Fragile X syndrome typically results from two abnormal changes at the FMR1 locus -- one genetic and theother epigenetic. The genetic change consists of a large expansion of CGG repeats in the promoter regionof FMR1. The epigenetic change includes dense methylation of the CpG island of FMR1 in both malesand females with fragile X. The presence and extent of abnormal methylation at FMR1 is the bestmolecular predictor of cognitive impairment in fragile X syndrome. In some individuals, expanded repeatalleles escape dense methylation. These individuals are partially protected from the most severedecrement in cognitive function that occurs in fragile X syndrome, but they are at risk for late-onset FragileX associated Tremor Ataxia Syndrome (FXTAS), which appears to result from transcriptional overexpressionof FMR1. Thus, both the presence and absence of methylation at the expanded FMR1 alleleare associated with disease. In the past grant period, we developed powerful new methods to analyzemethylation patterns on both strands of individual double-stranded DMA molecules. We determined thatcell-cell mosaicism is sometimes present in fragile X individuals who were previously reported not to bemosaic. We also excluded interstrand and intersite mosaicism as possible explanations for unexpectedlyhigh levels of FMR1 mRNA in some full mutation males. In the project proposed here, we seek tounderstand origins of the wide variation in the extent of abnormal methylation of FMR1 among individualswith expanded-repeat alleles, and among the cells of those individuals. Specifically, we will establishtrans-chromososomal fragile X/human ES cell lines to investigate the origin of abnormal methylation. Wewill ask whether or not genotype at the methyltetrahydrafolate reductase (MTHFR) locus, and the levels offolate and biochemically related compounds in tissue culture media of our trans-chromososomal fragileX/human ES cells, contribute to variation in the probability of dense methylation and/or the probability ofgenetic repeat expansion at FMR1. We will also analyze existing and additional clinical data from fragile Xfamilies to ask whether or not maternal, paternal, and/or patient genotypes at the MTHFR locus arecorrelated with repeat expansion and/or methylation at FMR1. Understanding the factors that contribute toepigenetic variation at FMR1 will further our understanding of patient risks for both fragile X and FXTAS.
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