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ItemIntragenic DNA methylation and neurodevelopmental outcomes in children with fragile X-related disordersArpone, Marta ( 2019)The type and severity of clinical involvement in children with fragile X syndrome (FXS) and disorders related to premutations (PM) of the fragile X mental retardation 1 gene (FMR1), herein collectively denoted as fragile X-related disorders (FXDs), is highly variable. Multiple molecular factors contribute to the heterogeneity of neurodevelopmental outcomes. Increased intragenic DNA methylation (DNAm) in blood of the fragile X-related epigenetic element 2 (FREE2) region, located at the FMR1 exon 1/intron 1 boundary, was associated with lower intellectual functioning in a cohort of female children and adults with FXS and with neuro-cognitive and psychiatric phenotypes in women with PM. Nevertheless, FREE2 DNAm has not yet been investigated in exclusively paediatric male and female FXDs cohorts. The overarching aim of this thesis was to explore FREE2 DNAm and neurodevelopmental outcomes of Australian male and female children with FXDs. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry and methylation specific-quantitative melt analysis were used to analyse FREE2 DNAm in venous blood, buccal epithelial cells (BEC) and retrieved newborn blood spots (NBS). In addition, FMR1 mRNA levels in blood were assessed using real-time polymerase chain reaction (PCR) relative standard curve method. The evaluation of the neurodevelopmental outcomes concentrated on direct clinical assessment of intellectual functioning and autism spectrum disorder (ASD) symptom severity. Intelligence Quotient (IQ) scores were corrected for floor effect using the Whitaker and Gordon (WG) extrapolation method. The findings highlighted the variability of the clinical presentation of children with PM. Results also showed that compared to sex-matched paediatric controls, children with FXS had significantly higher levels of FREE2 DNAm levels in blood and BEC and, within the FXS group, higher FREE2 DNAm levels in blood correlated with lower FMR1 mRNA levels. In children with FXS, the application of the WG method effectively addressed the floor effect inherent in standardised intelligence scales, unmasked inter-individual variability in IQ scores and uncovered significant associations between intragenic DNAm and neurodevelopmental outcomes. Strength and statistical significance of these epigenotype-phenotype relationships varied based on sex, position of the differentially methylated sites, tissue analysed, assay used and neurodevelopmental domain investigated. The most significant finding was in males with FXS, for whom higher levels of BEC FREE2 DNAm were associated with lower WG-corrected Full Scale IQ (cFSIQ) and Performance IQ (cPIQ) scores. Finally, findings showed that the best-performing FREE2 biomarker had sensitivity, specificity, negative and positive predictive values of 100% for detection of full mutation alleles in NBS of males and females with FXS. Additionally, this study revealed that for males with FXS, FREE2 DNAm in NBS was significantly associated with lower cFSIQ and cPIQ scores obtained in childhood and adolescence. This is the first study in any monogenic neurodevelopmental disorder associated with intellectual disability, showing that a perinatal epigenetic biomarker is significantly associated with paediatric neuropsychological outcomes. In conclusion, the results of this thesis contribute to the characterisation of the neurodevelopmental outcomes in children with FXDs, provide evidence that FREE2 DNAm is a sensitive epigenetic biomarker significantly associated with the variability of intellectual functioning in male children with FXS, and may have implications for the development of new methylation specific tests for earlier diagnosis with potential prognostic applications.