Cytogenomic Integrative Network Analysis of the Critical Region Associated with Wolf-Hirschhorn Syndrome

Abstract

Deletions in the 4p16.3 region are associated with Wolf-Hirschhorn syndrome (WHS), a contiguous gene deletion syndrome involving variable size deletions. In this study, we perform a cytogenomic integrative analysis combining classical cytogenetic methods, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and systems biology strategies, to establish the cytogenomic profile involving the 4p16.3 critical region and suggest WHS-related intracellular cell signaling cascades. The cytogenetic and clinical patient profiles were evaluated. We characterized 12 terminal deletions, one interstitial deletion, two ring chromosomes, and one classical translocation 4;8. CMA allowed delineation of the deletions, which ranged from 3.7 to 25.6 Mb with breakpoints from 4p16.3 to 4p15.33. Furthermore, the smallest region of overlapping (SRO) encompassed seven genes in a terminal region of 330 kb in the 4p16.3 region, suggesting a region of susceptibility to convulsions and microcephaly. Therefore, molecular interaction networks and topological analysis were performed to understand these WHS-related symptoms. Our results suggest that specific cell signaling pathways including dopamine receptor, NAD+ nucleosidase activity, and fibroblast growth factor-activated receptor activity are associated with the diverse pathological WHS phenotypes and their symptoms. Additionally, we identified 29 hub-bottlenecks (H-B) nodes with a major role in WHS.

Figure 1

Karyotype results of (a) case 1 showing one normal chromosome 4 (left) and a chromosome 4p (right); (b) case 2, both normal chromosomes 4; (c) case 4, a normal chromosome 4 (left) and a ring chromosome 4 (right) and fluorescence in situ hybridization (FISH) results with locus-specific probes for the Wolf-Hirschhorn syndrome critical region (WHSCR) 4p16.3 from (d) case 1, (e) case 2, and (f) case 4. Absence of red signal on one copy of chromosome 4 indicates deletion of critical region.

Figure 2

Cytogenomic profile of chromosome 4. (a) Red horizontal bars show extent of deleted segments on short arm of chromosome 4 in eight samples investigated using array- comparative genome hybridization (CGH). (b) Genes on 4p16.3p15.33 with haploinsufficiency effects associated with Wolf-Hirschhorn syndrome (WHS) clinical findings.

Figure 3

Smallest region of overlapping (SRO) associated with microcephaly and seizures. Bars show deletion sizes and genomic position on 4p. Red horizontal bars indicate seizures and microcephaly phenotype; green bars indicate absence of seizures and microcephaly, and two bars in gray represent critical regions of Wolf-Hirschhorn syndrome (WHS). The smallest region of susceptibility to microcephaly and seizures shown in this study is represented by blue bar, covering a 330 kb in size (1.8 to 2.13 Mb). WHSCR, Wolf-Hirschhorn syndrome critical region.

Figure 4

Graphs representing protein-protein interactions (PPI) network. (a) List of 343 genes was obtained from GENCODE V24-GRCh38/hg38-UCSC database. The data was used to construct networks using Cytoscape software processing. (b) Centralities parameters and topological analysis, using the CentiScaPe plugin; genes in small region overlapping (SRO) in our study are in red.