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Background: Despite the increasing number of clinical trials involving children with neurodevelopmental disorders, appropriate and objective outcome measures for behavioral symptoms are still required., Aim: This study assessed the agreement between parents' and clinical researchers' ratings of behavioral problem severity in children with fragile X syndrome (FXS) and chromosome 15 imprinting disorders., Methods and Procedures: The cohort comprised 123 children (64% males), aged 3-17 years, with FXS (n = 79), Prader-Willi (PWS; n = 19), Angelman (AS; n = 15), and Chromosome 15q duplication (n = 10) syndromes. Specific items from the Autism Diagnostic Observation Schedule-Second Edition and Aberrant Behavior Checklist-Community Edition mapping to corresponding behavioral domains were selected ad-hoc, to assess behavioral problems., Outcomes and Results: Inter-rater agreement for the cohort was slight for self-injury (Intraclass Correlation Coefficient (ICC) = 0.12), fair for tantrums/aggression (0.24) and mannerisms/stereotypies (0.25), and moderate for hyperactivity (0.48). When stratified by diagnosis, ICC ranged from poor (0; self-injury, AS and PWS) to substantial (0.48; hyperactivity, females with FXS)., Conclusions and Implications: The high level of inter-rater disagreement across most domains suggests that parents' and researchers' assessments led to discrepant appraisal of behavioral problem severity. These findings have implications for treatment targets and outcome measure selection in clinical trials, supporting a multi-informant approach., Competing Interests: Declarations of conflicting interests All authors declare no other competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2-17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene ( FMR1 ) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥ 200 CGG repeats, and a decrease in FMR1 mRNA and its protein. However, incomplete silencing from FM alleles has been associated with more severe autism features in FXS males. This study compared scores on the Aberrant Behavior Checklist-Community-FXS version (ABC-C FX ) in 62 males affected with FXS (3 to 32 years) stratified based on presence or absence of mosaicism and/or FMR1 mRNA silencing. Associations between ABC-C FX subscales and FMR1 mRNA levels, assessed using real-time PCR relative standard curve method, were also examined. The FXS group mosaic for premutation (PM: 55-199 CGGs) and FM alleles had lower irritability (p = 0.014) and inappropriate speech (p < 0.001) scores compared to males with only FM alleles and complete loss of FMR1 mRNA. The PM/FM mosaic group also showed lower inappropriate speech scores compared to the incomplete silencing (p = 0.002) group. Increased FMR1 mRNA levels were associated with greater irritability (p < 0.001), and lower health-related quality of life scores (p = 0.004), but only in the incomplete silencing FM-only group. The findings suggest that stratification based on CGG sizing and FMR1 mRNA levels may be warranted in future research and clinical trials utilising ABC-C FX subscales as outcome measures.

Background: Fragile X syndrome (FXS) is a common cause of intellectual disability and autism spectrum disorder (ASD) usually associated with a CGG expansion, termed full mutation (FM: CGG ≥ 200), increased DNA methylation of the FMR1 promoter and silencing of the gene. Mosaicism for presence of cells with either methylated FM or smaller unmethylated pre-mutation (PM: CGG 55-199) alleles in the same individual have been associated with better cognitive functioning. This study compares age- and sex-matched FM-only and PM/FM mosaic individuals on intellectual functioning, ASD features and maladaptive behaviours., Methods: This study comprised a large international cohort of 126 male and female participants with FXS (aged 1.15 to 43.17 years) separated into FM-only and PM/FM mosaic groups (90 males, 77.8% FM-only; 36 females, 77.8% FM-only). Intellectual functioning was assessed with age appropriate developmental or intelligence tests. The Autism Diagnostic Observation Schedule-2nd Edition was used to examine ASD features while the Aberrant Behavior Checklist-Community assessed maladaptive behaviours., Results: Comparing males and females (FM-only + PM/FM mosaic), males had poorer intellectual functioning on all domains (p < 0.0001). Although females had less ASD features and less parent-reported maladaptive behaviours, these differences were no longer significant after controlling for intellectual functioning. Participants with PM/FM mosaicism, regardless of sex, presented with better intellectual functioning and less maladaptive behaviours compared with their age- and sex-matched FM-only counterparts (p < 0.05). ASD features were similar between FM-only and PM/FM mosaics within each sex, after controlling for overall intellectual functioning., Conclusions: Males with FXS had significantly lower intellectual functioning than females with FXS. However, there were no significant differences in ASD features and maladaptive behaviours, after controlling for intellectual functioning, independent of the presence or absence of mosaicism. This suggests that interventions that primarily target cognitive abilities may in turn reduce the severity of maladaptive behaviours including ASD features in FXS.

Background: Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR 1 product (FMRP), mosaicism for active and inactive FMR1 alleles, including alleles termed premutation (PM: 55-199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete FMR1 mRNA silencing from FM alleles and/or levels of FMR1 mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS., Methods: The study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1-43 years. A control group of 14 females were used to establish control FMR1 mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. FMR1 mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method., Results: Females with FXS had significantly higher levels of FMR1 mRNA ( p  < 0.001) than males. FMR1 mRNA levels were positively associated with age ( p  < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM FMR1 mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced FMR1 ( p  = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of FMR1 mRNA were associated with decreased intellectual functioning in FXS males ( p  = 0.029), but not autism features, when combined with the PM/FM mosaic group., Conclusion: Incomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS., Competing Interests: All procedures were approved by The Royal Children’s Hospital and INTA Human Research Ethics Committees (HREC #33066 and #15, respectively). All parents/caregivers provided written informed consent and those who were deemed cognitively able also provided written informed consent.D. Godler is an inventor of the following patents: PCT/AU2010/001134; filing No. AU2010/903595; filing No. AU2011/902500; and filing No. 2013/900227, related to the technology described in this publication. All other authors have no conflicts of interest to declare.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

CGG repeat expansion >200 within FMR1, termed full mutation (FM), has been associated with promoter methylation, consequent silencing of gene expression and fragile X syndrome (FXS)-a common cause of intellectual disability and co-morbid autism. Unmethylated premutation (55-199 repeats) and FM alleles have been associated with fragile X related tremor/ataxia syndrome (FXTAS), a late onset neurodegenerative disorder. Here we present a 33-year-old male with FXS, with white matter changes and progressive deterioration in gait with cerebellar signs consistent with probable FXTAS; there was no evidence of any other cerebellar pathology. We show that he has tissue mosaicism in blood, saliva, and buccal samples for the size and methylation of his expanded alleles and a de novo, unmethylated microdeletion. This microdeletion involves a ∼80 bp sequence in the FMR1 promoter as well as complete loss of the CGG repeat in a proportion of cells. Despite FMR1 mRNA levels in blood within the normal range, the methylation and CGG sizing results are consistent with the diagnosis of concurrent FXS and probable FXTAS. The demonstrated presence of unmethylated FM alleles would explain the manifestation of milder than expected cognitive and behavioral impairments and early onset of cerebellar ataxia. Our case suggests that individuals with FXS, who manifest symptoms of FXTAS, may benefit from more detailed laboratory testing. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females., Methods: We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples., Results: In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA., Conclusions: MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes., (© 2014 The American Association for Clinical Chemistry.)

Fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥ 200 CGG repeats, and a decrease in FMR1 mRNA and its protein. However, incomplete silencing from FM alleles has been associated with more severe autism features in FXS males. This study compared scores on the Aberrant Behavior Checklist-Community-FXS version (ABC-CFX) in 62 males affected with FXS (3 to 32 years) stratified based on presence or absence of mosaicism and/or FMR1 mRNA silencing. Associations between ABC-CFX subscales and FMR1 mRNA levels, assessed using real-time PCR relative standard curve method, were also examined. The FXS group mosaic for premutation (PM: 55–199 CGGs) and FM alleles had lower irritability (p = 0.014) and inappropriate speech (p < 0.001) scores compared to males with only FM alleles and complete loss of FMR1 mRNA. The PM/FM mosaic group also showed lower inappropriate speech scores compared to the incomplete silencing (p = 0.002) group. Increased FMR1 mRNA levels were associated with greater irritability (p < 0.001), and lower health-related quality of life scores (p = 0.004), but only in the incomplete silencing FM-only group. The findings suggest that stratification based on CGG sizing and FMR1 mRNA levels may be warranted in future research and clinical trials utilising ABC-CFX subscales as outcome measures. [ABSTRACT FROM AUTHOR]

Mosaicism for FMR1 premutation (PM: 55-199 CGG)/full mutation (FM: >200 CGG) alleles or the presence of unmethylated FM (UFM) have been associated with a less severe fragile X syndrome (FXS) phenotype and fragile X associated tremor/ataxia syndrome (FXTAS)--a late onset neurodegenerative disorder. We describe a 38 year old male carrying a 100% methylated FM detected with Southern blot (SB), which is consistent with complete silencing of FMR1 and a diagnosis of fragile X syndrome. However, his formal cognitive scores were not at the most severe end of the FXS phenotype and he displayed tremor and ataxic gait. With the association of UFM with FXTAS, we speculated that his ataxia might be related to an undetected proportion of UFM alleles. Such UFM alleles were confirmed by more sensitive PCR based methylation testing showing FM methylation between 60% and 70% in blood, buccal, and saliva samples and real-time PCR analysis showing incomplete silencing of FMR1. While he did not meet diagnostic criteria for FXTAS based on MRI findings, the underlying cause of his ataxia may be related to UFM alleles not detected by SB, and follow-up clinical and molecular assessment are justified if his symptoms worsen. [ABSTRACT FROM AUTHOR]