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    The significance of low level mosaicism in Fragile X Syndrome.
    Aliaga, Solange ( 2017)
    Fragile X Syndrome (FXS) is the most common single disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD). FXS is fundamentally caused by a trinucleotide CGG sequence repeat expansion within the 5’ untranslated portion of the FMR1 gene, to more than 200 repeats which is called Full Mutation (FM). This is associated with abnormal methylation of this gene’s promoter and silencing of FMR1 expression. To date, the type and severity of the neurocognitive phenotype has been related more closely with the methylation status than with the number of CGG repeats in the FM range. Smaller CGG expansions are called premutation (PM) (55 and 200 CGGs), grey zone (GZ) (45-54 CGGs) and normal size (<44 CGGs) alleles, and are usually associated with presence of an unmethylated FMR1 promoter, and normal FMR1 expression. There is a proportion of FXS individuals in which two or more population of cells can be found, and who are called mosaic or mosaicism. These individuals possess cells with FM and smaller CGG sizes (e.g. Normal, GZ or PM), which lead to milder forms of the FXS phenotype. This PhD explores the clinical and diagnostic significance of low level mosaicism (LLM) and DNA methylation variation amongst different CpG sites within FMR1. The first aim of this study was to define the lower limit of detection LLM of six FMR1 tests currently used in diagnostic laboratories. This was achieved using three FMR1 PCR commercial kits (AmplideXTM, X-Sense and FastFraXTM) targeting the CGG region and three methylation tests (Methylation Specific- Quantitative Melt Analysis [MS-QMA], Sequenom®EpiTYPER system [MALDI-TOF MS] and mSouthern blot) targeting methylation of Exon1/Intron1 boundary region and specific CpG sites within FMR1 promoter. The second aim was to screen a large number of males with ID and ASD of unknown etiology referred for FXS testing to determine the prevalence of mosaicism containing FM together with a normal or GZ allele (cryptic FMR1-FM) who are not being identified as a part of standard FXS testing. This was achieved using MS-QMA and the positive results were confirmed with the FMR1 tests assessed in Aim 1. The third aim of this study was to examine the association between DNA methylation of FMR1 and mRNA levels and severity of intellectual functioning impairment in individuals with a cryptic FMR1-FM identified in Aim 2 and males and females with typical FXS (only FM and PM/FM alleles). Three significant contributions to the FXS field resulting from this PhD are: i) implementation of a new highly sensitive assay for detection LLM missed by standard testing; ii) determination of the prevalence of cryptic FM-FMR1 in males with idiopathic ID/ASD referred for FXS testing; iii) clinical and molecular characterization of LLM and its methylation characterization amongst tissues and CpG sites; and demonstration that LLM levels are significantly correlated with severity of cognitive impairment and ASD as well as FMR1 mRNA levels.