CLONAL HETEROGENEITY AT ALLELIC METHYLATION SITES DIAGNOSTIC FOR PRADER-WILLI-AND-ANGELMAN-SYNDROMES

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are development al disorders resulting from the absence of the paternal or maternal co ntribution to the 15q11-13 region, respectively. Allele-specific methy lation at D15S63 (PW71) has routinely been used as a diagnostic indica tor of PWS and AS in DNA samples derived from peripheral blood, Extens ive variation in allele-specific methylation patterns, however, has be en observed at this site in different tissues, but the frequency or me chanism of this variation has remained uncharacterized. Herein, we hav e investigated the cellular basis of variation in methylation patterns at four sites of allelic methylation near SNRPN by using DNA samples derived from a panel of primary T lymphocyte clones, Interclonal varia bility was observed at three of these sites, including the diagnostic PW71 site. Changes in allele-specific methylation patterns occurred at a frequency of about one change in 50% of the cells every 22-25 doubl ings, In contrast, stable allele-specific methylation was observed in these clonal populations at exon 1 of SNRPN and the androgen receptor locus on the inactive X chromosome, suggesting that methylation at som e CpG sites is more faithfully maintained than others, Clonal heteroge neity at PW71 was not an artifact of cell culture because the absence of allelic methylation was also observed in about 20% of the alleles i n unstimulated peripheral blood. These results demonstrate that variat ion in allele-specific methylation at PW71 and other sites in the PWS/ AS region appear to depend on the clonal complexity of the particular tissue and on the lack of strict maintenance of methylation within clo nes.