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Table S1. Comparison between males and females intellectual functioning (standard) scores. Table S2. Comparison between FM-only males with complete and incomplete FMR1 mRNA silencing on intellectual functioning (corrected) scores and autism features. Table S3. Relationship between intellectual functioning (standard) scores and FMR1 mRNA in males and females. Table S4. Relationship between intellectual functioning scores (corrected) and FMR1 mRNA in males and females with WISC-III (Chile) and MSEL removed. Table S5. Relationship between intellectual functioning (standard) scores with FMR1 mRNA in FM-only males with incomplete FMR1 mRNA silencing and PM/FM mosaic males. (DOCX 27 kb)

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.

Additional file 1. Note S1–S4 and Table S1–S15 Clinical Phenotype of FXS.

Additional file 1. Note S1–S4 and Table S1–S15 Clinical Phenotype of FXS.