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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.

The fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting a subset of carriers of the FMR1 (fragile X mental retardation 1) premutation. Penetrance and expression appear to be significantly higher in males than females. Although the most obvious aspect of the phenotype is the movement disorder that gives FXTAS its name, the disorder is also accompanied by progressive cognitive impairment. In this review, we address the cognitive neuropsychological and neurophysiological phenotype for males and females with FXTAS, and for male and female unaffected carriers. Despite differences in penetrance and expression, the cognitive features of the disorder appear similar for both genders, with impairment of executive functioning, working memory, and information processing the most prominent. Deficits in these functional systems may be largely responsible for impairment on other measures, including tests of general intelligence and declarative learning. FXTAS is to a large extent a white matter disease, and the cognitive phenotypes observed are consistent with what some have described as white matter dementia, in contrast to the impaired cortical functioning more characteristic of Alzheimer's disease and related disorders. Although some degree of impaired executive functioning appears to be ubiquitous among persons with FXTAS, the data suggest that only a subset of unaffected carriers of the premutation - both female and male - demonstrate such deficits, which typically are mild. The best-studied phenotype is that of males with FXTAS. The manifestations of cognitive impairment among asymptomatic male carriers, and among women with and without FXTAS, are less well understood, but have come under increased scrutiny.

Fragile X syndrome (FXS) is caused by hypermethylation of the FMR1 promoter due to the full mutation expansion (full mutation [FM]: CGG ≥ 200 repeats) and silencing of FMR1. Assessment of mosaicism for active‐unmethylated alleles has prognostic utility. This study examined relationships between FMR1 methylation in different tissues with FMR1 messenger ribonucleic acid (mRNA) and intellectual functioning in 87 males with FXS (1.89–43.17 years of age). Methylation sensitive Southern blot (mSB) and Methylation Specific‐Quantitative Melt Aanalysis (MS‐QMA) were used to examine FMR1 methylation. FMR1 mRNA levels in blood showed strong relationships with FMR1 methylation assessed using MS‐QMA in blood (n = 68; R2 = 0.597; p = 1.4 × 10−10) and buccal epithelial cells (BEC) (n = 62; R2 = 0.24; p = 0.003), with these measures also showing relationships with intellectual functioning scores (p < 0.01). However, these relationships were not as strong for mSB, with ~40% of males with only FM alleles that were 100% methylated and non‐mosaic by mSB, showing methylation mosaicism by MS‐QMA. This was confirmed through presence of detectable levels of FMR1 mRNA in blood. In summary, FMR1 methylation levels in blood and BEC examined by MS‐QMA were significantly associated with FMR1 mRNA levels and intellectual functioning in males with FXS. These relationships were not as strong for mSB, which underestimated prevalence of mosaicism. [ABSTRACT FROM AUTHOR]

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset condition characterised by cerebellar ataxia and intention tremor, usually found in individuals with FMR1 premutation alleles (PM--CGG expansion of 55-199 repeats). Population studies estimate that between 1 in 250 and 1 in 1600 men have a PM, with up to 45% of these men suggested to develop FXTAS by age 80. We used a Bayesian approach to compare the probability of finding a specific PM genotype in an ataxia population to a population control group and found an estimated penetrance of <1% (0.031%; CI 0.007% to 0.141%) for men with =70 CGGs. These findings suggest that men with a PM of =70 CGGs, who comprise the vast majority of those with a PM, have a much lower risk of being affected with FXTAS than previously suggested. This is an issue of growing importance for accurate genetic counselling, as those with a PM of =70 CGGs are increasingly detected through community carrier screening or neurodevelopmental assessment programmes. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

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.

Purpose: Developmental delay phenotypes have been associated with FMR1 premutation (PM: 55-200 CGG repeats) and "gray zone" (GZ: 45-54 CGG repeats) alleles. However, these associations have not been confirmed by larger studies to be useful in pediatric diagnostic or screening settings., Methods: This study determined the prevalence of PM and GZ alleles in two independent cohorts of 19,076 pediatric referrals to developmental delay diagnostic testing through Victorian Clinical Genetics Service (cohort 1: N = 10,235; cohort 2: N = 8841), compared with two independent general population cohorts (newborn screening N = 1997; carrier screening by the Victorian Clinical Genetics Service prepair program N = 14,249)., Results: PM and GZ prevalence rates were not significantly increased (p > 0.05) in either developmental delay cohort (male PM: 0.12-0.22%; female PM: 0.26-0.33%; male GZ: 0.68-0.69%; female GZ: 1.59-2.13-%) compared with general population cohorts (male PM: 0.20%; female PM: 0.27-0.82%; male GZ: 0.79%; female GZ: 1.43-2.51%). Furthermore, CGG size distributions were comparable across datasets, with each having a modal value of 29 or 30 and ~1/3 females and ~1/5 males having at least one allele with ≤26 CGG repeats., Conclusion: These data do not support the causative link between PM and GZ expansions and developmental-delay phenotypes in pediatric settings.