PCR-based methylation testing for Prader-Willi or Angelman syndromes usingarchived fixed-cell suspensions

All Prader-Willi syndrome (PWS) and 75% of Angelman syndrome (AS) patients have specific DNA methylation pattern alterations that can be used for diag nostic evaluation. The methylation testing identifies a significantly highe r proportion of patients as compared to fluorescence in situ hybridization (FISH)-based microdeletion analysis and is thus a useful diagnostic evaluat ion for clinically suspect, but FISH-negative, patients. We used two indepe ndent PCR-based protocols for methylation testing on fixed cell specimens a rchived after FISH analyses. Changes in DNA methylation due to the procedur e of cell fixation were ruled out by testing control specimens before and a fter fixation. Then methylation testing was carried out on 20 standard fixe d-cell suspensions from people suspected for PWS or AS. These fixed specime ns were stored after negative FISH analysis for up to 4 years at 4 degreesC in 3:1 methanol/acetic acid. Methylation patterns associated with AS (one specimen) and with PWS (one specimen) were identified for both protocols. T he observed methylation patterns were concordant with the phenotypes of the positive individuals and for the two protocols used. We have, thus, shown that archived fixed-cell suspensions from individuals suspected as PWS or AS that were negative for cytogenetic/FISH microdeletio ns, can now be re-evaluated with PCR-based methylation testing without the need for additional blood samples from the previously studied individuals.