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Background: Fragile X syndrome, with an approximate incidence rate of 1 in 4000 males to 1 in 8000 females, is the most prevalent genetic cause of heritable intellectual disability and the most common monogenic cause of autism spectrum disorder. The full mutation of the Fragile X Messenger Ribonucleoprotein-1 gene, characterized by an expansion of CGG trinucleotide repeats (>200 CGG repeats), leads to fragile X syndrome. Currently, there are no targeted treatments available for fragile X syndrome. In a recent large multi-site trial, FXLEARN, the effects of the mGluR5 negative allosteric modulator, AFQ056 (mavoglurant), were investigated, but did not show a significant impact of AFQ056 on language development in children with fragile X syndrome aged 3–6 years. Objectives: The current analyses from biospecimens collected in the FXLEARN study aimed to determine whether AFQ056 affects the level of potential biomarkers associated with Akt/mTOR and matrix metalloproteinase 9 signaling in young individuals with fragile X syndrome. Previous research has indicated that these biomarkers play crucial roles in the pathophysiology of fragile X syndrome. Design: A double-blind placebo-controlled parallel-group flexible-dose forced titration design. Methods: Blood samples for biomarkers were collected during the FXLEARN at baseline and subsequent visits (1- and 8-month visits). Biomarker analyses included fragile X messenger ribonucleoprotein-1 genotyping by Southern blot and PCR approaches, fragile X messenger ribonucleoprotein-1 mRNA levels determined by PCR, matrix metalloproteinase 9 levels’ detection using a magnetic bead panel, and targets of the Akt/mTOR signaling pathway with their phosphorylation levels detected. Results: This research revealed that administering AFQ056 does not affect the expression levels of the investigated blood biomarkers in young children with fragile X syndrome. Conclusion: Our findings of the lack of association between clinical improvement and biomarkers’ levels in the treatment group are in line with the lack of benefit observed in the FXLEARN study. These findings indicate that AFQ056 does not provide benefits as assessed by primary or secondary endpoints. Registration: ClincalTrials.gov NCT02920892.

Abstract The fragile X mental retardation (FMR1) gene contains an expansion-prone CGG repeat within its 5′ UTR. Alleles with 55–200 repeats are known as premutation (PM) alleles and confer risk for one or more of the FMR1 premutation (PM) disorders that include Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-Associated Neuropsychiatric Disorders (FXAND). PM alleles expand on intergenerational transmission, with the children of PM mothers being at risk of inheriting alleles with > 200 CGG repeats (full mutation FM) alleles) and thus developing Fragile X Syndrome (FXS). PM alleles can be somatically unstable. This can lead to individuals being mosaic for multiple size alleles. Here, we describe a detailed evaluation of somatic mosaicism in a large cohort of female PM carriers and show that 94% display some evidence of somatic instability with the presence of a series of expanded alleles that differ from the next allele by a single repeat unit. Using two different metrics for instability that we have developed, we show that, as with intergenerational instability, there is a direct relationship between the extent of somatic expansion and the number of CGG repeats in the originally inherited allele and an inverse relationship with the number of AGG interruptions. Expansions are progressive as evidenced by a positive correlation with age and by examination of blood samples from the same individual taken at different time points. Our data also suggests the existence of other genetic or environmental factors that affect the extent of somatic expansion. Importantly, the analysis of candidate single nucleotide polymorphisms (SNPs) suggests that two DNA repair factors, FAN1 and MSH3, may be modifiers of somatic expansion risk in the PM population as observed in other repeat expansion disorders.

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.

This study contributes to a greater understanding of the utility of molecular biomarkers to identify clinical phenotypes of fragile X syndrome (FXS). Correlations of baseline clinical trial data (molecular measures—FMR1 mRNA, CYFIP1 mRNA, MMP9 and FMRP protein expression levels, nonverbal IQ, body mass index and weight, language level, NIH Toolbox, adaptive behavior rating, autism, and other mental health correlates) of 59 participants with FXS ages of 6–32 years are reported. FMR1 mRNA expression levels correlated positively with adaptive functioning levels, expressive language, and specific NIH Toolbox measures. The findings of a positive correlation of MMP-9 levels with obesity, CYFIP1 mRNA with mood and autistic symptoms, and FMR1 mRNA expression level with better cognitive, language, and adaptive functions indicate potential biomarkers for specific FXS phenotypes. These may be potential markers for future clinical trials for targeted treatments of FXS.

Fragile X syndrome (FXS) is the most frequent cause of X-linked inherited intellectual disabilities (ID) and the most frequent monogenic form of autism spectrum disorders. It is caused by an expansion of a CGG trinucleotide repeat located in the 5′UTR of the FMR1 gene, resulting in the absence of the fragile X mental retardation protein, FMRP. Other mechanisms such as deletions or point mutations of the FMR1 gene have been described and account for approximately 1% of individuals with FXS. Here, we report a 7-year-old boy with FXS with a de novo deletion of approximately 1.1 Mb encompassing several genes, including the FMR1 and the ASFMR1 genes, and several miRNAs, whose lack of function could result in the observed proband phenotypes. In addition, we also demonstrate that FMR4 completely overlaps with ASFMR1, and there are no sequencing differences between both transcripts (i.e., ASFMR1/FMR4 throughout the article).

Abstract Fragile X associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele of 55–200 CGG repeats in the Fragile X mental retardation (FMR1) gene. It is currently unknown if and when an individual carrier of a premutation allele will develop FXTAS, as clinical assessment fails to identify carriers at risk before significant neurological symptoms are evident. The primary objective of this study was to investigate the alternative splicing landscape at the FMR1 locus in conjunction with brain measures in male individuals with a premutation allele enrolled in a very first longitudinal study, compared to age-matched healthy male controls, with the purpose of identifying biomarkers for early diagnosis, disease prediction and, a progression of FXTAS. Our findings indicate that increased expression of FMR1 mRNA isoforms, including Iso4/4b, Iso10/10b, as well as of the ASFMR1 mRNAs Iso131bp, are present in premutation carriers as compared to non-carrier healthy controls. More specifically, we observed a higher expression of Iso4/4b and Iso10/10b, which encode for truncated proteins, only in those premutation carriers who developed symptoms of FXTAS over time as compared to non-carrier healthy controls, suggesting a potential role in the development of the disorder. In addition, we found a significant association of these molecular changes with various measurements of brain morphology, including the middle cerebellar peduncle (MCP), superior cerebellar peduncle (SCP), pons, and midbrain, indicating their potential contribution to the pathogenesis of FXTAS. Interestingly, the high expression levels of Iso4/4b observed both at visit 1 and visit 2 and found to be associated with a decrease in mean MCP width only in those individuals who developed FXTAS over time, suggests their role as potential biomarkers for early diagnosis of FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele (55–200 CGG repeats; PM) in the fragile X mental retardation (FMR1) gene. It is currently unknown how the observed brain changes are associated with metabolic signatures in individuals who develop the disorder over time. The primary objective of this study was to investigate the correlation between longitudinal changes in the brain (area of the pons, midbrain, and MCP width) and the changes in the expression level of metabolic biomarkers of early diagnosis and progression of FXTAS in PM who, as part of an ongoing longitudinal study, emerged into two distinct categories. These included those who developed symptoms of FXTAS (converters, CON) at subsequent visits and those who did not meet the criteria of diagnosis (non-converters, NCON) and were compared to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern Blot and PCR analysis. Magnetic Resonance Imaging (MRIs) acquisition was obtained on a 3T Siemens Trio scanner and metabolomic profile was obtained by ultra-performance liquid chromatography, accurate mass spectrometer, and an Orbitrap mass analyzer. Our findings indicate that differential metabolite levels are linked with the area of the pons between healthy control and premutation groups. More specifically, we observed a significant association of ceramides and mannonate metabolites with a decreased area of the pons, both at visit 1 (V1) and visit 2 (V2) only in the CON as compared to the NCON group suggesting their potential role in the development of the disorder. In addition, we found a significant correlation of these metabolic signatures with the FXTAS stage at V2 indicating their contribution to the progression and pathogenesis of FXTAS. Interestingly, these metabolites, as part of lipid and sphingolipid lipids pathways, provide evidence of the role that their dysregulation plays in the development of FXTAS and inform us as potential targets for personalized therapeutic development.

Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition associated with intellectual disability and behavioral problems due to the lack of the Fragile X mental retardation protein (FMRP), which plays a crucial role in synaptic plasticity and memory. A desirable in vitro cell model to study FXS would be one that can be generated by simple isolation and culture method from a collection of a non-invasive donor specimen. Currently, the various donor-specific cells can be isolated mainly from peripheral blood and skin biopsy. However, they are somewhat invasive methods for establishing cell lines from the primary subject material. In this study, we characterized a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). The urine-derived cells expressed epithelial cell surface markers via fluorescence-activated cell sorting (FACS). We observed inter, and the intra-tissue CGG mosaicism in the PBMCs and the urine-derived cells from participants with FXS potentially related to the observed variations in the phenotypic and clinical presentation FXS. We characterized these urine-derived epithelial cells for FMR1 mRNA and FMRP expression and observed some expression in the lines derived from full mutation mosaic participants. Further, FMRP expression was localized in the cytoplasm of the urine-derived epithelial cells of healthy controls. Deficient FMRP expression was also observed in mosaic males, while, as expected, no expression was observed in cells derived from participants with a hypermethylated full mutation.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability (ID) and a known monogenic cause of autism spectrum disorder (ASD). It is a trinucleotide repeat disorder, in which more than 200 CGG repeats in the 5’ untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene causes methylation of the promoter with consequent silencing of the gene, ultimately leading to the loss of the encoded fragile X mental retardation 1 protein, FMRP. FMRP is an RNA binding protein that plays a primary role as a repressor of translation of various mRNAs, many of which are involved in the maintenance and development of neuronal synaptic function and plasticity. In addition to intellectual disability, patients with FXS face several behavioral challenges, including anxiety, hyperactivity, seizures, repetitive behavior, and problems with executive and language performance. Currently, there is no cure or approved medication for the treatment of the underlying causes of FXS, but in the past few years, our knowledge about the proteins and pathways that are dysregulated by the loss of FMRP has increased, leading to clinical trials and to the path of developing molecular biomarkers for identifying potential targets for therapies. In this paper, we review candidate molecular biomarkers that have been identified in preclinical studies in the FXS mouse animal model and are now under validation for human applications or have already made their way to clinical trials.