Prader-Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method.

The putative promoter region of the SNRPN gene contains a CpG island which is heavily methylated in the maternally derived allele and unmethylated in the paternally derived allele. In patients with Prader-Willi syndrome (PWS) only the methylated allele is present, while in those with Angelman syndrome (AS) only the unmethylated allele is present. The purpose of this paper is to report a polymerase chain reaction (PCR)-based assay to evaluate methylation status of the CpG island of the SNRPN gene and to show that this assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast, bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite-modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allele-specific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfite-modified DNA from 10 patients with PWS amplified only with the MET pair while modified DNA from 5 patients with AS amplified only with the UNMET pair. Modified DNA from 50 normal unrelated individuals amplified with both primer pairs. In that methylation-specific PCR (MSPCR) can detect all presently testable causes of PWS and AS in a rapid and cost-effective fashion, serious consideration should be given to the use of this test in the initial evaluation of all patients in which PWS or AS is being considered.