Derivation and investigation of the first human cell-based model of Beckwith-Wiedemann syndrome

Figures & data

Figure 1. Summary of reprogramming in this study

A schematic illustrating chromosome 11 with relative locations of IC1 and IC2 as well as regions of pUPD11 (open rectangles) of each pUPD11 fibroblast cell line (Patient 1, 2). pUPD11 break points are indicated next to regions of pUPD11 (Human Genome Build 37, hg19, 2009).Percent pUPD11 in the parental fibroblasts, total number of iPSC clones derived from each fibroblast line and, from patient fibroblasts, the number of non-pUPD11 and pUPD11 iPSC clones derived. NA: not applicable.

Figure 2. Methylation at BWS-related imprinting control regions in iPSC clones

(a) The imprinted region on chromosome 11p15. IC1 is methylated on the paternal allele, leading to the paternal allele-specific expression of IGF2 and the maternal allele-specific expression of H19. IC2 is methylated on the maternal allele; KCNQ1 and CDKN1C are expressed from the maternal allele while KCNQ1OT1 is expressed from the paternal allele. Black closed circles indicate the allele-specific methylation on each ICR. Arrows indicate the allele-specific expression of the genes. The small black bars below the region indicate primer location for the methylation assays (a: COBRA and direct sequencing of cloned bisulfilte treated DNA b: pyrosequencing, c: COBRA). Figure is not drawn to scale. (b) Methylation at IC1 measured by pyrosequencing (in IMR91 and patient 1 cell lines) and COBRA (in patient 2 cell lines). (c) Methylation at IC1 in select patient 1 iPSCs during extended culture. Bars are grouped by clones. ‘p’ indicates the passage number. (d) Methylation at IC2 measured by COBRA. (b-d) Each graph includes results from the parental fibroblast cell line (grey bar) and iPSC lines derived from the respective fibroblast cell line (black bars). pUPD11 clones are highlighted in grey boxes. Fib = fibroblasts.

Figure 3. RNA-Seq expression profiles in control lines, non-UPD11, and pUPD11 patient cell lines. (a) Venn diagram demonstrating the statistically significant differentially expressed genes (DGE) identified by DESeq2 when comparing all non-pUPD11 and pUPD11 patient-derived lines to control hepatocytes (blue oval) and those identified when comparing the pUPD11 iPSC lines from both patients to the non-pUPD11 lines from both patients grouped with the control lines (red oval). (b) Heatmap of transformed expression data for growth and liver cancer genes differentially expressed across groups identified through GSEA

Figure 4. Network of interactome among differentially expressed genes in BWS iPSC lines differentiated into hepatocyte lineages. pUPD11 lines were compared to non-UPD11 and control lines. Genes were identified by GSEA enrichment. Green clusters of nodes are largely components of insulin signalling and connect to IGF2. Cell proliferation pathways are indicated by blue and yellow clusters, with the yellow cluster specifically regulating cell cycle progression including CDKN1C. Edges between gene nodes indicate confidence in the interaction by the intensity of the line. Kmeans clustering was set to 8 and disconnected nodes were hidden