The cognitive neuropsychological phenotype of carriers of the FMR1 premutation

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

Neurocognitive and neuromotor evidence for the involvement of the fmr1 gene in female carriers of fragile x syndrome

Fragile X syndrome (FXS) is the most common single gene cause of intellectual disability and autism worldwide. FXS is caused by a long (>200) trinucleotide CGG-repeat expansion on the fragile X mental retardation 1 (FMR1) gene located on the long arm of the X chromosome, and through epigenetic gene silencing and alterations to the production of the fragile X mental retardation protein (FMRP), leads to a distinct neurological profile of abnormal synaptic structure and neuroplasticity. While FXS itself affects ~4000 individuals, it is estimated that as many as 1 in 209 females and 1 in 400 males are premutation (PM) ‘carriers’ of the FXS. For PM-carriers, the expanded CGG-repeat expansion (55-199) can lead to neurotoxic effects and progress to a late onset neurodegenerative disorder associated with executive dysfunction, dementia, tremor and ataxia, called fragile X-associated tremor/ataxia syndrome (FXTAS). Furthermore, female PM-carriers are at increased risk of developing premature menopause associated with fragile X-associated primary ovarian insufficiency (FXPOI). While much of the research to date has focussed on male PM-carriers and late-onset neurodegenerative disorders, the presence of neurocognitive and neurobehavioural manifestations among asymptomatic female PM-carriers is less well understood, but has been controversial. Although recent studies have reported difficulties in executive function, visuospatial processing and psychiatric functioning, and greater than hitherto expected prevalence of later dementia and parkinsonism-related symptoms, it remains unclear the extent to which subtle cognitive and motor manifestations are a forme fruste for later onset of more severe neurodegenerative decline, or a stable developmental phenotype. The overarching aim of this thesis was to investigate neurobehavioural profiles in adult female PM-carriers using hypothesis-driven neuromotor and neurocognitive measures that are known to be sensitive to subtle signs of dysfunction in prefrontal and cerebellar neural networks. Chapter 3 presented the first investigation of the effects of cognitive dual-task interference (counting backward by 3s or 7s) on spatiotemporal gait characteristics in female PM-carriers (22-55 years old) and age-matched controls with normal alleles, and explored relationships between dual-task gait interference and age and CGG-repeat length. The findings from Chapter 3 on gait control revealed significant dual-task costs on spatiotemporal gait characteristics in female PM-carriers compared to controls, and an interaction between age and CGG-repeat length for dual-task related gait variability. These findings indicated CGG-dose dependent effects on gait automaticity during dual-task performance, suggestive of dysfunction in cerebellar cognitive and motor networks in female PM-carriers. In Chapter 4, the extent to which these neuromotor at-risk profiles extended to postural control were investigated using hypothesis-driven measures of postural stability in response to manipulation of visual, proprioceptive and cognitive input. The results from Chapter 4 revealed significantly increased medio-lateral sway during concurrent performance of an excluded-letter-verbal-fluency task in female PM-carriers compared to controls, with CGG repeat length moderating the relationship between age and postural instability. Together, these findings suggest that measures of gait and postural control under dual-task interference may show clinical utility as outcome measures in future pharmaceutical interventions in the female PM. To further explore the role of cerebellar-cortico involvement in cognitive and psychiatric symptoms in female PM-carriers, the next section of the thesis (Chapter 5) examined the extent to which female PM-carriers showed deficits in specific subdomains of executive function, and their interrelationships with symptoms of ADHD, anxiety and depression. These findings demonstrated a core deficit in response inhibition alongside elevated symptoms of ADHD and social anxiety in females with the PM. Importantly, measures of response inhibition and working memory were significantly associated with self-reported psychiatric symptoms, and a large proportion of female carriers with poor executive functioning exceeded threshold markers for probable caseness of a mental disorder. While these findings raised the possibility that female PM-carriers may be at-risk of developing a cognitive-affective disorder, the range of cognitive, visuospatial and affective impairments is most consistent with cerebellar-cognitive affective syndrome. Chapter 6 explored implicit sequence learning impairments that may tie in a range of cognitive, visuospatial and affective symptoms. Although female PM-carriers showed preserved implicit learning, the slowed reaction time and poorer awareness of the repeating sequence were suggestive of reduced automaticity. Importantly, there were several important associations between sequence learning performance and a range of executive function, visuospatial and affective symptoms suggestive of cerebellar-cognitive affective syndrome in some females with the PM allele. The lack of age- or CGG-repeat length dependent associations with sequencing performance and cognitive-affective profiles suggests that this profile may arise from other developmental, molecular (e.g., FMRP, epigenetics) and/or environmental (psychosocial stress, carer burden) factors. The findings from this thesis converge to suggest at least two pathways, one in which developmental mechanisms may lead to a subtle cognitive-affective profile associated with disruption to cortico-cerebellar pathways, and the other to neurotoxic and ageing effects in those with long CGG-repeat lengths on neural regions underpinning stepping automaticity and postural stability. This novel hypothesis-driven approach to teasing apart cerebellar cognitive and motor profiles offers potential in identifying those PM-carrier women who are at increased risk for neuropsychiatric and neurodegenerative involvement. It will be important for future longitudinal studies to begin to isolate distinct subgroups and identify sensitive risk biomarkers in the female PM that might portend more severe neurological and neuropsychiatric impairments across the lifespan.Awards: Winner of the Mollie Holman Doctoral Medal for Excellence, Faculty of Art, Design and Architecture, [2014]

DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome

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

Neurocognitive and neuromotor evidence for the involvement of the fmr1 gene in female carriers of fragile x syndrome

Fragile X syndrome (FXS) is the most common single gene cause of intellectual disability and autism worldwide. FXS is caused by a long (>200) trinucleotide CGG-repeat expansion on the fragile X mental retardation 1 (FMR1) gene located on the long arm of the X chromosome, and through epigenetic gene silencing and alterations to the production of the fragile X mental retardation protein (FMRP), leads to a distinct neurological profile of abnormal synaptic structure and neuroplasticity. While FXS itself affects ~4000 individuals, it is estimated that as many as 1 in 209 females and 1 in 400 males are premutation (PM) ‘carriers’ of the FXS. For PM-carriers, the expanded CGG-repeat expansion (55-199) can lead to neurotoxic effects and progress to a late onset neurodegenerative disorder associated with executive dysfunction, dementia, tremor and ataxia, called fragile X-associated tremor/ataxia syndrome (FXTAS). Furthermore, female PM-carriers are at increased risk of developing premature menopause associated with fragile X-associated primary ovarian insufficiency (FXPOI). While much of the research to date has focussed on male PM-carriers and late-onset neurodegenerative disorders, the presence of neurocognitive and neurobehavioural manifestations among asymptomatic female PM-carriers is less well understood, but has been controversial. Although recent studies have reported difficulties in executive function, visuospatial processing and psychiatric functioning, and greater than hitherto expected prevalence of later dementia and parkinsonism-related symptoms, it remains unclear the extent to which subtle cognitive and motor manifestations are a forme fruste for later onset of more severe neurodegenerative decline, or a stable developmental phenotype. The overarching aim of this thesis was to investigate neurobehavioural profiles in adult female PM-carriers using hypothesis-driven neuromotor and neurocognitive measures that are known to be sensitive to subtle signs of dysfunction in prefrontal and cerebellar neural networks. Chapter 3 presented the first investigation of the effects of cognitive dual-task interference (counting backward by 3s or 7s) on spatiotemporal gait characteristics in female PM-carriers (22-55 years old) and age-matched controls with normal alleles, and explored relationships between dual-task gait interference and age and CGG-repeat length. The findings from Chapter 3 on gait control revealed significant dual-task costs on spatiotemporal gait characteristics in female PM-carriers compared to controls, and an interaction between age and CGG-repeat length for dual-task related gait variability. These findings indicated CGG-dose dependent effects on gait automaticity during dual-task performance, suggestive of dysfunction in cerebellar cognitive and motor networks in female PM-carriers. In Chapter 4, the extent to which these neuromotor at-risk profiles extended to postural control were investigated using hypothesis-driven measures of postural stability in response to manipulation of visual, proprioceptive and cognitive input. The results from Chapter 4 revealed significantly increased medio-lateral sway during concurrent performance of an excluded-letter-verbal-fluency task in female PM-carriers compared to controls, with CGG repeat length moderating the relationship between age and postural instability. Together, these findings suggest that measures of gait and postural control under dual-task interference may show clinical utility as outcome measures in future pharmaceutical interventions in the female PM. To further explore the role of cerebellar-cortico involvement in cognitive and psychiatric symptoms in female PM-carriers, the next section of the thesis (Chapter 5) examined the extent to which female PM-carriers showed deficits in specific subdomains of executive function, and their interrelationships with symptoms of ADHD, anxiety and depression. These findings demonstrated a core deficit in response inhibition alongside elevated symptoms of ADHD and social anxiety in females with the PM. Importantly, measures of response inhibition and working memory were significantly associated with self-reported psychiatric symptoms, and a large proportion of female carriers with poor executive functioning exceeded threshold markers for probable caseness of a mental disorder. While these findings raised the possibility that female PM-carriers may be at-risk of developing a cognitive-affective disorder, the range of cognitive, visuospatial and affective impairments is most consistent with cerebellar-cognitive affective syndrome. Chapter 6 explored implicit sequence learning impairments that may tie in a range of cognitive, visuospatial and affective symptoms. Although female PM-carriers showed preserved implicit learning, the slowed reaction time and poorer awareness of the repeating sequence were suggestive of reduced automaticity. Importantly, there were several important associations between sequence learning performance and a range of executive function, visuospatial and affective symptoms suggestive of cerebellar-cognitive affective syndrome in some females with the PM allele. The lack of age- or CGG-repeat length dependent associations with sequencing performance and cognitive-affective profiles suggests that this profile may arise from other developmental, molecular (e.g., FMRP, epigenetics) and/or environmental (psychosocial stress, carer burden) factors. The findings from this thesis converge to suggest at least two pathways, one in which developmental mechanisms may lead to a subtle cognitive-affective profile associated with disruption to cortico-cerebellar pathways, and the other to neurotoxic and ageing effects in those with long CGG-repeat lengths on neural regions underpinning stepping automaticity and postural stability. This novel hypothesis-driven approach to teasing apart cerebellar cognitive and motor profiles offers potential in identifying those PM-carrier women who are at increased risk for neuropsychiatric and neurodegenerative involvement. It will be important for future longitudinal studies to begin to isolate distinct subgroups and identify sensitive risk biomarkers in the female PM that might portend more severe neurological and neuropsychiatric impairments across the lifespan.<div><br></div><div> Awards: Winner of the Mollie Holman Doctoral Medal for Excellence, Faculty of Art, Design and Architecture, 2014.</div

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

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

Inter-group mRNA level comparison plots between 33 PM and 33 control females.

<p><i>FMR1</i> mRNA was normalised to either <b>(A)</b> <i>GUS</i> alone; <b>(B)</b> 3IC (<i>GUS</i>, <i>EIF4A2</i> and <i>SDHA</i>); or <b>(C)</b> 2IC (<i>EIF4A2</i> and <i>SDHA</i>, without <i>GUS</i>). <b>(D)</b> <i>GUS</i> mRNA levels were normalised to 2IC (<i>EIF4A2</i> and <i>SDHA</i>). <b>(E)</b> Variability in <i>EIF4A2</i> to <i>SDHA</i> mRNA ratio (<i>EIF4A2</i> and <i>SDHA)</i>, between groups is also presented. <b>Note:</b> Broken horizontal lines indicate the maximum control value for each plot with percentages above this line indicating the proportion of PM females with abnormally increased <i>FMR1</i> mRNA levels. Control and PM CGG groups reflect range in P values correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192151#pone.0192151.t001" target="_blank">Table 1</a> (30). Interquartile range (IQR); maximum value (MAX); minimum value (MIN).</p