PCR-Based Methylation Testing for Prader-Willi or Angelman Syndromes Using Archived Fixed-Cell Suspensions

Abstract

All Prader-Willi syndrome (PWS) and 75% of Angelman syndrome (AS) patients have specific DNA methylation pattern alterations that can be used for diagnostic evaluation. The methylation testing identifies a significantly higher proportion of patients as compared to fluorescence in situ hybridization (FISH)-based microdeletion analysis and is thus a useful diagnostic evaluation for clinically suspect, but FISH-negative, patients. We used two independent PCR-based protocols for methylation testing on fixed cell specimens archived after FISH analyses. Changes in DNA methylation due to the procedure of cell fixation were ruled out by testing control specimens before and after fixation. Then methylation testing was carried out on 20 standard fixed-cell supsensions from people suspected for PWS or AS. These fixed specimens were stored after negative FISH analysis for up to 4 years at 4°C in 3:1 methanol/acetic acid. Methylation patterns associated with AS (one specimen) and with PWS (one specimen) were identified for both protocols. The 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 microdeletions, can now be re-evaluated with PCR-based methylation testing without the need for additional blood samples from the previously studied individuals.