Prenatal detection of maternal UPD15 in a new case with i(15p) by Timing Replication Test (TRT) and methylation analysis

DNA replication kinetics of the Prader-Willi/Angelman Critical Region (PWACR) was studied with and without synchronisation in human amniotic cell cul- tures obtained from 20 cases with normal karyotype and 4 cases with a marker of chro- mosome 15, respectively. A Timing Replication Test (TRT) was performed by synchronisation of amniotic cell cultures and followed by interphase FISH to ana- lyse and compare the early/late replication patterns in SNRPN and UBE3A genes be- tween the homologues of chromosome 15. Asynchronous replication patterns of the analysed genes were observed in both amniotic cell cultures but the percentage of interphase nuclei presenting with asynchronous replication was significantly in- creased in the cultures with synchronisation (40-51%), as compared to those without synchronisation (20-23%). The evaluations, performed by means of TRT, showed asynchronous replication patterns on control values: between 39% and 46% of cells in all the cases with inv dup(15). In contrast, the percentage of cells with asynchronous replication in the case with i(15p) was significantly decreased (3-6%), as compared to the control value, and it may be indicated by uniparental disomy of chromosome 15 (UPD15). In addition, those results have been confirmed by molecular evaluation, us- ing the methylation diagnostic test for diagnosis of the Prader-Willi Syndrome.