Genetics of Hirschsprung disease:Rare variants, in vivo analysis and expression profiling

Hirschsprung disease (HSCR) is characterized by the absence of neurons in the distal region of the colon. Due to the lack of neuronal innervation, the muscles in the colon are unable to relax, causing life-threatening obstipation. HSCR is a heritable disease. Over 15 genes have been associated with the disease, but mutations in these genes are found in only a quarter of all patients. We aimed to identify new disease genes for HSCR by focusing on de novo mutations. De novo mutations are new genetic variations that are found in a child, but not in the parents. We investigated whether genes carrying de novo mutations in HSCR patients are involved in the development of the nervous system in the gut. To this end we inactivated these genes one by one in a zebrafish model in which the cells of the nervous system are fluorescently labeled. This led to the identification of four new genes for HSCR. We employed the same zebrafish model to show that overexpression of a particular gene on chromosome 21 possibly explains the high prevalence of HSCR among patients with Down syndrome. Additionally, we studied which genes are important for the development of the nervous system in the gastrointestinal tract of the mouse. We identified several genes that are critical for this developmental process, and these genes are excellent candidate genes for HSCR

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

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

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

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.ZonMWNetherlands Organization for Health Research and Development 40-00812-98-10042Maag Lever Darm stichting WO09-62NIHUnited States Department of Health & Human Services National Institute

Additional file 11: Table S10. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

gRNA and primers for zebrafish knockout and T7E1 assay. (XLSX 9 kb

Additional file 7: Table S6. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Gene recurrence and burden test. (XLSX 14 kb

Additional file 9: Table S8. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Characteristics of 116 ENS-related HSCR candidate genes. (XLSX 32 kb

Additional file 12: Table S11. of Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Primers for expression analysis in zebrafish. (XLSX 9 kb