Fig. 1 | Clinical Epigenetics

Fig. 1

From: DNA methylation impact on Fabry disease

Fig. 1

Different possible methods to analyze allele-specific methylation at GLA gene. Schematic representation of GLA gene showing promoter, exons and introns positions (not in scale) and exemplificative positions of hypothetical point mutations. The methylation state at promoter region is the best candidate factor able to regulate GLA gene expression. In order to identify single molecules containing information necessary to distinguish the two alleles and at the same time methylation profiles, different approaches can be used according to specific cases. In the case in which the mutation occurs close to promoter region (e.g., in exon 1), it is possible to perform amplicon bisulfite sequencing (a) with amplicon length ranging between 200 and 600 bp and then distinguish alleles by bioinformatic analyses, as performed by Echevarria et al. [16]. If mutation is located at downstream exons (e.g., exon 5), it is possible to analyze allele-specific DNA methylation at a region surrounding mutations that may be not informative for regulation of the gene (b), unless in these patients a polymorphisms in the promoter region may be associated with the mutated allele (e.g., following other members of the same family) as shown in De Riso et al. [56]. Alternatively, Nanopore sequencing may analyze much longer regions allowing to match methylation status of the promoter with mutation present in any position of the gene on the same molecule (c). Advantages of using this method are that it is possible to perform methylation analyses without PCR amplification and bisulfite treatment. However, it may be necessary to isolate the whole GLA genomic region by in vitro CRISPR Cas9 system [58]

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