Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.
Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function.



We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/-and RET-/-iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system.



ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs.



We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Frank Pui-Ling
FP
Lai
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Sin-Ting
ST
Lau
Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
John Kwong-Leong
JK
Wong
Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Hongsheng
H
Gui
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Reeson Xu
RX
Wang
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Tingwen
T
Zhou
Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Wing Hon
WH
Lai
Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Hung-Fat
HF
Tse
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Paul Kwong-Hang
PK
Tam
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Maria-Mercedes
MM
Garcia-Barcelo
Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong. Electronic address: engan@hku.hk.
Elly Sau-Wai
ES
Ngan
D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004252:DNA Mutational Analysis / Q000379:methods; D000072669:Gene Editing / Q000379:methods*; D006627:Hirschsprung Disease / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology; D009432:Neural Crest / Q000503:physiopathology*; D010641:Phenotype; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D016596:Vinculin / Q000235:genetics*
D016596:Vinculin; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human
Congenital; Enteric Nervous System; Neural Development; PSC-Based Model