Ultra-deep DNA methylation analysis of X-linked genes: GLA and AR as model genes

Recessive X-linked disorders may occasionally evolve in clinical manifestations of variable severity also in female carriers. For some of such diseases the frequency of symptoms appearance during women's life may be particularly relevant. This phenomenon has been largely attributed to the potential skewness of X-inactivation process leading to variable phenotypes. Nonetheless, in many cases, no correlation with X-inactivation unbalance was demonstrated. However, methods for analyzing skewness have been mainly limited to Human Androgen Receptor methylation analysis (HUMARA). Recently, the X-inactivation process has been largely revisited involving heterogeneity among loci in the epigenetic state within inactive and, possibly, active X-chromosomes. We reasoned that gene-specific and ultra-deep DNA methylation analyses could greatly help to unravel details of the X-inactivation process and the roles of specific X genes inactivation in disease manifestations. We recently provided evidence that studying DNA methylation at specific loci at single molecule resolution (epiallele distribution analysis) allows one to analyze cell-to-cell methylation differences in a given cell population.We here applied the epiallele analysis at two X-linked loci to investigate whether females shows allele-specific epiallelic patterns. Due to the high potential of this approach, the method allowed us to obtain clearly distinct allele-specific epiallele profiles.