Yunia Sribudiani1, Rajendra K Chauhan2, Maria M Alves2, Lucy Petrova3, Erwin Brosens2, Colin Harrison3, Tara Wabbersen3, Bianca M de Graaf2, Tim Rügenbrink2, Grzegorz Burzynski3, Rutger W W Brouwer4, Wilfred F J van IJcken4, Saskia M Maas5, Annelies de Klein2, Jan Osinga6, Bart J L Eggen7, Alan J Burns8, Alice S Brooks2, Iain T Shepherd3, Robert M W Hofstra9. 1. Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; Department of Biomedical Sciences, Division of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. 2. Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands. 3. Department of Biology, Emory University, Atlanta, Georgia. 4. Erasmus Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands. 5. Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 6. Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 7. Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 8. Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; Neural Development and Gastroenterology Units, UCL Institute of Child Health, London, UK. 9. Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; Neural Development and Gastroenterology Units, UCL Institute of Child Health, London, UK. Electronic address: r.hofstra@erasmusmc.nl.
Abstract
BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.
BACKGROUND & AIMS:Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.
Authors: Katherine C MacKenzie; Rhiana Garritsen; Rajendra K Chauhan; Yunia Sribudiani; Bianca M de Graaf; Tim Rugenbrink; Rutger Brouwer; Wilfred F J van Ijcken; Ivo de Blaauw; Alice S Brooks; Cornelius E J Sloots; Conny J H M Meeuwsen; René M Wijnen; Donald F Newgreen; Alan J Burns; Robert M W Hofstra; Maria M Alves; Erwin Brosens Journal: Int J Mol Sci Date: 2021-11-16 Impact factor: 5.923
Authors: Almira Zada; Laura E Kuil; Bianca M de Graaf; Naomi Kakiailatu; Jonathan D Windster; Alice S Brooks; Marjon van Slegtenhorst; Barbara de Koning; René M H Wijnen; Veerle Melotte; Robert M W Hofstra; Erwin Brosens; Maria M Alves Journal: Front Cell Dev Biol Date: 2022-07-08