Fig 1.
Analytical approach for disease gene discovery in a cohort of AVSD trios.
Protein altering variants meeting allele frequency, quality, and read depth cutoffs from 59 trios were sorted with an inheritance model consistent with rare disease which combines de novo, compound heterozygous, and rare homozygous variants to collate variants into a list of genes [16]. Three analytical approaches were applied to the final dataset. Primary Analysis. De novo variants in the AVSD trios were cross-referenced with the genes in a module highly enriched for CHD and cardiac development obtained from unsupervised weighted-gene coexpression network analysis to identify a novel AVSD gene. Secondary Analyses. Variants from the AVSD trios were mapped onto a protein interaction network followed by burden testing in a replication cohort (details in Fig 3). In a final analysis employing inheritance modeling, de novo, compound heterozygous, and rare homozygous loci observed in the AVSD trios were compared with a predefined list of genes associated with human or mouse cardiac malformations. Statistical comparisons were performed in a control group of 59 control trios without CHD.
Fig 2.
Mutation at a highly conserved arginine residue in the DNA binding domain of NR1D2 is associated with AVSD.
(a) Multiple alignment of amino acid residues in the C-terminal aspect of the DNA binding domain of NR1D2 show strong conservation through evolution to the Drosophila paralog Eip75B. (b) A 3d representation of the crystal structure of NR1D1 complexed to DNA. The NR1D1 and NR1D2 DNA binding domains display 96% sequence identity (S3 Fig), and the altered arginine residue (black arrow) contacts the minor groove of DNA in the C-terminal portion of the DNA binding domain (RCSB 1A6Y) [82]. (c) The p.R175W mutation shows increased transcriptional activity in a cell culture assay (p = 0.0022, two-tailed t-test). A wild-type or mutant Nr1d2 construct was transfected in HUVEC cells along with a vector containing a synthetic response element driving a GFP reporter. Mean and standard deviation for each condition are displayed. (d) Two representative cardiac lesions in Nr1d2tm1-Dgen -/- mouse hearts. An oblique coronal section of the heart of a spontaneously deceased P0 Nr1d2tm1-Dgen -/- pup reveals a primum ASD and suggests a common AV-valve (white arrow) indicative of AVSD (star). A coronal section of an e17.5 Nr1d2tm1-Dgen -/- heart shows an inlet VSD (plus sign) which is a type of VSD closely related to AVSDs in cardiac development [78,83]. Abbreviations: LV-left and RV-right ventricles, RA-right atrium, IVS- interventricular septum, VSD-ventricular septal defect, ASD-atrial septal defect.
Fig 3.
An illustration of discovery of protein interaction networks for AVSD and validation by burden testing in separate a replication cohort.
Filtered SNPs and CNVs from the AVSD trio probands are mapped to a protein interaction network (represented by grey dots and black lines), and the network is pruned to yield subnetworks (green dots and black lines). The subnetworks represent variants in genes which have verified interactions at the protein level, often constituting a portion of a signaling pathway or enzymatic complex [22]. To validate the disease association of the individual subnetworks discovered in the trios, we performed burden testing for each subnetwork in a replication cohort of 100 AVSD cases originating largely from a separate study of AVSDs [10] along with 533 controls without congenital heart disease.
Fig 4.
Protein interaction networks identify collagen genes putatively associated with atrioventricular septal defects.
(a) A network diagram displaying protein-protein interactions between genes. A greater number of networks (n = 86) and genes (n = 231) are detected by protein-interaction analysis in AVSD-trio subnetworks (green nodes) relative to the networks (n = 26) and genes (n = 60) detected in the Control-trio subnetworks (yellow nodes). A median p-value from a protein interaction network permutation procedure is reported for each group of subnetworks, which shows an enrichment of true protein-protein interactions in the AVSD-trio subnetworks (p = 0.01) that is not observed in the control trio subnetworks (p = 1). The black lines indicate a protein-protein interaction between two nodes in the subnetwork and the weight of the line corresponds to “heat” value output by the Hotnet2 algorithm. The number of protein interactions for each observed gene corresponds to the size of the node. The two collagen genes in the AVSD-trio subnetworks (COL2A1 and COL9A1) are labeled and the nodes highlighted in red. The networks from each group of trios are arranged in a circular layout. (b) A Venn diagram comparing genes/variants in the AVSD-trio subnetworks (green circle) and Control-trio subnetworks (yellow circle) to genes expressed during early mouse cardiac development (grey circle). The number of genes in each dataset and their overlap with other datasets is indicated and the p-value from a hypergeometric test is reported. A greater proportion of genes in the AVSD-trio subnetworks (60 of 231 genes, p = 9e-09) are found to be expressed in the early cardiac development dataset, compared to the control-trio subnetworks (10 of 60 genes, p = 0.34). (c) The subnetwork comprised of COL2A1 and COL9A1 are highlighted in a Manhattan plot of test statistics from the SKAT linear weight test for each of the 86 AVSD-trio subnetworks identifies an elevated burden of variation in a replication cohort of 100 AVSD subjects and 533 control subjects. The p-value is plotted for the chromosomal position of each gene within the subnetwork. A significance cutoff of 5.81e-04 was derived from the Bonferroni correction of 86 tests performed.
Table 1.
AVSD trio probands display de novo and inherited variation in genes related to human and mouse cardiac malformations.
Table 2.
Discovered CNVs associated with AVSDs and other cardiac malformations.