Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

ABSTRACTBeckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) of several imprinted loci (ICR1, ICR2, <i>H19</i>, PWS/AS-ICR, <i>GNASXL, GNAS1A, ZAC/PLAGL1</i>, and <i>MEST</i>) and of non-imprinted <i>MGMT</i> and <i>RASSF1A</i> promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, <i>GNASXL</i>, PWS/AS-ICR, and <i>MEST</i>. Conversely, the methylation levels of <i>H19</i> promoter, <i>GNAS1A and ZAC/PLAGL1</i> were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level at CVF/CVC of two BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not <i>H19</i>, could be reliable targets for prenatal BWS diagnosis by methylation test in CV and, also, after culture. In addition, PWS/AS-ICR, <i>GNASXL</i>, and <i>MEST</i>, but not <i>GNAS1A and ZAC/PLAGL1</i>, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.