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

Abstract

Beckwith-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) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 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 H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, 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.

Characterization of multi-locus imprinting disturbances and underlying genetic defects in patients with chromosome 11p15.5 related imprinting disorders

“…Different shades of yellow indicate 5% intervals of LOM (with light yellow corresponding to slight LOM and dark yellow corresponding to heavy LOM), while different shades of blue represent 5% intervals of GOM (from light blue corresponding to slight GOM to dark blue indicating heavy GOM). The last two columns contain ICR1/ICR2 methylation values determined by pyrosequencing, according to protocols previously described [41]. *t-test ICR1 MassARRAY vs. ICR1 pyrosequencing: P  = 0.2302.…”

“…All cases included in the study had a primary methylation defects at ICR1/ICR2, as confirmed by molecular diagnosis by pyrosequencing [41–43]. Genomic imbalances or UPD were excluded by SNP array (Supplementary materials and methods).…”

Prenatal molecular testing for Beckwith–Wiedemann and Silver–Russell syndromes: a challenge for molecular analysis and genetic counseling

“…It is well known that some DMRs are not finally established during the time of CVS in the 10-12th wp, 44 and methylation-specific testing at that time leads to false results, explaining the false-positive case in our cohort (Tables 3a and b). Mosaicism can cause a false-negative testing result as shown in our study cohort (Tables 3a-c).…”

“…Furthermore, in the case of early CVS (before 12 wp) the imprinting marks at the loci of interest might not be finally set. 44 As CVS is an extraembryonic tissue, it might not reflect the (epi)genetic constitution of the fetus. Although the analysis of cells derived from the fetus itself offers the best chance to detect altered imprinting marks, our data show that even in this case the risk of false-positive results cannot be prevented (Table 3b).…”

DNA Methylation in the Diagnosis of Monogenic Diseases

“…An accurate molecular prenatal diagnosis is often challenged by heterogeneity and frequent mosaicism of the genetic/epigenetic alterations [7]. In addition, the offered tests must take into account the source of fetal cells, because methylation patterns of embryonic and extraembryonic tissues may differ from those of adult tissues [165,166]. For these reasons, prior to offering prenatal diagnosis based on methylation testing, a detailed presentation of the technological limitations should be discussed with the parents; in particular, they should be made aware that a normal result does not necessarily exclude the suspected diagnosis.…”