Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.
OBJECTIVE
Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis.


METHODS
We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging.


RESULTS
We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity.


CONCLUSIONS
We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.
Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Jessica Ai-Jia
JA
Liu
Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Frank Pui-Ling
FP
Lai
Department of Psychiatry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong; Center for Genomic Sciences, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Hong-Sheng
HS
Gui
Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Mai-Har
MH
Sham
Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Paul Kwong-Hang
PK
Tam
Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
Maria-Mercedes
MM
Garcia-Barcelo
Program in Developmental and Stem Cell Biology, The Hospital for Sick Children and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Chi-Chung
CC
Hui
Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong. Electronic address: engan@hku.hk.
Elly Sau-Wai
ES
Ngan
D000818:Animals; D016022:Case-Control Studies; D019070:Cell Lineage; D002465:Cell Movement; D004252:DNA Mutational Analysis / Q000379:methods; D004195:Disease Models, Animal; D017615:Enteric Nervous System / Q000002:abnormalities* / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D020022:Genetic Predisposition to Disease; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006627:Hirschsprung Disease / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009432:Neural Crest / Q000378:metabolism / Q000473:pathology*; D055495:Neurogenesis; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D055757:SOXE Transcription Factors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D051155:Wnt1 Protein / Q000235:genetics / Q000378:metabolism; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3
C400551:GLI1 protein, human; C082593:GLI2 protein, human; C065683:GLI3 protein, human; C465571:Gli3 protein, mouse; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C527923:SOX10 protein, human; D055757:SOXE Transcription Factors; C490764:SUFU protein, human; C527924:Sox10 protein, mouse; C490763:Sufu protein, mouse; D014157:Transcription Factors; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3
Aganglionic Megacolon; HSCR; Mouse Model; Nervous System Development