De novo variants in congenital diaphragmatic hernia identify MYRF as a new syndrome and reveal genetic overlaps with other developmental disorders

Abstract

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.

Fig 1. De novo coding variants in MYRF and their functional impact on transcriptome.

(a) Schematic diagram of the MYRF protein structure. DBD: DNA binding domain; ICA: Intramolecular chaperone auto-processing domain; Pro-rich: proline-rich region; TM: transmembrane helix. The position of DBD and ICA were based on the annotation from InterPro, and Pro-Rich and TM were from SwissProt. The coordinates are given with respect to the canonical isoform (1151 amino acids). The relative position of 12 de novo coding variants are displayed, including 6 discovered in the current study (shown in red), and five from published studies of congenital heart disease (CHD) [29, 30] (shown in blue). LGD variants are shown on top of the protein; and missense variants are on the other side. Shown below the protein structure is the density of missense variants in gnomAD (http://gnomad.broadinstitute.org/). A missense constraint region [37] is highlighted in red (observed/expected number of missense variants = 0.31) (b) Z-score for each gene is the standardized expression level across samples. Mean Z-scores of MYRF target genes in three MYRF variant carriers were shifted to the lower end as compared with other genes. (c) Gene-set enrichment analysis (GESA) was applied to genes ranked by the estimated fold change of expression level comparing MYRF variant carriers with other cases. The MYRF target genes tend to have lower ranks and majority of them were down-regulated in MYRF variant carriers (NES = -2.10, P<5.0E-4).

Fig 2. Genetic overlap with other developmental disorders.

(a) Venn diagram shows 25 genes implicated in developmental disorders and congenital heart disease (DD and CHD genes, Materials and Methods) that are affected by damaging variants in CDH. (b) Enrichment of LGD and D-mis variants in DD and CHD genes. Enrichment was evaluated by comparing the observed number of de novo damaging variants in DD and CHD genes with the expected number of hits by randomly scattering the same number of variants to the exome while controlling for the number of variants, gene length, and sequence context (Materials and Methods). (c) Expression percentile ranks in the developing diaphragm [34], heart [70] and brain [70] are shown for all genes (green density), highlighting DD and CHD genes listed in (b). Smaller ranks correspond to higher expression levels.

Fig 3. Burden of damaging de novo variants in different gene sets and sub classes of CDH.

(a) In complex cases, LGD variants were dramatically (4.6 fold) enriched in genes highly expressed (ranked in the top quartile) in mouse developing diaphragm (MDD) [34], and showed no enrichment in other quartiles. By comparison in isolated cases, LGD variants showed similar enrichment (~2 fold) across expression levels. (b) D-mis variants carried by complex cases also showed highest (2.4 fold) enrichment in the top quartile of MDD expression. Enrichment was evaluated by comparing observed number of variants to the baseline expectation[23, 70] using a one-sided Poisson test. Bars represent the 95% confidence intervals of estimated fold enrichment.

Fig 4. A functional enrichment map of genes affected by de novo damaging variants in CDH.

(a) Enrichment results were visualized by a network of gene sets, where node size is proportional to the number of genes in each gene set and the thickness of edge represents the overlaps between gene sets. The significance of enrichment (p-value) is indicated by the color gradient. Functionally related gene sets are circled and manually labeled. Sub-clusters of network with similar functional annotations are grouped together as functional modules. (b) Mapping genes affected by damaging variants in CDH to the enriched functional groups shown in (a).