Ultra-Deep DNA Methylation Analysis of X-Linked Genes: GLA and AR as Model Genes

Abstract

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 the symptoms’ appearance during women’s life may be particularly relevant. This phenomenon has been largely attributed to the potential skewness of the 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, highlighting the heterogeneity existing 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 autosomic loci at a single-molecule resolution (epiallele distribution analysis) allows one to analyze cell-to-cell methylation differences in a given cell population. We here apply the epiallele analysis at two X-linked loci to investigate whether females show allele-specific epiallelic patterns. Due to the high potential of this approach, the method allows us to obtain clearly distinct allele-specific epiallele profiles.

DNA methylation impact on Fabry disease

“…Experiments performed to establish the GLA methylation state, distinguishing mutant and non-mutant alleles, included the use of methylation-sensitive restriction enzymes or bisulfite treatment of genomic DNA followed by PCR amplification of GLA promoter [ 17 , 18 , 21 , 44 – 46 ]. The results of these experiments revealed that the amount of methylation of non-mutant allele correlated with the severity of the disease [ 55 ]. Single molecule methylation analysis and epiallele distribution analysis, obtained by high coverage bisulfite sequencing of GLA gene, promise to help to predict the evolution of expression potential overtime.…”

“…[ 45 ] 4 FD women Fibroblast from Skin tissue Allele-specific GLA expression (RT-PCR) mRNA expression level of the GLA mutant allele correlated with disease severity De Riso et al . [ 55 ] 3 FD women Peripheral blood High coverage-amplicon bisulfite sequencing (HC-ABS) versus HUMARA Substantial concordance in direction and entity of the methylation imbalance between AR and GLA genes. Clearly distinct allele-specific epiallele profiles were obtained by epiallele distribution analysis Rossanti et al .…”

“…The epiallele distribution analysis was performed by high coverage-amplicon bisulfite sequencing ( HC-ABS) consisting in the frequency determination of each methylated CpG combination among hundred thousands molecules analyzed after bisulfite treatment, amplification of specific loci and high coverage sequencing by next generation methods. Such ultra-deep DNA methylation analysis was subsequently applied to track two X-linked loci, GLA and AR, and to investigate whether females showed allele-specific epiallelic patterns [ 55 ]. The peripheral human whole blood from 3 women was analyzed in parallel by HUMARA and High Coverage-Amplicon Bisulfite Sequencing (HC-ABS) approaches targeting AR and GLA genes.…”

DNA Methylation Profiles of Tph1A and BDNF in Gut and Brain of L. Rhamnosus-Treated Zebrafish

“…Indeed, this approach mimics a single-cell analysis since it provides “epiallele” distribution profiles. As we and others have previously reported in detail [ 28 , 31 , 32 , 33 ], the term “epiallele” refers to a specific arrangement of methyl-CpG in a given gene region. Combining together all the epialleles that bear the same number of methylated CpGs regardless of their position, it is possible to obtain the amount of a specific “epiallele class”.…”

Modeling DNA Methylation Profiles through a Dynamic Equilibrium between Methylation and Demethylation

“…Using high-coverage deep bisulfite amplicon sequencing (HC-ABS) [ 30 , 31 ], we performed an in-depth DNA methylation profiling of two non-housekeeping genes, DDO and DAO, from two different species (mouse and human). For each gene, specific genomic regions near the promoter were chosen, for which we previously had provided evidence of a relationship between the DNA methylation and the gene transcriptional status [ 25 , 32 ].…”