Origins of Variarion in Abnormal FMR1 Methylation in Fragile X Syndrome

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