Organoid models of breathing disorders reveal patterning defect of hindbrain neurons caused by PHOX2B-PARMs.

Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2B-polyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells.

Transcriptomics of Hirschsprung disease patient-derived enteric neural crest cells reveals a role for oxidative phosphorylation.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung.

Whole genome sequencing reveals epistasis effects within RET for Hirschsprung disease.

Common variants in RET and NRG1 have been associated with Hirschsprung disease (HSCR), a congenital disorder characterised by incomplete innervation of distal gut, in East Asian (EA) populations. However, the allelic effects so far identified do not fully explain its heritability, suggesting the presence of epistasis, where effect of one genetic variant differs depending on other (modifier) variants. Few instances of epistasis have been documented in complex diseases due to modelling complexity and data challenges.

Human Pluripotent Stem Cell-Based Models for Hirschsprung Disease: From 2-D Cell to 3-D Organoid Model.

Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. Due to the oligogenic nature of the disease, some HSCR conditions could not be phenocopied in animal models.

New insights empowered by single-cell sequencing: From neural crest to enteric nervous system.

With the rapid development of single-cell sequencing technologies, it has become a powerful strategy for the discovery of rare cells and delineating the molecular basis underlying various biological processes. Use of single-cell multimodal sequencing to explore the chromatin accessibility, gene expression and spatial transcriptome has propelled us to success in untangling the unknowns in the enteric nervous system (ENS) and provided unprecedented resources for building new diagnostic framework for enteric neuropathies. Here, we summarize the recent findings of single-cell multimodal sequencing, especially focusing on the most commonly used single-cell RNA sequencing (scRNA-seq) on ENS cells, ranged from the progenitors, neural crest (NC) cells, to the mature ENS circuit, in both human and mouse.

BACE2 variant identified from HSCR patient causes AD-like phenotypes in hPSC-derived brain organoids.

β-site APP-cleaving enzyme 2 (BACE2) is a homolog of BACE1, which is considered as the most promising therapeutic target for Alzheimer's disease (AD). However, the expression and functional role of BACE2 in central nervous system (CNS) remain obscured. Previously, we identified several BACE2 rare variants in Hirschsprung disease (HSCR) patients and proved that BACE2-mediated APP cleavage might represent a novel HSCR pathogenesis mechanism in enteric nervous system.

Ciliary protein Kif7 regulates Gli and Ezh2 for initiating the neuronal differentiation of enteric neural crest cells during development.

Gastrointestinal motility disorders occur frequently in patients with ciliopathy, but the underlying genetic link is unclear. The ciliary protein Kif7 can positively or negatively regulate Hedgehog signaling in different cellular contexts. Mice with neural crest cell (NCC)–specific deficiency show a marked reduction of enteric NOS inhibitory neurons.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways.

Activation of Hedgehog Signaling Promotes Development of Mouse and Human Enteric Neural Crest Cells, Based on Single-Cell Transcriptome Analyses.

Background & Aims: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs.

Methods: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation.

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

Background & Aims: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants.

Methods: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls).

Update on the Role of Stem Cells in the Treatment of Hirschsprung Disease.

Stem cells possess the ability of self-renewal and the potency to differentiate into multiple cell lineages. Somatic stem cells are present in adult tissues, but they usually exhibit limited differentiation capacity and life span. On the other hand, somatic cells from adult tissues can be reprogrammed into induced pluripotent stem cells (iPSCs) that retain a full differentiation capacity with unlimited self-renewal ability.

Uncovering the genetic lesions underlying the most severe form of Hirschsprung disease by whole-genome sequencing.

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

Background & Aims: 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.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

Background & Aims: 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.

Dysregulation of clathrin promotes thyroid cell growth and contributes to multinodular goiter pathogenesis.

A germline mutation (A339V) in thyroid transcription factor-1 (TITF1/NKX2.1) was shown to be associated with multinodular goiter (MNG) and papillary thyroid carcinoma (PTC) pathogenesis. The overexpression of A339V TTF1 significantly promoted hormone-independent growth of the normal thyroid cells, representing a cause of MNG and/or PTC.

Hedgehog and Notch signaling in enteric nervous system development.

The enteric nervous system (ENS) in mammals is derived from a small pool of progenitor cells, namely enteric neural crest cells (NCCs). These precursor cells proliferate extensively to expand, migrate over a long distance to fully colonize the developing gut and differentiate into millions of neurons and glia to form a functional ENS for regulating the complex behaviors of the gut. This developmental process relies on a precise regulation of the neuronal and glial differentiation and requires an appropriate balance between the migration, proliferation and differentiation of enteric NCCs and their progeny.

Common genetic variants regulating ADD3 gene expression alter biliary atresia risk.

Background & Aims: Biliary atresia (BA) is a rare and most severe cholestatic disease in neonates, but the pathogenic mechanisms are unknown. Through a previous genome wide association study (GWAS) on Han Chinese, we discovered association of the 10q24.2 region encompassing ADD3 and XPNPEP1 genes, which was replicated in Chinese and Thai populations.

Potential use of skin-derived precursors (SKPs) in establishing a cell-based treatment model for Hirschsprung's disease.

Background: Hirschsprung's (HSCR) disease is characterized by absence of ganglia in the distant bowel. Skin-derived precursor cells (SKPs) are somatic stem cells located in the bulge of hair follicles with high neural plasticity. In this study, we elucidated the therapeutic potential of SKPs for replenishing absent ganglia in HSCR bowel.

RET and NRG1 interplay in Hirschsprung disease.

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex genetic disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. While rare variants (RVs) in the coding sequence (CDS) of several genes involved in ENS development lead to disease, the association of common variants (CVs) with HSCR has only been reported for RET (the major HSCR gene) and NRG1. Importantly, RVs in the CDS of these two genes are also associated with the disorder.

Pluripotent stem cell for modeling neurological diseases.

The availability of human pluriopotent stem cells, embryonic (ESC) and induced pluriopotent (iPSC) stem cells, not only can be a renewable source for investigating the early human development, etiology and progression of different diseases but also recapitulating the disease with the same genomic materials of the patient. In particular, specific neuronal subtypes generated from the patient ESC/iPSCs has become a source for studying disease mechanisms underlying different neurological disorders and allowed drug discovery. In this review, we summarize the recent advances in establishing patient ESC/iPSC to model various neurological diseases.

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3.

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.