Zhong G et al. (2022), Identification and validation of candidate risk...

XB-ART-59096

HGG Adv 2022 Jul 14;33:100107. doi: 10.1016/j.xhgg.2022.100107.

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Identification and validation of candidate risk genes in endocytic vesicular trafficking associated with esophageal atresia and tracheoesophageal fistulas.

Zhong G , Ahimaz P , Edwards NA , Hagen JJ , Faure C , Lu Q , Kingma P , Middlesworth W , Khlevner J , El Fiky M , Schindel D , Fialkowski E , Kashyap A , Forlenza S , Kenny AP , Zorn AM , Shen Y , Chung WK .

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Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify the genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein-altering de novo coding variants in complex cases (p = 3.3 × 10-4), especially in genes that are intolerant of loss-of-function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein-altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. Our results suggest significant genetic heterogeneity of EA/TEF and may have implications for the mechanisms of other rare congenital anomalies.

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Species referenced: Xenopus laevis
Genes referenced: abra add1 amer3 ap1g2 apc2 arhgap17 arhgap21 celsr2 disp1 eftud2 itgb4 itsn1 map4k3 pam pcdh1 ptpn14 rab3gap2 rapgef3 smad6 sox2 tyr
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gRNAs referenced: abra gRNA1 add1 gRNA1 amer3 gRNA1 ap1g2 gRNA1 apc2 gRNA1 arhgap17 gRNA1 arhgap21 gRNA1 celsr2 gRNA1 disp1 gRNA1 eftud2 gRNA1 eftud2 gRNA2 itgb4 gRNA1 itsn1 gRNA2 itsn1 gRNA3 map4k3 gRNA1 pcdh1 gRNA1 ptpn14 gRNA1 rab3gap2 gRNA1 rapgef3 gRNA1 smad6 gRNA1 smad6 gRNA2 sox2 gRNA1 sox2 gRNA3 tyr gRNA17

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	Figure 1. Pathway enrichment analysis (A) Volcano plot. Each dot represents a pathway. X axis represents the enrichment rate in log scale, and Y axis is the Poisson test p value in log10 scale. The horizontal dashed line marks family-wise error rate (FWER) of 0.05. Significant pathways (FWER < 0.05) are colored by the percentage of LGD variants, and other pathways are colored gray. (B) Pathway overlaps. Each circle represents a pathway with FWER < 0.05. Circle size is proportional to the number of observed de novo variants in the pathway; circle color represents the FWER; edge width is determined by the Jaccard index between two pathways, and edge color represents the correlation coefficient of the two pathways under the null in simulations.
	Figure 2. StringDB of LGD and missense genes in complex cases Dots are colored to indicate whether it is involved in one of the significant pathways. Constrained genes (pLI ≥ 0.5) with LGD mutations are colored black. Edge width represents the StringDB score. Genes not involved in any of the annotation groups were not shown.
	Figure 3. CRISPR-mutation of candidate risk genes in Xenopus disrupts trachea-esophagus morphogenesis (A–G) Representative confocal microscopy images of NF44 foregut from Xenopus CRISPR mutants. Sox2 F0 CRISPR mutants (C) have the same trachea-esophageal phenotype as sox2−/− F2 germline mutants (B), validating the F0 screen. Compared to control tyr mutations in which the trachea (t) and esophagus (e) have completely separated (A), mutation of 13/18 genes caused a failure of the foregut to separate into distinct trachea and esophagus (D–E and H–L) and/or resulted in a disrupted esophagus with multiple lumens (F and G). Dashed lines indicate the esophagus, trachea, and foregut lumens. Arrows point to a tracheoesophageal cleft. The number of embryos with a TED phenotype/total injected. Scale bars represent 50 μm.
	Supplemental Figure 1. Deconvolution of CRISPR-mediated indels in Xenopus embryos. CRISPR-Cas9-injected Xenopus embryos were genotyped by PCR amplification of the target region followed by Sanger sequencing. Sanger sequencing traces were analyzed using ICE software (Synthego, USA) to deconvolute the proportion and sequence of each indel mutation in each embryo. Representative sample traces are shown for gene editing of itsn1, disp1, and arhgap17 (traces generated by ICE). The gRNA sequence is indicated by black lines and the PAM sequence by dotted red lines. Dashed vertical black lines indicate the predicted location of the Cas9 cut site. Bar graphs indicate the percent of sequences with the indicated insertion or deletion size (in base pairs) ± standard error of the mean (n=5-10 embryos analyzed per graph). F0 CRISPR editing of embryos results in mosaic indels however each gRNA tends to generate similar mutation profiles in embryos within a given experiment (e.g. itsn1 gRNA tends to produce -2 and +1 bp indels).

References [+] :

Abyzov, CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. 2011, Pubmed