid evidence supporting ASD in FXS as a distinctive entity is emerging, neurobiological and molecular data are still scarce. Animal model studies have not been particularly revealing about ASD in FXS either. Nevertheless, recent studies provide intriguing new leads and suggest that a better understanding of the bases of ASD will require the integration of multidisciplinary data from FXS and other genetic disorders.

Gaboxadol in Fragile X Syndrome: A 12-Week Randomized, Double-Blind, Parallel-Group, Phase 2a Study.

Background: Fragile X syndrome (FXS), the most common single-gene cause of intellectual disability and autism spectrum disorder (ASD), is caused by a >200-trinucleotide repeat expansion in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Individuals with FXS can present with a range of neurobehavioral impairments including, but not limited to: cognitive, language, and adaptive deficits; ASD; anxiety; social withdrawal and avoidance; and aggression. Decreased expression of the γ-aminobutyric acid type A (GABAA) receptor δ subunit and deficient GABAergic tonic inhibition could be associated with symptoms of FXS. Gaboxadol (OV101) is a δ-subunit-selective, extrasynaptic GABAA receptor agonist that enhances GABAergic tonic inhibition, providing the rationale for assessment of OV101 as a potential targeted treatment of FXS. No drug is approved in the United States for the treatment of FXS. Methods: This 12-weeks, randomized (1:1:1), double-blind, parallel-group, phase 2a study was designed to assess the safety, tolerability, efficacy, and optimal daily dose of OV101 5 mg [once (QD), twice (BID), or three-times daily (TID)] when administered for 12 weeks to adolescent and adult men with FXS. Safety was the primary study objective, with key assessments including treatment-emergent adverse events (TEAEs), treatment-related adverse events leading to study discontinuation, and serious adverse events (SAEs). The secondary study objective was to evaluate the effect of OV101 on a variety of problem behaviors. Results: A total of 23 participants with FXS (13 adolescents, 10 adults) with moderate-to-severe neurobehavioral phenotypes (Full Scale Intelligence Quotient, 41.5 ± 3.29; ASD, 82.6%) were randomized to OV101 5 mg QD (n = 8), 5 mg BID (n = 8), or 5 mg TID (n = 7) for 12 weeks. OV101 was well tolerated across all 3 treatment regimens. The most common TEAEs were upper respiratory tract infection (n = 4), headache (n = 3), diarrhea (n = 2), and irritability (n = 2). No SAEs were reported. Improvements from baseline to end-of-treatment were observed on several efficacy endpoints, and 60% of participants were identified as treatment responders based on Clinical Global Impressions-Improvement. Conclusions: Overall, OV101 was safe and well tolerated. Efficacy results demonstrate an initial signal for OV101 in individuals with FXS. These results need to be confirmed in a larger, randomized, placebo-controlled study with optimal outcomes and in the most appropriate age group. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03697161.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability and autism spectrum disorder. Individuals with FXS often present with a wide range of cognitive deficits and problem behaviors. Educational, behavioral and pharmacological interventions are used to manage these and other complex issues affecting individuals with FXS. Despite the success of preclinical models and early-phase drug clinical studies in FXS, large-scale randomized-controlled trials have failed to meet primary endpoints. Currently, no targeted or disease-modifying treatments for FXS have received regulatory approval. Here, we examined the placebo response in FXS clinical trials conducted between 2006 and 2018. Specifically, we performed a meta-analysis of placebo-treated groups in eight double-blind, randomized controlled trials. Placebo groups demonstrated significant improvements on caregiver-rated efficacy endpoints, which were greater in adolescents and adults than in children. Among the latter measures, the Visual Analog Scale scores displayed the greatest improvements, whereas the positive effects on the Vineland-II Adaptive Behavior Composite and the Aberrant Behavior Checklist-Community/fragile X version were statistically significant in both children and adolescents/adults. Although the Clinical Global Impression scale Improvement appears to have exhibited a substantial placebo effect in multiple clinical trials in FXS, limited data availability for meta-analysis, prevented us from drawing conclusions. No placebo-related improvements were observed in performance-rated measures. These findings raise substantial concerns about placebo effects in outcome measures commonly used in the randomized-controlled trials in FXS and suggest several potential improvements in the study design and implementation of such trials. Considering the small number of trials available for this study, larger and more detailed follow up meta-analyses are needed. Meanwhile, efforts to improve the measurement properties of endpoints and rater training in drug trials in FXS should be prioritized.

Reduced Expression of Cerebral Metabotropic Glutamate Receptor Subtype 5 in Men with Fragile X Syndrome.

Glutamatergic receptor expression is mostly unknown in adults with fragile X syndrome (FXS). Favorable behavioral effects of negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5) in fmr1 knockout (KO) mouse models have not been confirmed in humans with FXS. Measurement of cerebral mGluR5 expression in humans with FXS exposed to NAMs might help in that effort. We used positron emission tomography (PET) to measure the mGluR5 density as a proxy of mGluR5 expression in cortical and subcortical brain regions to confirm target engagement of NAMs for mGluR5s. The density and the distribution of mGluR5 were measured in two independent samples of men with FXS (N = 9) and typical development (TD) (N = 8). We showed the feasibility of this complex study including MRI and PET, meaning that this challenging protocol can be accomplished in men with FXS with an adequate preparation. Analysis of variance of estimated mGluR5 expression showed that mGluR5 expression was significantly reduced in cortical and subcortical regions of men with FXS in contrast to age-matched men with TD.