Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Abstract

Background: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human.

Results: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS.

Conclusions: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.

Keywords: De novo mutations; ENS; Hirschsprung disease; Neural crest.

Fig. 1

Pathogenicity analysis in vivo by morpholino gene knockdown and CRISPR/Cas9 knockout in zebrafish. Morpholino knockdown of ckap2l, dennd3, ncl1, nup98, and tbata resulted in a HSCR-like phenotype when compared to control (a–j). Kaede-expressing enteric neurons were absent in the distal intestine at 5 dpf. The number of embryos with phenotype out of the total number of embryos observed is shown. Co-injection of p53 morpholino reproduced the phenotype except ckap2l, indicating the loss of enteric neurons in dennd3, ncl1, nup98, and tbata knockdown was not the result of p53-induced apoptosis (k–o). The results were verified by CRISPR/Cas9 knockout of ckap2l, dennd3a and b, ncl1, nup98, and tbata, in which the HSCR-like phenotype was reproduced (p–t). Dotted lines outline the intestines. Asterisks indicate the position of the anus. Arrows indicate the position where the aganglionic region begins. Scale bar = 200 μm. MO morpholino, nt nucleotide

Fig. 2

Temporal and spatial expression patterns of zebrafish orthologs. Whole mount in situ hybridized embryos hybridized with antisense riboprobes for dennd3a (a–d), dennd3b (e–h), ncl1 (i–l), nup98 (m–p), and tbata (q–t) at the indicated developmental stages. All columns show lateral views. Intestinal expression for all genes is apparent from 48 hpf onwards. Scale bar = 500 μm

Fig. 3

Newly identified genes in ENS development. All symbols represent proteins coded by genes known to be involved in HSCR or novel genes identified in this study. The effect of NUP98 is shown by protein NUP96. The interaction effects between different proteins are illustrated by four different lines representing binding, secreted/express, phosphorylation, and activation. ENCC enteric neural crest cell