Figure 5.

CpG methylation status at codon 468. (A) DHPLC profiles showing missing of PTPN11 exon 12 PCR product in HeLa cells treated with the DNA demethylating agent 5-azadC and digested with the methylation-sensitive enzyme HpyCH4 IV. (B) DHPLC profiles showing amplification of PTPN11 exon 12 from genomic DNA obtained from peripheral lymphocites of a subject heterozygous for the LS-causing C1403T mutation and unaffected parents, digested or not with the HpyCH4 IV endonuclease. Efficiency of the treatment was confirmed by digestion of an amplified genomic fragment encompassing exon 12 and subsequent re-amplification of the digested product. (C) Methylation-specific PCR assay performed on genomic DNA obtained from the same members of the family. Amplification was carried out using primers opportunely designed to amplify either the methylated (M) or the unmethylated (UM) alleles.