PMC4910218_01
PMC4910218_02
Characterization of the duplications. a-c Characterization of the 0.88 Mb duplication in family 1. a Analysis of 11p15 microsatellite markers showing the segregation of the duplication in three generations. The haplotype of the chromosome carrying the duplication is shadowed. Gray color of II-2 and III-2 indicates a growth restriction observed only in childhood. b DNA methylation analysis of ICR1 and ICR2 as determined by Pyrosequencing. Line chart reporting the methylation level (%) of seven CpGs of ICR2 and three CpGs of ICR1.The placenta DNAs of the proband (III-3) and a healthy control (Plac. N 1) and leukocyte DNAs of the parents (II-1 and II-2) and a healthy control (normal Ctrl 1) showed similar methylation patterns at both ICRs. Two BWS patients carrying a duplication of the entire domain, Dupl Ctrl, have been analyzed as controls. c Real-time messenger RNA (mRNA) expression analysis of CDKN1C and PHLDA2 normalized to the GAPDH control gene in the placenta cells of the proband (III-3) and three normal controls (Ctrl 1, 2, 3). Experiments were performed in triplicate and statistical significance determined by Student's t test. d-e Characterization of the 1.13 Mb duplication in family 2. d DNA methylation analysis at ICR1 and ICR2 determined by Pyrosequencing in the trio as described in b. The proband (III-1) and his mother (II-2) show ICR2 methylation level similar to two BWS patients carrying ICR2 duplications (Dupl Ctrl 1 and 2) and lower than the father (II-1) and two healthy controls. ICR1 methylation of the proband and his parents is comparable to that of two healthy controls (normal Ctrl 1 and 2). e Analysis of 11p15 microsatellite markers showing a de novo paternal duplication in the mother and a maternally inherited duplication in the proband. The haplotype of the chromosome carrying the duplication is shadowed
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PMC4910218_02_13148_2016_236_Fig2_HTML.jpg
Fig2
As shown in Fig. 2d, the methylation profiles of both ICRs were normal in the father, while the proband and his mother showed normal methylation of ICR1 but hypomethylation of ICR2 at a level comparable with other previously described ICR2 duplication carriers. The segregation and signal intensity of the D11S4088 marker, located in the duplicated region, confirmed that the duplication was maternally inherited in the proband and demonstrated that it originated de novo from the paternal chromosome in the mother (Fig. 2e and Additional file 7: Figure S7).