Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

Abstract

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre-B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

Molecular pathogenesis of congenital diaphragmatic hernia revealed by exome sequencing, developmental data, and bioinformatics

“…First, we focused on genes derived from a PPF expression array profiling, which we have previously shown to be significantly enriched for CDH-causing genes (10). Second, we generated a network of protein interaction partners with known CDHcausing genes, using the interactome-based affiliation scoring (IBAS) algorithm (18), which predicted 250 proteins as having statistically significant first-or second-order interactions ( Fig.…”

“…additional developmental pathways must also be important. A comprehensive transcriptome analysis of the developing diaphragm, which includes genes in the RA pathway, was made available recently by our laboratory (10) and can serve as a useful tool for evaluating CDH candidate genes.…”

Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia

“…The genes Aldh1a2, Crabp2, Cyp26a1, Cyp26b1, Rara, Rarb, and Rarg all contain RA response elements and are components and transcriptional targets of the RA-signaling pathway (12). Because a complex of Meis homeobox 2 and Pre B-cell leukemia homeobox 1,2 (Pbx1,2) regulates the expression of Aldh1a2 and may be important for diaphragm development (12,34), we assayed the expression of these genes to determine if they were also up-regulated in mutant diaphragms. Meis homeobox 2 and Pbx1,2 were expressed normally in mutant diaphragms (Fig.…”

PBX transcription factors drive pulmonary vascular adaptation to birth

“…Cre is not active in the diaphragm (28), the diaphragm of Pbx1/2-CKO mice developed normally (Supplemental Figure 1H), unlike the diaphragmatic defects observed in Pbx1 global mutants (26). A previous study showed that conditional inactivation of Pbx1 in the lung, heart, brain and other tissues using Islet-1…”

“…Among them are Pbx genes that encode homeodomain-containing transcription factors best known to act as Hox cofactors (24,25). Global inactivation of Pbx1 in mice led to diaphragm malformation, supporting its involvement in CDH (26). In this study, we set out to address the role of Pbx in pulmonary vascular development, a process that is frequently impaired in patients with CDH.…”