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The fragile X mental retardation 1 gene (FMR1)-related disorder fragile X syndrome (FXS) is the most common heritable form of cognitive impairment and the second most common cause of comorbid autism. FXS usually results when a premutation trinucleotide CGG repeat in the 5' untranslated region of the FMR1 gene (CGG 55-200) expands over generations to a full mutation allele (CGG >200). This expansion is associated with silencing of the FMR1 promoter via an epigenetic mechanism that involves DNA methylation of the CGG repeat and the surrounding regulatory regions. Decrease in FMR1 transcription is associated with loss of the FMR1 protein that is needed for typical brain development. The past decade has seen major advances in our understanding of the genetic and epigenetic processes that underlie FXS. Here we review these advances and their implications for diagnosis and treatment for individuals who have FMR1-related disorders. WHAT THIS PAPER ADDS: Improved analysis of DNA methylation allows better epigenetic evaluation of the fragile X gene. New testing techniques have unmasked interindividual variation among children with fragile X syndrome. New testing methods have also detected additional cases of fragile X. [ABSTRACT FROM AUTHOR]

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55–199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10−11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships. [ABSTRACT FROM AUTHOR]

Highlights: [•] Female premutation carriers have a defective CGG trinucleotide expansion on the FMR1 gene. [•] This CGG expansion is associated with a high risk for ataxia motor symptoms. [•] We showed enhanced dual-task postural displacement in asymptomatic female carriers. [•] Dual-task postural displacement also associated with age- and CGG-length. [•] Postural control paradigms may identify early cerebellar changes in female carriers. [Copyright &y& Elsevier]

Numerous variants of verbal fluency tasks exist within clinical and research domains that purport to measure "executive function." However, to date, there has been a paucity of research examining what specific abilities are measured by these tasks. In this study, the relationships between a select group of cognitive constructs and phonemic, semantic, alternating, and excluded-letter verbal fluency tests were examined in 93 young healthy individuals (aged 18 to 35 years old). Forward-selection multiple regression analyses were performed for each fluency task. Phonemic fluency was associated with verbal intellectual function and processing speed; semantic fluency was associated with working memory and semantic word retrieval; excluded-letter fluency was associated with processing speed; and alternating fluency was associated with semantic word retrieval. These results highlight verbal intellectual function, processing speed, and semantic word-retrieval contributions to verbal fluency performances. The main conclusion from this study is that the abilities associated with verbal fluency performance in a young healthy population are multifactorial and differ across fluency variants. These findings progress our theoretical understanding of what is measured by different verbal fluency tasks and will assist interpretation of performance. [ABSTRACT FROM AUTHOR]

Abstract: For years, premutation-carriers of fragile X-syndrome (FXS) were assumed free from any deleterious phenotype. In this review, we discuss the current literature on neurocognitive, emotional and neuromotor profiles emerging in females with the fragile-X premutation, and discuss phenotypic profiles in male premutation-carriers to gain insights into possible underlying mechanisms associated with FMR1 gene expression. We contend that this emerging phenotypic profile in females with the fragile-X premutation needs further investigation using experimentally-driven tasks sensitive to neural networks especially vulnerable to FMR1 gene expression. Further investigation of developmental aspects of the female carrier profile is needed to determine the extent to which emotional, cognitive and neurobehavioural challenges indicate at-risk profiles for later degenerative decline, or rather a stable developmental phenotype. These future research avenues will provide critical new information which will enable identification of women at greatest risk for subtle age-dependent neurobehavioural changes well before the onset of more serious clinical consequences alongside the identification of biomarkers which may be useful in establishing the efficacy of future therapeutic interventions. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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]

Abstract: Recent studies report a higher risk of dementia and motor symptoms in females with the fragile X mental retardation 1 premutation (PM-carriers) than has hitherto been appreciated. Here, we use dual-task gait paradigms to identify potential markers of cognitive and motor decline in female PM-carriers. Spatiotemporal gait characteristics and variability of gait were assessed during single- and dual-task conditions in 28 female PM-carriers (mean age 41.32 ± 8.03 years) and 31 female controls with normal fragile X mental retardation 1 alleles (mean age 41.61 ± 8.30 years). Despite comparable gait characteristics at baseline, gait performance was significantly poorer for PM-carriers when performing concurrent working memory tasks (counting backwards by 3′s or 7′s) when compared with controls. Correlational analyses showed that low working memory capacity was significantly associated with dual-task interference for the gait domains of pace (speed, step length) and variability (step time, swing time) in PM-carriers. Multiple regression analyses further showed that the interaction between age and CGG repeat length was strongly predictive of gait variability during dual-task performance. These findings indicate for the first time that vulnerability in specific domains of gait control may act as sensitive surrogate markers of future decline in female PM-carriers. [Copyright &y& Elsevier]

There is now growing evidence of cognitive weakness in female premutation carriers (between 55 and 199 CGG repeats) of the fragile X mental retardation gene, including impairments associated with executive function. While an age-related decline in assessments of executive function has been found for male premutation carriers, few studies have explored whether female carriers show a similar trajectory with age. A total of 20 female premutation carriers and 21 age- and IQ-matched healthy controls completed a battery of tasks assessing executive function tasks, including the behavioural dyscontrol scale (BDS), symbol digit modalities test (SDMT), paced auditory serial addition test (PASAT), Haylings sentence completion test and the digit span task (forward and backward). Performance was compared between premutation carriers and healthy controls, and the association between task performance and age was also ascertained. Compared to controls, female premutation carriers had significant impairment on the BDS, SDMT, PASAT, and Haylings sentence completion task, all of which rely on quick, or timed, responses. Further analyses revealed no significant association between age and task performance for either premutation carriers or controls. This study demonstrates that a cohort of female premutation carriers have deficits on a range of tasks of executive function that require the rapid temporal resolution of responses. We propose that the understanding of the phenotype of premutation carriers will be advanced through use of such measures. [ABSTRACT FROM AUTHOR]

Despite their widespread use in research and clinical practice, the cognitive abilities purportedly assessed by different verbal fluency task variants remain unclear and may vary across different healthy and clinical populations. The overarching aim of this study was to identify which cognitive abilities contribute to phonemic, semantic, excluded letter, and alternating verbal fluency tasks and whether these contributions differ across younger and older healthy adults. Method: Ninety-six younger (18–36 years) and 83 older (65–87 years) healthy participants completed measures of estimated verbal intelligence, semantic retrieval, processing speed, working memory, and inhibitory control, in addition to phonemic, semantic, excluded letter, and alternating fluency tasks. Eight hierarchical multiple regressions were conducted across the four fluency variants and two age groups to identify which cognitive variables uniquely contributed to these fluency tasks. Results: In the younger group, verbal intelligence and processing speed contributed to phonemic fluency, semantic retrieval to semantic fluency, processing speed and working memory to excluded letter fluency, and semantic retrieval to alternating fluency. In contrast, in the older group, verbal intelligence contributed to phonemic fluency, no cognitive variables contributed to semantic fluency, inhibition to excluded letter fluency, and verbal intelligence to alternating fluency. Conclusions: Our findings highlight that both lower and higher order cognitive skills contribute to verbal fluency tasks; however, these contributions vary considerably across fluency variants and age groups. The heterogeneity of cognitive determinants of verbal fluency, across variants and age, may explain why older people performed less proficiently on semantic and excluded letter fluency tasks while no age effects were found for phonemic and alternating fluency. Interpretation of verbal fluency performances need to be tailored according to which verbal fluency variant and age group are used. [ABSTRACT FROM AUTHOR]

Highlights: [•] Similar behavioural self-regulation profile found in female PM-carriers as males. [•] Female PM-carriers committed more inhibitory errors than controls. [•] Equivalent saccade profiles for PM-carriers and controls. [•] Negative correlation between CGG genetic data and inhibitory errors. [Copyright &y& Elsevier]

Fragile X mental retardation 1 (FMR1) premutation carriers (PM-carriers) are characterised as having mid-sized expansions of between 55 and 200 CGG repeats in the 5′ untranslated region of the FMR1 gene. While there is evidence of executive dysfunction in PM-carriers, few studies have explicitly explored working memory capabilities in female PM-carriers. 14 female PM-carriers and 13 age- and IQ-matched healthy controls completed an ocular motor n-back working memory paradigm. This task examined working memory ability and the effect of measured increases in cognitive load. Female PM-carriers were found to have attenuated working memory capabilities. Increasing the cognitive load did not elicit the expected reciprocal increase in the task errors for female PM-carriers, as it did in controls. However female PM-carriers took longer to respond than controls, regardless of the cognitive load. Further, FMR1 mRNA levels were found to significantly predict PM-carrier response time. Although preliminary, these findings provide further evidence of executive dysfunction, specifically disruption to working memory processes, which were found to be associated with increases in FMR1 mRNA expression in female PM-carriers. With future validation, ocular motor paradigms such as the n-back paradigm will be critical to the development of behavioural biomarkers for identification of PM-carrier cognitive-affective phenotypes. [ABSTRACT FROM AUTHOR]

Background: Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the FMR1 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. [ABSTRACT FROM AUTHOR]