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Xiao J, Feng C, Zhu T, Zhang X, Chen X, Li Z, You J, Wang Q, Zhuansun D, Meng X, Wang J, Xiang L, Yu X, Zhou B, Tang W, Tou J, Wang Y, Yang H, Yu L, Liu Y, Jiang X, Ren H, Yu M, Chen Q, Yin Q, Liu X, Xu Z, Wu D, Yu D, Wu X, Yang J, Xiong B, Chen F, Hao X, Feng J. Rare and common genetic variants underlying the risk of Hirschsprung's disease. Hum Mol Genet 2025; 34:586-598. [PMID: 39817569 DOI: 10.1093/hmg/ddae205] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 12/17/2024] [Accepted: 12/27/2024] [Indexed: 01/18/2025] Open
Abstract
Hirschsprung's disease (HSCR) is a congenital enteric neuropathic disorder characterized by high heritability (>80%) and polygenic inheritance (>20 genes). The previous genome-wide association studies (GWAS) identified several common variants associated with HSCR and demonstrated increased predictive performance for HSCR risk in Europeans using a genetic risk score, there remains a notable gap in knowledge regarding Chinese populations. We conducted whole exome sequencing in a HSCR case cohort in Chinese. By using the common controls (505 controls from 1KG EAS and 10 588 controls from ChinaMAP), we conducted GWAS for the common variants in the exome and gene-based association for rare variants. We further validated the associated variants and genes in replicated samples and in vitro and vivo experiments. We identified one novel gene PLK5 by GWAS and suggested 45 novel putative genes based the gene-based test. By using genetic variant at RET and PLK5, we constructed a genetic risk score that could identify the individuals with very high genetic risk for HSCR. Compared with patients with zero or one risk allele from the three variants, the risk for HSCR was 36.61 times higher with six alleles. In addition, we delineated a HSCR risk gene landscape that encompasses 57 genes, which explains 88.5% and 54.5% of HSCR in Chinese and European, respectively. In summary, this study improved the understanding of genetic architecture of HSCR and provided a risk prediction approach for HSCR in the Chinese.
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Affiliation(s)
- Jun Xiao
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Chenzhao Feng
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Tianqi Zhu
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Xuan Zhang
- Department of Pediatric Surgery, Pingshan District Maternal & Child Healthcare Hospital of Shenzhen, No. 6 Longtian South Road, Longtian Subdistrict, Pingshan District, Shenzhen, Guangdong 518122, China
| | - Xuyong Chen
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Zejian Li
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Jingyi You
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Qiong Wang
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Didi Zhuansun
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Xinyao Meng
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Jing Wang
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Lei Xiang
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Xiaosi Yu
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Bingyan Zhou
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Weibing Tang
- Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, No. 72 Guangzhou Road, Gulou District, Nanjing, Jiangsu 210008, China
| | - Jinfa Tou
- Department of General Surgery, Children's Hospital, Zhejiang University School of Medicine, No. 3333 Binsheng Road, Binjiang District, Hangzhou, Zhejiang 310003, China
| | - Yi Wang
- Department of General and Neonatal Surgery, Children's Hospital of Chongqing Medical University, No. 136, Zhongshan 2nd Road, Yuzhong District, Chongqing 400014, China
| | - Heying Yang
- Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, No. 1 Renmin Road, Erqi District, Henan 450052, China
| | - Lei Yu
- Department of Neonatal Surgery, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 100 Hong Kong Road, Jiang'an District, Wuhan, Hubei 430030, China
| | - Yuanmei Liu
- Department of Pediatric Surgery, The Affiliated Hospital of Zunyi Medical University, No. 149 Dalian Road, Huichuan District, Zunyi, Guizhou 563000, China
| | - Xuewu Jiang
- Department of Pediatric Surgery, The Second Affiliated Hospital of Shantou University Medical College, No. 69, Dongxia North Road, Jinping District, Shantou, Guangdong 515041, China
| | - Hongxia Ren
- Department of Neonatal Surgery, Children's Hospital of Shanxi, No. 13 Xinminbei Street, Xinhualing district, Taiyuan, Shanxi 030013, China
| | - Mei Yu
- Department of Pediatric Surgery, Guiyang Maternal and Child Health Hospital, No. 63 Ruijin South Road, Nanming district, Guiyang, Guizhou 550002, China
| | - Qi Chen
- Department of Pediatric Surgery, The Third Affiliated Hospital of Zhengzhou University, No. 7 Kangfuqian Street, Erqi District, Zhengzhou 450052, Henan, China
| | - Qiang Yin
- Department of General Surgery, Hunan Children's Hospital, No. 86 Ziyuan Road, Yuhua District, Changsha, Hunan 515041, China
| | - Xiang Liu
- Department of Pediatric Surgery, Anhui Provincial Children's Hospital, No. 39 Wangjiang East Road, Wuhu Road Subdistrict, Hefei, Anhui 230051, China
| | - Zhilin Xu
- Department of Pediatric Surgery, The First Affiliated Hospital of Harbin Medical University, No. 199 Dazhi Street, Nangang district, Harbin, Heilongjiang 150001, China
| | - Dianming Wu
- Department of Pediatric Surgery, Fujian Maternity and Child Health Hospital, Fujian Medical University, No. 18 Daoshan Road, Gulou District, Fuzhou, Fujian 350001, China
| | - Donghai Yu
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Xiaojuan Wu
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Jixin Yang
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
| | - Bo Xiong
- Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, No. 13 Hangkong Road, Qiaokou District, Wuhan, Hubei 430030, China
| | - Feng Chen
- Department of Pediatric Surgery, Union Hospital, Fujian Medical University, No. 29, Xinquan Road, Gulou District, Fuzhou, Fujian 350001, China
| | - Xingjie Hao
- Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College Huazhong University of Science and Technology, No. 13 Hangkong Road, Qiaokou District, Wuhan, Hubei 430030, China
| | - Jiexiong Feng
- Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
- Hubei Clinical Center of Hirschsprung's disease and allied disorders, No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei 430030, China
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Tjaden NEB, Liou MJ, Sax SE, Lassoued N, Lou M, Schneider S, Beigel K, Eisenberg JD, Loeffler E, Anderson SE, Yan G, Litichevskiy L, Dohnalová L, Zhu Y, Jin DMJC, Raab J, Furth EE, Thompson Z, Rubenstein RC, Pilon N, Thaiss CA, Heuckeroth RO. Dietary manipulation of intestinal microbes prolongs survival in a mouse model of Hirschsprung disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.10.637436. [PMID: 39990395 PMCID: PMC11844371 DOI: 10.1101/2025.02.10.637436] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/25/2025]
Abstract
Enterocolitis is a common and potentially deadly manifestation of Hirschsprung disease (HSCR) but disease mechanisms remain poorly defined. Unexpectedly, we discovered that diet can dramatically affect the lifespan of a HSCR mouse model ( Piebald lethal , sl/sl ) where affected animals die from HAEC complications. In the sl/sl model, diet alters gut microbes and metabolites, leading to changes in colon epithelial gene expression and epithelial oxygen levels known to influence colitis severity. Our findings demonstrate unrecognized similarity between HAEC and other types of colitis and suggest dietary manipulation could be a valuable therapeutic strategy for people with HSCR. Abstract Hirschsprung disease (HSCR) is a birth defect where enteric nervous system (ENS) is absent from distal bowel. Bowel lacking ENS fails to relax, causing partial obstruction. Affected children often have "Hirschsprung disease associated enterocolitis" (HAEC), which predisposes to sepsis. We discovered survival of Piebald lethal ( sl/sl ) mice, a well-established HSCR model with HAEC, is markedly altered by two distinct standard chow diets. A "Protective" diet increased fecal butyrate/isobutyrate and enhanced production of gut epithelial antimicrobial peptides in proximal colon. In contrast, "Detrimental" diet-fed sl/sl had abnormal appearing distal colon epithelium mitochondria, reduced epithelial mRNA involved in oxidative phosphorylation, and elevated epithelial oxygen that fostered growth of inflammation-associated Enterobacteriaceae . Accordingly, selective depletion of Enterobacteriaceae with sodium tungstate prolonged sl/sl survival. Our results provide the first strong evidence that diet modifies survival in a HSCR mouse model, without altering length of distal colon lacking ENS. Highlights Two different standard mouse diets alter survival in the Piebald lethal ( sl/sl ) mouse model of Hirschsprung disease, without impacting extent of distal colon aganglionosis (the region lacking ENS). Piebald lethal mice fed the "Detrimental" diet had many changes in colon epithelial transcriptome including decreased mRNA for antimicrobial peptides and genes involved in oxidative phosphorylation. Detrimental diet fed sl/sl also had aberrant-appearing mitochondria in distal colon epithelium, with elevated epithelial oxygen that drives lethal Enterobacteriaceae overgrowth via aerobic respiration. Elimination of Enterobacteriaceae with antibiotics or sodium tungstate improves survival of Piebald lethal fed the "Detrimental diet". Graphical abstract
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Fries LE, Grullon G, Berk-Rauch HE, Chakravarti A, Chatterjee S. Synergistic effects of Ret coding and enhancer loss-of-function alleles cause progressive loss of inhibitory motor neurons in the enteric nervous system. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.23.634550. [PMID: 39896597 PMCID: PMC11785208 DOI: 10.1101/2025.01.23.634550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 02/04/2025]
Abstract
Coding and enhancer variants of the RET receptor tyrosine kinase gene contribute to ~50% of Hirschsprung disease (HSCR) risk, a congenital disorder of disrupted enteric nervous system (ENS) development. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. To uncover the cellular basis of RET-mediated aganglionosis, we used CRISPR/Cas9 to delete (Δ) the homologous mouse enhancer (mcs+9.7). We used single cell RNA sequencing and high-resolution immunofluorescence to demonstrate four significant features of the developing E14.5 gut of Δmcs+9.7/Δmcs+9.7 embryos: (1) a small (5%) yet significant reduction in Ret gene expression in only two major cell types - early differentiating neurons and fate-restricted inhibitory motor neurons; (2) no significant cellular loss in the ENS; and, (3) loss of expression of 19 cell cycle regulator genes suggesting a proliferative defect. To identify the Ret functional threshold for normal ENS development, we also generated, in combination with the Ret CFP null allele, (4) Δmcs+9.7/CFP double heterozygote mice which reduced Ret gene expression in the ENS to 42% with severe loss of inhibitory motor neurons, an effect restricted to the hindgut and driven by proliferative loss. Thus, Ret gene expression drives proliferation of ENS progenitor cells and hindgut-specific inhibitory motor neuron development, and that HSCR aganglionosis arises from a cascade of cellular defects triggered by >50% loss of Ret function.
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Affiliation(s)
- Lauren E Fries
- Center for Human Genetics & Genomics, New York University Grossman School of Medicine, New York, NY 10016
| | - Gabriel Grullon
- Center for Human Genetics & Genomics, New York University Grossman School of Medicine, New York, NY 10016
| | - Hanna E Berk-Rauch
- Center for Human Genetics & Genomics, New York University Grossman School of Medicine, New York, NY 10016
| | - Aravinda Chakravarti
- Center for Human Genetics & Genomics, New York University Grossman School of Medicine, New York, NY 10016
- Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY 10016
| | - Sumantra Chatterjee
- Center for Human Genetics & Genomics, New York University Grossman School of Medicine, New York, NY 10016
- Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY 10016
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Poltavski DM, Cunha AT, Tan J, Sucov HM, Makita T. Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development. eLife 2024; 13:RP96424. [PMID: 39641974 PMCID: PMC11623925 DOI: 10.7554/elife.96424] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/07/2024] Open
Abstract
Two major ligand-receptor signaling axes - endothelin Edn3 and its receptor Ednrb, and glial-derived neurotrophic factor (GDNF) and its receptor Ret - are required for migration of enteric nervous system (ENS) progenitors to the hindgut. Mutations in either component cause colonic aganglionosis, also called Hirschsprung disease. Here, we have used Wnt1Cre and Pax2Cre in mice to show that these driver lines label distinct ENS lineages during progenitor migration and in their terminal hindgut fates. Both Cre lines result in Hirschsprung disease when combined with conditional Ednrb or conditional Ret alleles. In vitro explant assays and analysis of lineage-labeled mutant embryos show that GDNF but not Edn3 is a migration cue for cells of both lineages. Instead, Edn3-Ednrb function is required in both for GDNF responsiveness albeit in different ways: by expanding the Ret+ population in the Pax2Cre lineage, and by supporting Ret function in Wnt1Cre-derived cells. Our results demonstrate that two distinct lineages of progenitors give rise to the ENS, and that these divergently utilize endothelin signaling to support migration to the hindgut.
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Affiliation(s)
- Denise M Poltavski
- Department of Regenerative Medicine and Cell Biology, Medical University of South CarolinaCharlestonUnited States
| | - Alexander T Cunha
- Department of Regenerative Medicine and Cell Biology, Medical University of South CarolinaCharlestonUnited States
| | - Jaime Tan
- Department of Regenerative Medicine and Cell Biology, Medical University of South CarolinaCharlestonUnited States
| | - Henry M Sucov
- Department of Regenerative Medicine and Cell Biology, Medical University of South CarolinaCharlestonUnited States
| | - Takako Makita
- Department of Regenerative Medicine and Cell Biology, Medical University of South CarolinaCharlestonUnited States
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Elkrewi EZ, Al Abdulqader AA, Khasanov R, Maas-Omlor S, Boettcher M, Wessel LM, Schäfer KH, Tapia-Laliena MÁ. Role of Inflammation and the NF-κB Signaling Pathway in Hirschsprung's Disease. Biomolecules 2024; 14:992. [PMID: 39199380 PMCID: PMC11352745 DOI: 10.3390/biom14080992] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2024] [Revised: 07/24/2024] [Accepted: 08/05/2024] [Indexed: 09/01/2024] Open
Abstract
Hirschsprung's disease (HSCR, incidence 1/5000 live births) is caused by the failure of neural crest-derived precursors to migrate, survive, proliferate, or differentiate during the embryonic development of the Enteric Nervous System (ENS), which could be disrupted by many factors, including inflammatory processes. The NF-κB family controls several biological processes, including inflammation, neurogenesis, and cell migration. With the aim of studying the potential role of NF-κB in HSCR, we have analyzed the expression of the NF-κB main subunits and other NF-κB-related genes by RT-qPCR in HSCR tissue samples (sub-divided into ganglionic and aganglionic segments). We found decreased gene expression of the NF-κB main subunit RELA but also of NFKBIA, TNFA, TFGBR2, and ERBB3 in the pathologic distal aganglionic segments compared to the proximal ganglionic segments. Moreover, we could also confirm the lower protein expression of RelA/p65 in the aganglionic distal segments by immunofluorescence staining. Further, we show that the expression of RelA/p65 protein in the proximal segments concurs with lymphocyte infiltration in the bowel tissue, indicating a pro-inflammatory activation of p65 in the proximal ganglionic HSCR tissue in the patients analyzed. All in all, our findings suggest that the modulation of NF-κB signaling in the neuro-enteric system does obviously contribute to the pathological effects of HSCR.
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Affiliation(s)
- Enas Zoheer Elkrewi
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
| | - Ahmad A. Al Abdulqader
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
- Department of Surgery, College of Medicine, King Faisal University, Al Hofuf 31982, Saudi Arabia
| | - Rasul Khasanov
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
| | - Silke Maas-Omlor
- Working Group Enteric Nervous Systems (AGENS), University of Applied Sciences Kaiserslautern, Amerikastrasse 1,66482 Zweibrücken, Germany (K.-H.S.)
| | - Michael Boettcher
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
| | - Lucas M. Wessel
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
| | - Karl-Herbert Schäfer
- Working Group Enteric Nervous Systems (AGENS), University of Applied Sciences Kaiserslautern, Amerikastrasse 1,66482 Zweibrücken, Germany (K.-H.S.)
| | - María Ángeles Tapia-Laliena
- Department of Pediatric Surgery, Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
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Vincent E, Chatterjee S, Cannon GH, Auer D, Ross H, Chakravarti A, Goff LA. Ret deficiency decreases neural crest progenitor proliferation and restricts fate potential during enteric nervous system development. Proc Natl Acad Sci U S A 2023; 120:e2211986120. [PMID: 37585461 PMCID: PMC10451519 DOI: 10.1073/pnas.2211986120] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Accepted: 07/18/2023] [Indexed: 08/18/2023] Open
Abstract
The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.
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Affiliation(s)
- Elizabeth Vincent
- McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Sumantra Chatterjee
- Center for Human Genetics and Genomics, New York University Grossman School of Medicine, New York, NY10016
- Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY10016
| | - Gabrielle H. Cannon
- McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Dallas Auer
- McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Holly Ross
- McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Aravinda Chakravarti
- Center for Human Genetics and Genomics, New York University Grossman School of Medicine, New York, NY10016
- Department of Neuroscience and Physiology, New York University Grossman School of Medicine, New York, NY10016
| | - Loyal A. Goff
- McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD21205
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD21205
- Kavli Neurodiscovery Institute, Johns Hopkins University, Baltimore, MD21205
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Genetic Background Influences Severity of Colonic Aganglionosis and Response to GDNF Enemas in the Holstein Mouse Model of Hirschsprung Disease. Int J Mol Sci 2021; 22:ijms222313140. [PMID: 34884944 PMCID: PMC8658428 DOI: 10.3390/ijms222313140] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 11/30/2021] [Accepted: 12/03/2021] [Indexed: 12/13/2022] Open
Abstract
Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.
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Mesenteric Neural Crest Cells Are the Embryological Basis of Skip Segment Hirschsprung's Disease. Cell Mol Gastroenterol Hepatol 2020; 12:1-24. [PMID: 33340715 PMCID: PMC8082118 DOI: 10.1016/j.jcmgh.2020.12.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 12/14/2020] [Accepted: 12/14/2020] [Indexed: 12/22/2022]
Abstract
BACKGROUND & AIMS Defective rostrocaudal colonization of the gut by vagal neural crest cells (vNCCs) results in Hirschsprung's disease (HSCR), which is characterized by aganglionosis in variable lengths of the distal bowel. Skip segment Hirschsprung's disease (SSHD), referring to a ganglionated segment within an otherwise aganglionic intestine, contradicts HSCR pathogenesis and underscores a significant gap in our understanding of the development of the enteric nervous system. Here, we aimed to identify the embryonic origin of the ganglionic segments in SSHD. METHODS Intestinal biopsy specimens from HSCR patients were prepared via the Swiss-roll technique to search for SSHD cases. NCC migration from the neural tube to the gut was spatiotemporally traced using targeted cell lineages and gene manipulation in mice. RESULTS After invading the mesentery surrounding the foregut, vNCCs separated into 2 populations: mesenteric NCCs (mNCCs) proceeded to migrate along the mesentery, whereas enteric NCCs invaded the foregut to migrate along the gut. mNCCs not only produced neurons and glia within the gut mesentery, but also continuously complemented the enteric NCC pool. Two new cases of SSHD were identified from 183 HSCR patients, and Ednrb-mutant mice, but not Ret-/- mice, showed a high incidence rate of SSHD-like phenotypes. CONCLUSIONS mNCCs, a subset of vNCCs that migrate into the gut via the gut mesentery to give rise to enteric neurons, could provide an embryologic explanation for SSHD. These findings lead to novel insights into the development of the enteric nervous system and the etiology of HSCR.
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Cardinal T, Bergeron KF, Soret R, Souchkova O, Faure C, Guillon A, Pilon N. Male-biased aganglionic megacolon in the TashT mouse model of Hirschsprung disease involves upregulation of p53 protein activity and Ddx3y gene expression. PLoS Genet 2020; 16:e1009008. [PMID: 32898154 PMCID: PMC7500598 DOI: 10.1371/journal.pgen.1009008] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 09/18/2020] [Accepted: 07/23/2020] [Indexed: 02/07/2023] Open
Abstract
Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.
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Affiliation(s)
- Tatiana Cardinal
- Molecular Genetics of Development Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
| | - Karl-Frédérik Bergeron
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
- Lipid Metabolism Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
| | - Rodolphe Soret
- Molecular Genetics of Development Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
| | - Ouliana Souchkova
- Molecular Genetics of Development Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
| | - Christophe Faure
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
- Département de pédiatrie, Université de Montréal, Montréal, Québec, Canada
- Division de gastroentérologie, hépatologie et nutrition pédiatrique, Centre hospitalier universitaire Sainte-Justine, Montréal, Québec, Canada
| | - Amélina Guillon
- Molecular Genetics of Development Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
| | - Nicolas Pilon
- Molecular Genetics of Development Laboratory, Département des Sciences Biologiques, Université du Québec à Montréal (UQAM), Montréal, Québec, Canada
- Centre d'excellence en recherche sur les maladies orphelines-Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montréal, Québec, Canada
- Département de pédiatrie, Université de Montréal, Montréal, Québec, Canada
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Kostouros A, Koliarakis I, Natsis K, Spandidos DA, Tsatsakis A, Tsiaoussis J. Large intestine embryogenesis: Molecular pathways and related disorders (Review). Int J Mol Med 2020; 46:27-57. [PMID: 32319546 PMCID: PMC7255481 DOI: 10.3892/ijmm.2020.4583] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2020] [Accepted: 04/08/2020] [Indexed: 02/07/2023] Open
Abstract
The large intestine, part of the gastrointestinal tract (GI), is composed of all three germ layers, namely the endoderm, the mesoderm and the ectoderm, forming the epithelium, the smooth muscle layers and the enteric nervous system, respectively. Since gastrulation, these layers develop simultaneously during embryogenesis, signaling to each other continuously until adult age. Two invaginations, the anterior intestinal portal (AIP) and the caudal/posterior intestinal portal (CIP), elongate and fuse, creating the primitive gut tube, which is then patterned along the antero‑posterior (AP) axis and the radial (RAD) axis in the context of left‑right (LR) asymmetry. These events lead to the formation of three distinct regions, the foregut, midgut and hindgut. All the above‑mentioned phenomena are under strict control from various molecular pathways, which are critical for the normal intestinal development and function. Specifically, the intestinal epithelium constitutes a constantly developing tissue, deriving from the progenitor stem cells at the bottom of the intestinal crypt. Epithelial differentiation strongly depends on the crosstalk with the adjacent mesoderm. Major molecular pathways that are implicated in the embryogenesis of the large intestine include the canonical and non‑canonical wingless‑related integration site (Wnt), bone morphogenetic protein (BMP), Notch and hedgehog systems. The aberrant regulation of these pathways inevitably leads to several intestinal malformation syndromes, such as atresia, stenosis, or agangliosis. Novel theories, involving the regulation and homeostasis of intestinal stem cells, suggest an embryological basis for the pathogenesis of colorectal cancer (CRC). Thus, the present review article summarizes the diverse roles of these molecular factors in intestinal embryogenesis and related disorders.
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Affiliation(s)
- Antonios Kostouros
- Laboratory of Anatomy-Histology-Embryology, Medical School, University of Crete, 71110 Heraklion
| | - Ioannis Koliarakis
- Laboratory of Anatomy-Histology-Embryology, Medical School, University of Crete, 71110 Heraklion
| | - Konstantinos Natsis
- Department of Anatomy and Surgical Anatomy, Medical School, Aristotle University of Thessaloniki, 54124 Thessaloniki
| | | | - Aristidis Tsatsakis
- Laboratory of Toxicology, Medical School, University of Crete, 71409 Heraklion, Greece
| | - John Tsiaoussis
- Laboratory of Anatomy-Histology-Embryology, Medical School, University of Crete, 71110 Heraklion
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Green DJ, Sallah SR, Ellingford JM, Lovell SC, Sergouniotis PI. Variability in Gene Expression is Associated with Incomplete Penetrance in Inherited Eye Disorders. Genes (Basel) 2020; 11:genes11020179. [PMID: 32050448 PMCID: PMC7074066 DOI: 10.3390/genes11020179] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2019] [Revised: 01/29/2020] [Accepted: 02/06/2020] [Indexed: 12/21/2022] Open
Abstract
Inherited eye disorders (IED) are a heterogeneous group of Mendelian conditions that are associated with visual impairment. Although these disorders often exhibit incomplete penetrance and variable expressivity, the scale and mechanisms of these phenomena remain largely unknown. Here, we utilize publicly-available genomic and transcriptomic datasets to gain insights into variable penetrance in IED. Variants in a curated set of 340 IED-implicated genes were extracted from the Human Gene Mutation Database (HGMD) 2019.1 and cross-checked with the Genome Aggregation Database (gnomAD) 2.1 control-only dataset. Genes for which >1 variants were encountered in both HGMD and gnomAD were considered to be associated with variable penetrance (n = 56). Variability in gene expression levels was then estimated for the subset of these genes that was found to be adequately expressed in two relevant resources: the Genotype-Tissue Expression (GTEx) and Eye Genotype Expression (EyeGEx) datasets. We found that genes suspected to be associated with variable penetrance tended to have significantly more variability in gene expression levels in the general population (p = 0.0000015); this finding was consistent across tissue types. The results of this study point to the possible influence of cis and/or trans-acting elements on the expressivity of variants causing Mendelian disorders. They also highlight the potential utility of quantifying gene expression as part of the investigation of families showing evidence of variable penetrance.
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Affiliation(s)
- David J. Green
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester M13 9PT, UK; (D.J.G.); (S.R.S.); (J.M.E.); (S.C.L.)
| | - Shalaw R. Sallah
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester M13 9PT, UK; (D.J.G.); (S.R.S.); (J.M.E.); (S.C.L.)
| | - Jamie M. Ellingford
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester M13 9PT, UK; (D.J.G.); (S.R.S.); (J.M.E.); (S.C.L.)
- Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester M13 9WL, UK
| | - Simon C. Lovell
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester M13 9PT, UK; (D.J.G.); (S.R.S.); (J.M.E.); (S.C.L.)
| | - Panagiotis I. Sergouniotis
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicines and Health, University of Manchester, Manchester M13 9PT, UK; (D.J.G.); (S.R.S.); (J.M.E.); (S.C.L.)
- Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester M13 9WL, UK
- Manchester Royal Eye Hospital, Manchester University NHS Foundation Trust, Manchester M13 9WL, UK
- Correspondence: ; Tel.: +44-(0)161-27-55748
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12
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Chatterjee S, Nandakumar P, Auer DR, Gabriel SB, Chakravarti A. Gene- and tissue-level interactions in normal gastrointestinal development and Hirschsprung disease. Proc Natl Acad Sci U S A 2019; 116:26697-26708. [PMID: 31818953 PMCID: PMC6936708 DOI: 10.1073/pnas.1908756116] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The development of the gut from endodermal tissue to an organ with multiple distinct structures and functions occurs over a prolonged time during embryonic days E10.5-E14.5 in the mouse. During this process, one major event is innervation of the gut by enteric neural crest cells (ENCCs) to establish the enteric nervous system (ENS). To understand the molecular processes underpinning gut and ENS development, we generated RNA-sequencing profiles from wild-type mouse guts at E10.5, E12.5, and E14.5 from both sexes. We also generated these profiles from homozygous Ret null embryos, a model for Hirschsprung disease (HSCR), in which the ENS is absent. These data reveal 4 major features: 1) between E10.5 and E14.5 the developmental genetic programs change from expression of major transcription factors and its modifiers to genes controlling tissue (epithelium, muscle, endothelium) specialization; 2) the major effect of Ret is not only on ENCC differentiation to enteric neurons but also on the enteric mesenchyme and epithelium; 3) a muscle genetic program exerts significant effects on ENS development; and 4) sex differences in gut development profiles are minor. The genetic programs identified, and their changes across development, suggest that both cell autonomous and nonautonomous factors, and interactions between the different developing gut tissues, are important for normal ENS development and its disorders.
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Affiliation(s)
- Sumantra Chatterjee
- Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205
- Center for Human Genetics and Genomics, New York University School of Medicine, New York, NY 10016
| | - Priyanka Nandakumar
- Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205
| | - Dallas R. Auer
- Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205
- Center for Human Genetics and Genomics, New York University School of Medicine, New York, NY 10016
| | - Stacey B. Gabriel
- Genomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142
| | - Aravinda Chakravarti
- Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205
- Center for Human Genetics and Genomics, New York University School of Medicine, New York, NY 10016
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13
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Chatterjee S, Chakravarti A. A gene regulatory network explains RET-EDNRB epistasis in Hirschsprung disease. Hum Mol Genet 2019; 28:3137-3147. [PMID: 31313802 PMCID: PMC7275776 DOI: 10.1093/hmg/ddz149] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 05/22/2019] [Accepted: 06/21/2019] [Indexed: 12/31/2022] Open
Abstract
Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (HSCR), a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TFs) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10, and in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.
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Affiliation(s)
- Sumantra Chatterjee
- Center for Human Genetics and Genomics, New York University School of Medicine, New York, NY, USA
| | - Aravinda Chakravarti
- Center for Human Genetics and Genomics, New York University School of Medicine, New York, NY, USA
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14
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Rylaarsdam L, Guemez-Gamboa A. Genetic Causes and Modifiers of Autism Spectrum Disorder. Front Cell Neurosci 2019; 13:385. [PMID: 31481879 PMCID: PMC6710438 DOI: 10.3389/fncel.2019.00385] [Citation(s) in RCA: 293] [Impact Index Per Article: 48.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2019] [Accepted: 08/06/2019] [Indexed: 12/18/2022] Open
Abstract
Autism Spectrum Disorder (ASD) is one of the most prevalent neurodevelopmental disorders, affecting an estimated 1 in 59 children. ASD is highly genetically heterogeneous and may be caused by both inheritable and de novo gene variations. In the past decade, hundreds of genes have been identified that contribute to the serious deficits in communication, social cognition, and behavior that patients often experience. However, these only account for 10-20% of ASD cases, and patients with similar pathogenic variants may be diagnosed on very different levels of the spectrum. In this review, we will describe the genetic landscape of ASD and discuss how genetic modifiers such as copy number variation, single nucleotide polymorphisms, and epigenetic alterations likely play a key role in modulating the phenotypic spectrum of ASD patients. We also consider how genetic modifiers can alter convergent signaling pathways and lead to impaired neural circuitry formation. Lastly, we review sex-linked modifiers and clinical implications. Further understanding of these mechanisms is crucial for both comprehending ASD and for developing novel therapies.
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Affiliation(s)
| | - Alicia Guemez-Gamboa
- Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States
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15
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Null mutation of the endothelin receptor type B gene causes embryonic death in the GK rat. PLoS One 2019; 14:e0217132. [PMID: 31170185 PMCID: PMC6553694 DOI: 10.1371/journal.pone.0217132] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Accepted: 05/06/2019] [Indexed: 12/28/2022] Open
Abstract
The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis.
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Heuckeroth RO. Even When You Know Everything, There Is Still More to Learn About Hirschsprung Disease. Gastroenterology 2018; 155:1681-1684. [PMID: 30419210 DOI: 10.1053/j.gastro.2018.11.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Affiliation(s)
- Robert O Heuckeroth
- Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania and The Children's Hospital of Philadelphia - Research Institute, Philadelphia, Pennsylvania.
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17
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Bondurand N, Dufour S, Pingault V. News from the endothelin-3/EDNRB signaling pathway: Role during enteric nervous system development and involvement in neural crest-associated disorders. Dev Biol 2018; 444 Suppl 1:S156-S169. [PMID: 30171849 DOI: 10.1016/j.ydbio.2018.08.014] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2018] [Revised: 08/27/2018] [Accepted: 08/27/2018] [Indexed: 01/08/2023]
Abstract
The endothelin system is a vertebrate-specific innovation with important roles in regulating the cardiovascular system and renal and pulmonary processes, as well as the development of the vertebrate-specific neural crest cell population and its derivatives. This system is comprised of three structurally similar 21-amino acid peptides that bind and activate two G-protein coupled receptors. In 1994, knockouts of the Edn3 and Ednrb genes revealed their crucial function during development of the enteric nervous system and melanocytes, two neural-crest derivatives. Since then, human and mouse genetics, combined with cellular and developmental studies, have helped to unravel the role of this signaling pathway during development and adulthood. In this review, we will summarize the known functions of the EDN3/EDNRB pathway during neural crest development, with a specific focus on recent scientific advances, and the enteric nervous system in normal and pathological conditions.
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Affiliation(s)
- Nadege Bondurand
- Laboratory of Embryology and Genetics of Congenital Malformations, INSERM U1163, Institut Imagine, Paris, France; Paris Descartes-Sorbonne Paris Cité University, Institut Imagine, Paris, France.
| | - Sylvie Dufour
- INSERM, U955, Equipe 06, Créteil 94000, France; Université Paris Est, Faculté de Médecine, Créteil 94000, France
| | - Veronique Pingault
- Laboratory of Embryology and Genetics of Congenital Malformations, INSERM U1163, Institut Imagine, Paris, France; Paris Descartes-Sorbonne Paris Cité University, Institut Imagine, Paris, France; Service de Génétique Moléculaire, Hôpital Necker-Enfants Malades, Assistance Publique - Hôpitaux de Paris, Paris, France
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18
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Chaudhury A, Dendi VSR, Chaudhury M, Jain A, Kasarla MR, Panuganti K, Jain G, Ramanujam A, Rena B, Koyagura SR, Fogla S, Kumar S, Shekhawat NS, Maddur S. HSV1/2 Genital Infection in Mice Cause Reversible Delayed Gastrointestinal Transit: A Model for Enteric Myopathy. Front Med (Lausanne) 2018; 5:176. [PMID: 30065927 PMCID: PMC6056620 DOI: 10.3389/fmed.2018.00176] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Accepted: 05/21/2018] [Indexed: 12/20/2022] Open
Abstract
In an interesting investigation by Khoury-Hanold et al. (1), genital infection of mice with herpes simplex virus 1 (HSV1) were reported to cause multiple pelvic organ involvement and obstruction. A small subset of mice succumbed after the first week of HSV1 infection. The authors inferred that the mice died due to toxic megacolon. In a severe form of mechanical and/or functional obstruction involving gross dilation of the colon and profound toxemia, the presentation is called "toxic megacolon." The representative observations by Khoury-Hanold likely do not resemble toxic megacolon. The colon was only slightly dilated and benign appearing. Importantly, HSV1 infection affected the postjunctional mechanisms of smooth muscle relaxation like the sildenafil-response proteins, which may have been responsible for defective nitrergic neurotransmission and the delayed transit. Orally administered polyethylene glycol reversed the gastrointestinal "obstruction," suggesting a mild functional type of slowed luminal transit, resembling constipation, rather than toxic megacolon, which cannot be reversed by an osmotic laxative without perforating the gut. The authors suggest that the mice did not develop HSV1 encephalitis, the commonly known cause of mortality. The premature death of some of the mice could be related to the bladder outlet obstruction, whose backflow effects may alter renal function, electrolyte abnormalities and death. Muscle strip recordings of mechanical relaxation after electrical field stimulation of gastrointestinal, urinary bladder or cavernosal tissues shall help obtain objective quantitative evidence of whether HSV infection indeed cause pelvic multi-organ dysfunction and impairment of autonomic neurotransmission and postjunctional electromechanical relaxation mechanisms of these organs.
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Affiliation(s)
| | | | | | - Astha Jain
- Wanderful Media/University of Southern California, Los Angeles, CA, United States
| | | | | | - Gaurav Jain
- Berkshire Medical Center, Pittsfield, MA, United States
| | | | - Bhavin Rena
- Xenco Laboratories, Houston, TX, United States
| | | | - Sumit Fogla
- Beaumont Hospital, Grosse Pointe, MI, United States
| | - Sunil Kumar
- Neshoba County General Hospital, Philadelphia, MS, United States
| | | | - Srinivas Maddur
- All India Institute of Medical Sciences, New Delhi, India
- ESIC Medical College, Sanathnagar, India
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Hirschsprung disease - integrating basic science and clinical medicine to improve outcomes. Nat Rev Gastroenterol Hepatol 2018; 15:152-167. [PMID: 29300049 DOI: 10.1038/nrgastro.2017.149] [Citation(s) in RCA: 179] [Impact Index Per Article: 25.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Hirschsprung disease is defined by the absence of enteric neurons at the end of the bowel. The enteric nervous system (ENS) is the intrinsic nervous system of the bowel and regulates most aspects of bowel function. When the ENS is missing, there are no neurally mediated propulsive motility patterns, and the bowel remains contracted, causing functional obstruction. Symptoms of Hirschsprung disease include constipation, vomiting, abdominal distension and growth failure. Untreated disease usually causes death in childhood because bloodstream bacterial infections occur in the context of bowel inflammation (enterocolitis) or bowel perforation. Current treatment is surgical resection of the bowel to remove or bypass regions where the ENS is missing, but many children have problems after surgery. Although the anatomy of Hirschsprung disease is simple, many clinical features remain enigmatic, and diagnosis and management remain challenging. For example, the age of presentation and the type of symptoms that occur vary dramatically among patients, even though every affected child has missing neurons in the distal bowel at birth. In this Review, basic science discoveries are linked to clinical manifestations of Hirschsprung disease, including partial penetrance, enterocolitis and genetics. Insights into disease mechanisms that might lead to new prevention, diagnostic and treatment strategies are described.
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Hirst CS, Stamp LA, Bergner AJ, Hao MM, Tran MX, Morgan JM, Dutschmann M, Allen AM, Paxinos G, Furlong TM, McKeown SJ, Young HM. Kif1bp loss in mice leads to defects in the peripheral and central nervous system and perinatal death. Sci Rep 2017; 7:16676. [PMID: 29192291 PMCID: PMC5709403 DOI: 10.1038/s41598-017-16965-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Accepted: 11/19/2017] [Indexed: 12/29/2022] Open
Abstract
Goldberg-Shprintzen syndrome is a poorly understood condition characterized by learning difficulties, facial dysmorphism, microcephaly, and Hirschsprung disease. GOSHS is due to recessive mutations in KIAA1279, which encodes kinesin family member 1 binding protein (KIF1BP, also known as KBP). We examined the effects of inactivation of Kif1bp in mice. Mice lacking Kif1bp died shortly after birth, and exhibited smaller brains, olfactory bulbs and anterior commissures, and defects in the vagal and sympathetic innervation of the gut. Kif1bp was found to interact with Ret to regulate the development of the vagal innervation of the stomach. Although newborn Kif1bp−/− mice had neurons along the entire bowel, the colonization of the gut by neural crest-derived cells was delayed. The data show an essential in vivo role for KIF1BP in axon extension from some neurons, and the reduced size of the olfactory bulb also suggests additional roles for KIF1BP. Our mouse model provides a valuable resource to understand GOSHS.
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Affiliation(s)
- Caroline S Hirst
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Lincon A Stamp
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Annette J Bergner
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Marlene M Hao
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Mai X Tran
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Jan M Morgan
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia
| | - Matthias Dutschmann
- Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia
| | - Andrew M Allen
- Department of Physiology, The University of Melbourne, Victoria, 3010, Australia
| | - George Paxinos
- Neuroscience Research Australia and School of Medical Sciences, The University of New South Wales, 2031, NSW, Australia
| | - Teri M Furlong
- Neuroscience Research Australia and School of Medical Sciences, The University of New South Wales, 2031, NSW, Australia
| | - Sonja J McKeown
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia. .,Cancer Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Victoria, 3800, Australia.
| | - Heather M Young
- Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, 3010, Australia.
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Kapoor A, Auer DR, Lee D, Chatterjee S, Chakravarti A. Testing the Ret and Sema3d genetic interaction in mouse enteric nervous system development. Hum Mol Genet 2017; 26:1811-1820. [PMID: 28334784 DOI: 10.1093/hmg/ddx084] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Accepted: 03/02/2017] [Indexed: 12/20/2022] Open
Abstract
For most multigenic disorders, clinical manifestation (penetrance) and presentation (expressivity) are likely to be an outcome of genetic interaction between multiple susceptibility genes. Here, using gene knockouts in mice, we evaluated genetic interaction between loss of Ret and loss of Sema3d, two Hirschsprung disease susceptibility genes. We intercrossed Ret and Sema3d double null heterozygotes to generate mice with the nine possible genotypes and assessed survival by counting various genotypes, myenteric plexus presence by acetylcholinesterase staining and embryonic day 12.5 (E12.5) intestine transcriptome by RNA-sequencing. Survival rates of Ret wild-type, null heterozygote and null homozygote mice at E12.5, birth and weaning were not influenced by the genotypes at Sema3d locus and vice versa. Loss of myenteric plexus was observed only in all Ret null homozygotes, irrespective of the genotypes at Sema3d locus, and Sema3d null heterozygote and homozygote mice had normal intestinal innervation. As compared with wild-type mice intestinal gene expression, loss of Ret in null homozygotes led to differential expression of ∼300 genes, whereas loss of Sema3d in null homozygotes had no major consequence and there was no evidence supporting major interaction between the two genes influencing intestine transcriptome. Overall, given the null alleles and phenotypic assays used, we did not find evidence for genetic interaction between Ret and Sema3d affecting survival, presence of myenteric plexus or intestine transcriptome.
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Affiliation(s)
- Ashish Kapoor
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Dallas R Auer
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Dongwon Lee
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Sumantra Chatterjee
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Aravinda Chakravarti
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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22
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Nagy N, Goldstein AM. Enteric nervous system development: A crest cell's journey from neural tube to colon. Semin Cell Dev Biol 2017; 66:94-106. [PMID: 28087321 DOI: 10.1016/j.semcdb.2017.01.006] [Citation(s) in RCA: 145] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Revised: 01/03/2017] [Accepted: 01/09/2017] [Indexed: 12/31/2022]
Abstract
The enteric nervous system (ENS) is comprised of a network of neurons and glial cells that are responsible for coordinating many aspects of gastrointestinal (GI) function. These cells arise from the neural crest, migrate to the gut, and then continue their journey to colonize the entire length of the GI tract. Our understanding of the molecular and cellular events that regulate these processes has advanced significantly over the past several decades, in large part facilitated by the use of rodents, avians, and zebrafish as model systems to dissect the signals and pathways involved. These studies have highlighted the highly dynamic nature of ENS development and the importance of carefully balancing migration, proliferation, and differentiation of enteric neural crest-derived cells (ENCCs). Proliferation, in particular, is critically important as it drives cell density and speed of migration, both of which are important for ensuring complete colonization of the gut. However, proliferation must be tempered by differentiation among cells that have reached their final destination and are ready to send axonal extensions, connect to effector cells, and begin to produce neurotransmitters or other signals. Abnormalities in the normal processes guiding ENCC development can lead to failure of ENS formation, as occurs in Hirschsprung disease, in which the distal intestine remains aganglionic. This review summarizes our current understanding of the factors involved in early development of the ENS and discusses areas in need of further investigation.
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Affiliation(s)
- Nandor Nagy
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; Center for Neurointestinal Health, Massachusetts General Hospital, Boston, MA, United States; Department of Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis University, Budapest, Hungary
| | - Allan M Goldstein
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; Center for Neurointestinal Health, Massachusetts General Hospital, Boston, MA, United States.
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23
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Heuckeroth RO, Schäfer KH. Gene-environment interactions and the enteric nervous system: Neural plasticity and Hirschsprung disease prevention. Dev Biol 2016; 417:188-97. [PMID: 26997034 PMCID: PMC5026873 DOI: 10.1016/j.ydbio.2016.03.017] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2016] [Revised: 03/04/2016] [Accepted: 03/14/2016] [Indexed: 12/12/2022]
Abstract
Intestinal function is primarily controlled by an intrinsic nervous system of the bowel called the enteric nervous system (ENS). The cells of the ENS are neural crest derivatives that migrate into and through the bowel during early stages of organogenesis before differentiating into a wide variety of neurons and glia. Although genetic factors critically underlie ENS development, it is now clear that many non-genetic factors may influence the number of enteric neurons, types of enteric neurons, and ratio of neurons to glia. These non-genetic influences include dietary nutrients and medicines that may impact ENS structure and function before or after birth. This review summarizes current data about gene-environment interactions that affect ENS development and suggests that these factors may contribute to human intestinal motility disorders like Hirschsprung disease or irritable bowel syndrome.
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Affiliation(s)
- Robert O Heuckeroth
- Department of Pediatrics, The Children's Hospital of Philadelphia Research Institute, USA; The Perelman School of Medicine at the University of Pennsylvania, Abramson Research Center, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.
| | - Karl-Herbert Schäfer
- ENS Group, University of Applied Sciences Kaiserslautern/Zweibrücken, Germany; University of Heidelberg, Paediatric Surgery Mannheim, Germany
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24
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Brosens E, Burns AJ, Brooks AS, Matera I, Borrego S, Ceccherini I, Tam PK, García-Barceló MM, Thapar N, Benninga MA, Hofstra RMW, Alves MM. Genetics of enteric neuropathies. Dev Biol 2016; 417:198-208. [PMID: 27426273 DOI: 10.1016/j.ydbio.2016.07.008] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Revised: 07/13/2016] [Accepted: 07/13/2016] [Indexed: 12/23/2022]
Abstract
Abnormal development or disturbed functioning of the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is associated with the development of neuropathic gastrointestinal motility disorders. Here, we review the underlying molecular basis of these disorders and hypothesize that many of them have a common defective biological mechanism. Genetic burden and environmental components affecting this common mechanism are ultimately responsible for disease severity and symptom heterogeneity. We believe that they act together as the fulcrum in a seesaw balanced with harmful and protective factors, and are responsible for a continuum of symptoms ranging from neuronal hyperplasia to absence of neurons.
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Affiliation(s)
- Erwin Brosens
- Department of Clinical Genetics, Erasmus University Medical Centre - Sophia Children's Hospital, Rotterdam, The Netherlands.
| | - Alan J Burns
- Department of Clinical Genetics, Erasmus University Medical Centre - Sophia Children's Hospital, Rotterdam, The Netherlands; Stem Cells and Regenerative Medicine, Birth Defects Research Centre, UCL Institute of Child Health, London, UK
| | - Alice S Brooks
- Department of Clinical Genetics, Erasmus University Medical Centre - Sophia Children's Hospital, Rotterdam, The Netherlands
| | - Ivana Matera
- UOC Medical Genetics, Istituto Giannina Gaslini, Genova, Italy
| | - Salud Borrego
- Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), Seville, Spain; Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain
| | | | - Paul K Tam
- Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine of the University of Hong Kong, Hong Kong, China
| | - Maria-Mercè García-Barceló
- State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Reproduction, Development, and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Nikhil Thapar
- Stem Cells and Regenerative Medicine, Birth Defects Research Centre, UCL Institute of Child Health, London, UK
| | - Marc A Benninga
- Pediatric Gastroenterology, Emma Children's Hospital/Academic Medical Center, Amsterdam, The Netherlands
| | - Robert M W Hofstra
- Department of Clinical Genetics, Erasmus University Medical Centre - Sophia Children's Hospital, Rotterdam, The Netherlands; Stem Cells and Regenerative Medicine, Birth Defects Research Centre, UCL Institute of Child Health, London, UK
| | - Maria M Alves
- Department of Clinical Genetics, Erasmus University Medical Centre - Sophia Children's Hospital, Rotterdam, The Netherlands
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25
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Bondurand N, Southard-Smith EM. Mouse models of Hirschsprung disease and other developmental disorders of the enteric nervous system: Old and new players. Dev Biol 2016; 417:139-57. [PMID: 27370713 DOI: 10.1016/j.ydbio.2016.06.042] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Revised: 06/27/2016] [Accepted: 06/27/2016] [Indexed: 12/18/2022]
Abstract
Hirschsprung disease (HSCR, intestinal aganglionosis) is a multigenic disorder with variable penetrance and severity that has a general population incidence of 1/5000 live births. Studies using animal models have contributed to our understanding of the developmental origins of HSCR and the genetic complexity of this disease. This review summarizes recent progress in understanding control of enteric nervous system (ENS) development through analyses in mouse models. An overview of signaling pathways that have long been known to control the migration, proliferation and differentiation of enteric neural progenitors into and along the developing gut is provided as a framework for the latest information on factors that influence enteric ganglia formation and maintenance. Newly identified genes and additional factors beyond discrete genes that contribute to ENS pathology including regulatory sequences, miRNAs and environmental factors are also introduced. Finally, because HSCR has become a paradigm for complex oligogenic diseases with non-Mendelian inheritance, the importance of gene interactions, modifier genes, and initial studies on genetic background effects are outlined.
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Affiliation(s)
- Nadege Bondurand
- INSERM, U955, Equipe 6, F-94000 Creteil, France; Universite Paris-Est, UPEC, F-94000 Creteil, France.
| | - E Michelle Southard-Smith
- Vanderbilt University Medical Center, Department of Medicine, 2215 Garland Ave, Nashville, TN 37232, USA.
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26
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Schill EM, Lake JI, Tusheva OA, Nagy N, Bery SK, Foster L, Avetisyan M, Johnson SL, Stenson WF, Goldstein AM, Heuckeroth RO. Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse. Dev Biol 2016; 409:473-88. [PMID: 26586201 PMCID: PMC4862364 DOI: 10.1016/j.ydbio.2015.09.023] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2015] [Revised: 08/31/2015] [Accepted: 09/07/2015] [Indexed: 12/17/2022]
Abstract
Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/- mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.
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Affiliation(s)
- Ellen Merrick Schill
- Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Jonathan I Lake
- Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Olga A Tusheva
- Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Nandor Nagy
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St., Boston, MA 02114, USA; Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary
| | - Saya K Bery
- Department of Pediatrics, The Children's Hospital of Philadelphia Research Institute and the Perelman School of Medicine at the University of Pennsylvania, Abramson Research Center, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA
| | - Lynne Foster
- Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Marina Avetisyan
- Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Stephen L Johnson
- Department of Genetics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - William F Stenson
- Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Allan M Goldstein
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St., Boston, MA 02114, USA
| | - Robert O Heuckeroth
- Department of Pediatrics, The Children's Hospital of Philadelphia Research Institute and the Perelman School of Medicine at the University of Pennsylvania, Abramson Research Center, 3615 Civic Center Blvd, Philadelphia, PA 19104, USA.
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Abstract
PURPOSE Hirschsprung disease (HSCR) is a congenital and heterogeneous disorder, which is caused by no neuronal ganglion cells in part or all of distal gastrointestinal tract. Recently, our genome-wide association study has identified solute carrier family 6, proline IMINO transporter, member 20 (SLC6A20) as one of the potential risk factors for HSCR development. This study performed a replication study for the association of SLC6A20 polymorphisms with HSCR and an extended analysis to investigate further associations for subgroups and haplotypes. METHODS For the replication study, a total of 40 single nucleotide polymorphisms (SNPs) of SLC6A20 were genotyped in 187 HSCR subjects composed of 121 short-segment HSCR, 45 long-segment HSCR (L-HSCR), 21 total colonic aganglionosis, and 283 unaffected controls. Imputation was performed using genotype data from our genome-wide association study and this replication study. RESULTS Imputed meta-analysis revealed that 13 SLC6A20 SNPs (minimum P = 0.0002 at rs6770261) were significantly associated with HSCR even after correction for multiple comparisons using false discovery rate (FDR) (minimum PFDR = .005). In further subgroup analysis, SLC6A20 polymorphisms appeared to have increased associations with L-HSCR. Moreover, haplotype analysis also showed significant associations between 2 haplotypes (BL3_ht2 and BL4_ht2) and HSCR susceptibility (PFDR < .05). CONCLUSIONS Although further replications and functional evaluations are required, our results suggest that SLC6A20 may have roles in HSCR development and in the extent of aganglionic segment during enteric nervous system development.
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Heuckeroth RO. Hirschsprung's disease, Down syndrome, and missing heritability: too much collagen slows migration. J Clin Invest 2015; 125:4323-6. [PMID: 26571392 PMCID: PMC4665790 DOI: 10.1172/jci85003] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Hirschsprung's disease (HSCR) causes functional intestinal obstruction due to the absence of the enteric nervous system (ENS) in the distal bowel and is usually diagnosed shortly after birth or during childhood. While several genetic and nongenetic factors have been linked to HSCR, the underlying mechanisms that prevent ENS precursors from colonizing distal bowel during fetal development are not completely understood in many affected children. In this issue of the JCI, Soret and colleagues identify a new mechanism that causes HSCR-like disease in mice and involves deposition of excess collagen VI in the intestine by migrating ENS precursors as they colonize fetal bowel. Remarkably, their findings may explain some of the so-called missing heritability of HSCR and suggest a mechanism for increased HSCR incidence in children with Down syndrome (trisomy 21).
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Abstract
PURPOSE During the past two decades several genes have been identified that control morphogenesis and differentiation of the enteric neuron system (ENS). These genes, when mutated or deleted, interfere with ENS development. RET gene is the major gene causing Hirschsprung's disease (HD). Mutations in RET gene are responsible for 50% of familial HD cases and 15-20% of sporadic cases. The aim of this meta-analysis was to determine the incidence of RET gene mutations in patients with HD and to correlate RET mutations with the extent of aganglionosis. METHODS A systematic literature-based search for relevant cohorts was performed using the terms "Hirschsprung's disease AND RET Proto-oncogene", "Hirschsprung's disease AND genetic polymorphism" and "RET Gene". The relevant cohorts of HD were systematically searched for reported mutations in the RET gene (RET+). Data on mutation site, phenotype, and familial or sporadic cases were extracted. Combined odds ratio (OR) with 95% CI was calculated to estimate the strength of the different associations. RESULTS In total, 23 studies concerning RET with 1270 individuals affected with HD were included in this study. 228 (18%) of these HDs were RET+. Of these 228, 96 (42%) presented as rectosigmoid, 81 (36%) long segment, 18 (8%) as TCA, 16 (7%) as total intestinal aganglionosis and 17 (7%) individuals were RET+ but no extent of aganglionosis was not reported. In the rectosigmoid group, no significant association between phenotype and RET mutation could be shown (P = 0.006), whereas a clear association could be shown between long-segment disease, total colonic- and total intestinal aganglionosis and RET mutations (P = 0.0002). Mutations most often occurred in Exon 13 (24) and showed significant association with rectosigmoid disease (P = 0.004). No significance could be shown between RET+ and sporadic cases (P = 0.53), albeit a trend towards RET+ and Familial cases could be observed (P = 0.38). CONCLUSIONS The association with the RET gene and HD is well recognized. This study showed a clear association between RET+ mutations and the long-segment, total colonic- and total intestinal aganglionosis. Exon 13 appears to be a mutational "hot spot" in rectosigmoid disease.
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30
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Gasc JM, Clemessy M, Corvol P, Kempf H. A chicken model of pharmacologically-induced Hirschsprung disease reveals an unexpected role of glucocorticoids in enteric aganglionosis. Biol Open 2015; 4:666-71. [PMID: 25836673 PMCID: PMC4434818 DOI: 10.1242/bio.201410454] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The enteric nervous system originates from neural crest cells that migrate in chains as they colonize the embryonic gut, eventually forming the myenteric and submucosal plexus. Failure of the neural crest cells to colonize the gut leads to aganglionosis in the terminal gut, a pathological condition called Hirschsprung disease (HSCR) in humans, also known as congenital megacolon or intestinal aganglionosis. One of the characteristics of the human HSCR is its variable penetrance, which may be attributable to the interaction between genetic factors, such as the endothelin-3/endothelin receptor B pathway, and non-genetic modulators, although the role of the latter has not well been established. We have created a novel HSCR model in the chick embryo allowing to test the ability of non-genetic modifiers to alter the HSCR phenotype. Chick embryos treated by phosphoramidon, which blocks the generation of endothelin-3, failed to develop enteric ganglia in the very distal bowel, characteristic of an HSCR-like phenotype. Administration of dexamethasone influenced the phenotype, suggesting that glucocorticoids may be environmental modulators of the penetrance of the aganglionosis in HSCR disease.
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Affiliation(s)
- Jean-Marie Gasc
- Centre Interdisciplinaire de Recherche Biomédicale (CIRB), Collège de France, 75005 Paris, France Chaire de Médecine Expérimentale, Collège de France, 75005 Paris, France
| | - Maud Clemessy
- Centre Interdisciplinaire de Recherche Biomédicale (CIRB), Collège de France, 75005 Paris, France Chaire de Médecine Expérimentale, Collège de France, 75005 Paris, France Centre de Recherche St-Antoine UMRS-938, INSERM-Université Pierre et Marie Curie, Paris 6, 75012 Paris, France
| | - Pierre Corvol
- Centre Interdisciplinaire de Recherche Biomédicale (CIRB), Collège de France, 75005 Paris, France Chaire de Médecine Expérimentale, Collège de France, 75005 Paris, France
| | - Hervé Kempf
- Centre Interdisciplinaire de Recherche Biomédicale (CIRB), Collège de France, 75005 Paris, France Chaire de Médecine Expérimentale, Collège de France, 75005 Paris, France UMR 7365 CNRS-Université de Lorraine, IMoPA, Faculté de Médecine, 54500 Vandoeuvre-lès-Nancy, France
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31
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Jiang Q, Arnold S, Heanue T, Kilambi K, Doan B, Kapoor A, Ling A, Sosa M, Guy M, Jiang Q, Burzynski G, West K, Bessling S, Griseri P, Amiel J, Fernandez R, Verheij J, Hofstra R, Borrego S, Lyonnet S, Ceccherini I, Gray J, Pachnis V, McCallion A, Chakravarti A. Functional loss of semaphorin 3C and/or semaphorin 3D and their epistatic interaction with ret are critical to Hirschsprung disease liability. Am J Hum Genet 2015; 96:581-96. [PMID: 25839327 DOI: 10.1016/j.ajhg.2015.02.014] [Citation(s) in RCA: 97] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2014] [Accepted: 02/20/2015] [Indexed: 10/23/2022] Open
Abstract
Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.
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Huang J, Dang R, Torigoe D, Lei C, Lan X, Chen H, Sasaki N, Wang J, Agui T. Identification of genetic loci affecting the severity of symptoms of Hirschsprung disease in rats carrying Ednrbsl mutations by quantitative trait locus analysis. PLoS One 2015; 10:e0122068. [PMID: 25790447 PMCID: PMC4366197 DOI: 10.1371/journal.pone.0122068] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Accepted: 02/12/2015] [Indexed: 01/17/2023] Open
Abstract
Hirschsprung’s disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrbsl/sl, resistant F344-Ednrbsl/sl, and LEH-Ednrbsl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F2 intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F2 intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis.
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Affiliation(s)
- Jieping Huang
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Ruihua Dang
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
- * E-mail: (RD); (CL)
| | - Daisuke Torigoe
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Chuzhao Lei
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
- * E-mail: (RD); (CL)
| | - Xianyong Lan
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Hong Chen
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Nobuya Sasaki
- Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Aomori, Japan
| | - Jinxi Wang
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Takashi Agui
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
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Bergeron KF, Cardinal T, Touré AM, Béland M, Raiwet DL, Silversides DW, Pilon N. Male-biased aganglionic megacolon in the TashT mouse line due to perturbation of silencer elements in a large gene desert of chromosome 10. PLoS Genet 2015; 11:e1005093. [PMID: 25786024 PMCID: PMC4364714 DOI: 10.1371/journal.pgen.1005093] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2014] [Accepted: 02/23/2015] [Indexed: 01/13/2023] Open
Abstract
Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line—named TashT—that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung’s disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a “tipping point” of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation—Gdnf/Ret and Edn3/Ednrb—and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung’s disease but also provides important new insights into its male sex bias. Hirschsprung’s disease (also known as aganglionic megacolon) is a severe congenital defect of the enteric nervous system (ENS) resulting in complete failure to pass stools. It is characterized by the absence of neural ganglia (aganglionosis) in the distal gut due to incomplete colonization of the embryonic intestines by neural crest cells (NCC), the ENS precursors. Hirschsprung’s disease has an incidence of 1 in 5000 newborns and a 4:1 male sex bias. Although many genes have been associated with this complex genetic disease, most of its heritability as well as its male sex bias remain unexplained. Here, we describe an insertional mutant mouse line (“TashT”) in which virtually all homozygotes display colonic aganglionosis due to defective migration of enteric NCC, but in which only a subset of homozygotes develops megacolon. Surprisingly, this group is almost exclusively male. The TashT ENS defect stems, at least in part, from the disruption of long-range interactions between evolutionarily conserved elements with silencer activity and Fam162b, resulting in NCC-specific upregulation of this uncharacterized protein coding gene. Global analysis of gene expression further revealed that several hundreds of genes are significantly deregulated in TashT enteric NCC. Interestingly, this dataset includes multiple X-linked candidate genes potentially underlying the male sex bias. Taken together, our data pave the way for a clearer understanding of the intriguing male sex bias of Hirschsprung’s disease.
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Affiliation(s)
- Karl-F. Bergeron
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada
| | - Tatiana Cardinal
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada
| | - Aboubacrine M. Touré
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada
| | - Mélanie Béland
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada
| | - Diana L. Raiwet
- Veterinary Genetics Laboratory, Faculty of Veterinary Medicine, University of Montreal, Quebec, Canada
| | - David W. Silversides
- Veterinary Genetics Laboratory, Faculty of Veterinary Medicine, University of Montreal, Quebec, Canada
| | - Nicolas Pilon
- Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed Research Center, University of Quebec at Montreal (UQAM), Quebec, Canada
- * E-mail:
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Kabouridis PS, Pachnis V. Emerging roles of gut microbiota and the immune system in the development of the enteric nervous system. J Clin Invest 2015; 125:956-64. [PMID: 25729852 DOI: 10.1172/jci76308] [Citation(s) in RCA: 82] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The enteric nervous system (ENS) consists of neurons and glial cells that differentiate from neural crest progenitors. During embryogenesis, development of the ENS is controlled by the interplay of neural crest cell-intrinsic factors and instructive cues from the surrounding gut mesenchyme. However, postnatal ENS development occurs in a different context, which is characterized by the presence of microbiota and an extensive immune system, suggesting an important role of these factors on enteric neural circuit formation and function. Initial reports confirm this idea while further studies in this area promise new insights into ENS physiology and pathophysiology.
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Moore SW, Zaahl M. Clinical and genetic correlations of familial Hirschsprung's disease. J Pediatr Surg 2015; 50:285-8. [PMID: 25638620 DOI: 10.1016/j.jpedsurg.2014.11.016] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Accepted: 11/02/2014] [Indexed: 02/04/2023]
Abstract
BACKGROUND The risk of familial transmission in Hirschsprung's disease (HSCR) currently lacks correlation between the clinical phenotype and the underlying genetic factors. The aim of this study was to clinically evaluate familial HSCR transmission and to correlate with the genetic background. METHODS Clinical and gene analysis of familial HSCR patients were explored. DNA from 45 patients (35 kindreds) was screened for genetic variations of the RET, and EDNRB genes were screened for genetic variation by semi-automated bi-directional sequencing analysis and matched to controls. MAIN RESULTS Male:female ratio (3:1) had a female proband in 4 families. Aganglionosis was significantly more frequent with total colonic aganglionosis (TCA) in 40% familial cases (viz: 17/43 (43%) vs. 19/342 non-familial patients (5.6%) (p<0.01)). Transmission of S-HSCR was observed in 13 (31%), which was associated with EDNRB variation. RET gene promoter variation correlated with extended aganglionosis in 6/35 kindreds (17%). In 3 kindreds, both significant EDNRB and RET mutations were identified and where present were associated with increased penetrance in succeeding generations. An increased penetrance with succeeding generations occurred in 6 (14%). In a further 3 generation family, extensive variations in exon 6, 13, and 18 affected 3 males with progressive penetration and aganglionic length, including total intestinal aganglionosis in the further offspring. RET and MEN association was noted in 5 kindreds (14.3%) related to RET variations at Cysteine sites. CONCLUSIONS Cumulative effects of the RET and EDNRB genes contribute to long-segment and total colonic aganglionosis.
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Affiliation(s)
- Sam W Moore
- Division of Paediatric Surgery, University of Stellenbosch, Tygerberg, Western Cape, South Africa.
| | - Monique Zaahl
- Division of Paediatric Surgery, University of Stellenbosch, Tygerberg, Western Cape, South Africa
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Liu H, Luo Y, Li S, Wang S, Wang N, Jin X. Expression profiles of HA117 and its neighboring gene DPF3 in different colon segments of Hirschsprung's disease. INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY 2014; 7:3966-3974. [PMID: 25120773 PMCID: PMC4129008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Accepted: 06/12/2014] [Indexed: 06/03/2023]
Abstract
Hirschsprung's disease (HSCR) is characterized by the absence of enteric ganglion cells along variable regions of the colon. Established theory demonstrates that HSCR is the consequence caused by the abnormal arrest of the migration and differentiation of neural crest-derived stem cells (NCSCs). And retinoid signaling was considered to be involved. We speculated that, HA117, a retinoid-related transcript of a long noncoding RNA (LncRNA), may be involved in the genesis of HSCR. In current research, colon specimens were collected from 25 HSCR patients and grouped into 3 segments: proximal anastomosis, dilated segment and stenotic segment. Real-Time PCR was used to analyze the expression profiles of HA117 and its neighboring gene DPF3 in different colon segments. Fluorescence in situ hybridization (FISH) was employed to detect the distribution of HA117 in the gut wall. Immunohistochemistry was performed to analyze the protein expression of DPF3 in different colon segments. HA117 expression in stenotic segment was higher compared to proximal anastomosis and dilated segment (p < 0.05). Whereas DPF3b mRNA was lower in stenotic segment than that in two other segments (p < 0.05). FISH detected HA117 was distributed in mucosa and muscle layer, mainly present in stenotic segment. Immunohistochemical staining showed that intensive DPF3 staining occurred in proximal anastomosis and the positive staining was hardly observed in stenotic segment. The results suggested that HA117 may be a factor exerting an anti-differentiation or or anti-maturation role in the genesis of HSCR. This gave us a novel cue for better understanding the etiology of HSCR.
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Affiliation(s)
- Hang Liu
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
| | - Yuanyuan Luo
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
| | - Shuangshuang Li
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
| | - Shiqi Wang
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
| | - Ning Wang
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
| | - Xianqing Jin
- Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqing, China
- Department of Pediatric Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, China
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Chakravarti A. 2013 William Allan Award: My multifactorial journey. Am J Hum Genet 2014; 94:326-33. [PMID: 24607382 PMCID: PMC3951947 DOI: 10.1016/j.ajhg.2013.11.014] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2013] [Accepted: 11/18/2013] [Indexed: 11/23/2022] Open
Affiliation(s)
- Aravinda Chakravarti
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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Gisser JM, Cohen AR, Yin H, Gariepy CE. A novel bidirectional interaction between endothelin-3 and retinoic acid in rat enteric nervous system precursors. PLoS One 2013; 8:e74311. [PMID: 24040226 PMCID: PMC3767828 DOI: 10.1371/journal.pone.0074311] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2012] [Accepted: 08/02/2013] [Indexed: 01/31/2023] Open
Abstract
BACKGROUND Signaling through the endothelin receptor B (EDNRB) is critical for the development of the enteric nervous system (ENS) and mutations in endothelin system genes cause Hirschsprung's aganglionosis in humans. Penetrance of the disease is modulated by other genetic factors. Mutations affecting retinoic acid (RA) signaling also produce aganglionosis in mice. Thus, we hypothesized that RA and endothelin signaling pathways may interact in controlling development of the ENS. METHODS Rat immunoselected ENS precursor cells were cultured with the EDNRB ligand endothelin-3, an EDNRB-selective antagonist (BQ-788), and/or RA for 3 or 14 days. mRNA levels of genes related to ENS development, RA- and EDNRB-signaling were measured at 3 days. Proliferating cells and cells expressing neuronal, glial, and myofibroblast markers were quantified. RESULTS Culture of isolated ENS precursors for 3 days with RA decreases expression of the endothelin-3 gene and that of its activation enzyme. These changes are associated with glial proliferation, a higher percentage of glia, and a lower percentage of neurons compared to cultures without RA. These changes are independent of EDNRB signaling. Conversely, EDNRB activation in these cultures decreases expression of RA receptors β and γ mRNA and affects the expression of the RA synthetic and degradative enzymes. These gene expression changes are associated with reduced glial proliferation and a lower percentage of glia in the culture. Over 14 days in the absence of EDNRB signaling, RA induces the formation of a heterocellular plexus replete with ganglia, glia and myofibroblasts. CONCLUSIONS A complex endothelin-RA interaction exists that coordinately regulates the development of rat ENS precursors in vitro. These results suggest that environmental RA may modulate the expression of aganglionosis in individuals with endothelin mutations.
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Affiliation(s)
- Jonathan M. Gisser
- The Center for Molecular and Human Genetics, the Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, United States of America
- Department of Pediatrics, the Ohio State University, Columbus, Ohio, United States of America
| | - Ariella R. Cohen
- The Center for Molecular and Human Genetics, the Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, United States of America
| | - Han Yin
- The Biostatistics Shared Resources, Nationwide Children’s Hospital, Columbus, Ohio, United States of America
| | - Cheryl E. Gariepy
- The Center for Molecular and Human Genetics, the Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, United States of America
- Department of Pediatrics, the Ohio State University, Columbus, Ohio, United States of America
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Lake JI, Heuckeroth RO. Enteric nervous system development: migration, differentiation, and disease. Am J Physiol Gastrointest Liver Physiol 2013; 305:G1-24. [PMID: 23639815 PMCID: PMC3725693 DOI: 10.1152/ajpgi.00452.2012] [Citation(s) in RCA: 254] [Impact Index Per Article: 21.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The enteric nervous system (ENS) provides the intrinsic innervation of the bowel and is the most neurochemically diverse branch of the peripheral nervous system, consisting of two layers of ganglia and fibers encircling the gastrointestinal tract. The ENS is vital for life and is capable of autonomous regulation of motility and secretion. Developmental studies in model organisms and genetic studies of the most common congenital disease of the ENS, Hirschsprung disease, have provided a detailed understanding of ENS development. The ENS originates in the neural crest, mostly from the vagal levels of the neuraxis, which invades, proliferates, and migrates within the intestinal wall until the entire bowel is colonized with enteric neural crest-derived cells (ENCDCs). After initial migration, the ENS develops further by responding to guidance factors and morphogens that pattern the bowel concentrically, differentiating into glia and neuronal subtypes and wiring together to form a functional nervous system. Molecules controlling this process, including glial cell line-derived neurotrophic factor and its receptor RET, endothelin (ET)-3 and its receptor endothelin receptor type B, and transcription factors such as SOX10 and PHOX2B, are required for ENS development in humans. Important areas of active investigation include mechanisms that guide ENCDC migration, the role and signals downstream of endothelin receptor type B, and control of differentiation, neurochemical coding, and axonal targeting. Recent work also focuses on disease treatment by exploring the natural role of ENS stem cells and investigating potential therapeutic uses. Disease prevention may also be possible by modifying the fetal microenvironment to reduce the penetrance of Hirschsprung disease-causing mutations.
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Affiliation(s)
- Jonathan I. Lake
- 1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; and
| | - Robert O. Heuckeroth
- 1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; and ,2Department of Developmental, Regenerative, and Stem Cell Biology, Washington University School of Medicine, St. Louis, Missouri
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Cui L, Wong EHM, Cheng G, Firmato de Almeida M, So MT, Sham PC, Cherny SS, Tam PKH, Garcia-Barceló MM. Genetic Analyses of a Three Generation Family Segregating Hirschsprung Disease and Iris Heterochromia. PLoS One 2013; 8:e66631. [PMID: 23840513 PMCID: PMC3694150 DOI: 10.1371/journal.pone.0066631] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2013] [Accepted: 05/08/2013] [Indexed: 12/15/2022] Open
Abstract
We present the genetic analyses conducted on a three-generation family (14 individuals) with three members affected with isolated-Hirschsprung disease (HSCR) and one with HSCR and heterochromia iridum (syndromic-HSCR), a phenotype reminiscent of Waardenburg-Shah syndrome (WS4). WS4 is characterized by pigmentary abnormalities of the skin, eyes and/or hair, sensorineural deafness and HSCR. None of the members had sensorineural deafness. The family was screened for copy number variations (CNVs) using Illumina-HumanOmni2.5-Beadchip and for coding sequence mutations in WS4 genes (EDN3, EDNRB, or SOX10) and in the main HSCR gene (RET). Confocal microscopy and immunoblotting were used to assess the functional impact of the mutations. A heterozygous A/G transition in EDNRB was identified in 4 affected and 3 unaffected individuals. While in EDNRB isoforms 1 and 2 (cellular receptor) the transition results in the abolishment of translation initiation (M1V), in isoform 3 (only in the cytosol) the replacement occurs at Met91 (M91V) and is predicted benign. Another heterozygous transition (c.-248G/A; -predicted to affect translation efficiency-) in the 5'-untranslated region of EDN3 (EDNRB ligand) was detected in all affected individuals but not in healthy carriers of the EDNRB mutation. Also, a de novo CNVs encompassing DACH1 was identified in the patient with heterochromia iridum and HSCR Since the EDNRB and EDN3 variants only coexist in affected individuals, HSCR could be due to the joint effect of mutations in genes of the same pathway. Iris heterochromia could be due to an independent genetic event and would account for the additional phenotype within the family.
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Affiliation(s)
- Long Cui
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Emily Hoi-Man Wong
- Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Guo Cheng
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | | | - Man-Ting So
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Pak-Chung Sham
- Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Center for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Centre for Reproduction, Development, and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong SAR, China
| | - Stacey S. Cherny
- Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Center for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong SAR, China
| | - Paul Kwong-Hang Tam
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Centre for Reproduction, Development, and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Maria-Mercè Garcia-Barceló
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Center for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Centre for Reproduction, Development, and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- * E-mail:
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Alves MM, Sribudiani Y, Brouwer RWW, Amiel J, Antiñolo G, Borrego S, Ceccherini I, Chakravarti A, Fernández RM, Garcia-Barcelo MM, Griseri P, Lyonnet S, Tam PK, van Ijcken WFJ, Eggen BJL, te Meerman GJ, Hofstra RMW. Contribution of rare and common variants determine complex diseases-Hirschsprung disease as a model. Dev Biol 2013; 382:320-9. [PMID: 23707863 DOI: 10.1016/j.ydbio.2013.05.019] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2012] [Revised: 05/13/2013] [Accepted: 05/15/2013] [Indexed: 12/22/2022]
Abstract
Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.
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Affiliation(s)
- Maria M Alves
- Department of Clinical Genetics, Dr. Molewaterplein, 50, Rotterdam, The Netherlands.
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Watanabe Y, Broders-Bondon F, Baral V, Paul-Gilloteaux P, Pingault V, Dufour S, Bondurand N. Sox10 and Itgb1 interaction in enteric neural crest cell migration. Dev Biol 2013; 379:92-106. [PMID: 23608456 DOI: 10.1016/j.ydbio.2013.04.013] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2013] [Revised: 04/10/2013] [Accepted: 04/12/2013] [Indexed: 01/11/2023]
Abstract
SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with β1-integrins. To this end, we crossed the Sox10(lacZ/+) mice with the conditional Ht-PA::Cre; beta1(neo/+) and beta1(fl/fl) mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10(lacZ/+); Ht-PA::Cre; beta1(neo/fl) double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of β1-integrins upon SOX10 overexpression or in Sox10(lacZ/+) mice was also analyzed. The modulation of SOX10 expression altered β1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.
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Affiliation(s)
- Yuli Watanabe
- INSERM U955, Equipe 11, F-94000 Créteil, France; Université Paris-Est, UMR_S955, UPEC, F-94000 Créteil, France
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Sasselli V, Boesmans W, Vanden Berghe P, Tissir F, Goffinet AM, Pachnis V. Planar cell polarity genes control the connectivity of enteric neurons. J Clin Invest 2013; 123:1763-72. [PMID: 23478408 DOI: 10.1172/jci66759] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2012] [Accepted: 01/14/2013] [Indexed: 12/31/2022] Open
Abstract
A highly complex network of intrinsic enteric neurons is required for the digestive and homeostatic functions of the gut. Nevertheless, the genetic and molecular mechanisms that regulate their assembly into functional neuronal circuits are currently unknown. Here we report that the planar cell polarity (PCP) genes Celsr3 and Fzd3 are required during murine embryogenesis to specifically control the guidance and growth of enteric neuronal projections relative to the longitudinal and radial gut axes. Ablation of these genes disrupts the normal organization of nascent neuronal projections, leading to subtle changes of axonal tract configuration in the mature enteric nervous system (ENS), but profound abnormalities in gastrointestinal motility. Our data argue that PCP-dependent modules of connectivity established at early stages of enteric neurogenesis control gastrointestinal function in adult animals and provide the first evidence that developmental deficits in ENS wiring may contribute to the pathogenesis of idiopathic bowel disorders.
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Affiliation(s)
- Valentina Sasselli
- Division of Molecular Neurobiology, MRC National Institute for Medical Research, London, United Kingdom
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44
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Bergeron KF, Silversides DW, Pilon N. The developmental genetics of Hirschsprung's disease. Clin Genet 2012; 83:15-22. [PMID: 23043324 DOI: 10.1111/cge.12032] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2012] [Revised: 10/03/2012] [Accepted: 10/03/2012] [Indexed: 12/31/2022]
Abstract
Hirschsprung's disease (HSCR), also known as aganglionic megacolon, derives from a congenital malformation of the enteric nervous system (ENS). It displays an incidence of 1 in 5000 live births with a 4:1 male to female sex ratio. Clinical signs include severe constipation and distended bowel due to a non-motile colon. If left untreated, aganglionic megacolon is lethal. This severe congenital condition is caused by the absence of colonic neural ganglia and thus lack of intrinsic innervation of the colon due in turn to improper colonization of the developing intestines by ENS progenitor cells. These progenitor cells are derived from a transient stem cell population called neural crest cells (NCC). The genetics of HSCR is complex and can involve mutations in multiple genes. However, it is estimated that mutations in known genes account for less than half of the cases of HSCR observed clinically. The male sex bias is currently unexplained. The objective of this review is to provide an overview of the pathophysiology and genetics of HSCR, within the context of our current knowledge of NCC development, sex chromosome genetics and laboratory models.
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Affiliation(s)
- K-F Bergeron
- Molecular Genetics of Development Laboratory, Department of Biological Sciences, University of Quebec at Montreal, Canada
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Evangelisti C, Bianco F, Pradella LM, Puliti A, Goldoni A, Sbrana I, Rossi M, Vargiolu M, Seri M, Romeo G, Stanghellini V, de Giorgio R, Bonora E. Apolipoprotein B is a new target of the GDNF/RET and ET-3/EDNRB signalling pathways. Neurogastroenterol Motil 2012; 24:e497-508. [PMID: 22897442 DOI: 10.1111/j.1365-2982.2012.01998.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
BACKGROUND GDNF/RET and Endothelin-3 (ET-3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest-derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1. METHODS Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest-derived cell development, along with a mouse knock-in for the Hirschsprung-associated mutation Ret(C620R). Silencing for Apob and Ret has been performed via shRNA. KEY RESULTS GDNF/RET and ET-3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the Ret(C620R) mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53-dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression. CONCLUSIONS & INFERENCES Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.
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Affiliation(s)
- C Evangelisti
- Medical Genetics Unit, St.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
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46
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Hitch MC, Leinicke JA, Wakeman D, Guo J, Erwin CR, Rowland KJ, Merrick EC, Heuckeroth RO, Warner BW. Ret heterozygous mice have enhanced intestinal adaptation after massive small bowel resection. Am J Physiol Gastrointest Liver Physiol 2012; 302:G1143-50. [PMID: 22421622 PMCID: PMC3362098 DOI: 10.1152/ajpgi.00296.2011] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2011] [Accepted: 03/12/2012] [Indexed: 01/31/2023]
Abstract
Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 ± 46.0 μm vs. WT 306.5 ± 7.7 μm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 μm vs. WT 82.4 ± 3.1 μm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P = 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon-like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P = 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome.
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Affiliation(s)
- Meredith C Hitch
- Department of Pediatrics, Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Washington University School of Medicine, St. Louis Children’s Hospital, St. Louis, MO 63110, USA
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47
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McKeown SJ, Stamp L, Hao MM, Young HM. Hirschsprung disease: a developmental disorder of the enteric nervous system. WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY 2012; 2:113-29. [PMID: 23799632 DOI: 10.1002/wdev.57] [Citation(s) in RCA: 106] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Hirschsprung disease (HSCR), which is also called congenital megacolon or intestinal aganglionosis, is characterized by an absence of enteric (intrinsic) neurons from variable lengths of the most distal bowel. Because enteric neurons are essential for propulsive intestinal motility, infants with HSCR suffer from severe constipation and have a distended abdomen. Currently the only treatment is surgical removal of the affected bowel. HSCR has an incidence of around 1:5,000 live births, with a 4:1 male:female gender bias. Most enteric neurons arise from neural crest cells that emigrate from the caudal hindbrain and then migrate caudally along the entire gut. The absence of enteric neurons from variable lengths of the bowel in HSCR results from a failure of neural crest-derived cells to colonize the affected gut regions. HSCR is therefore regarded as a neurocristopathy. HSCR is a multigenic disorder and has become a paradigm for understanding complex factorial disorders. The major HSCR susceptibility gene is RET. The penetrance of several mutations in HSCR susceptibility genes is sex-dependent. HSCR can occur as an isolated disorder or as part of syndromes; for example, Type IV Waardenburg syndrome is characterized by deafness and pigmentation defects as well as intestinal aganglionosis. Studies using animal models have shown that HSCR genes regulate multiple processes including survival, proliferation, differentiation, and migration. Research into HSCR and the development of enteric neurons is an excellent example of the cross fertilization of ideas that can occur between human molecular geneticists and researchers using animal models. WIREs Dev Biol 2013, 2:113-129. doi: 10.1002/wdev.57 For further resources related to this article, please visit the WIREs website.
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Affiliation(s)
- Sonja J McKeown
- Department of Anatomy & Cell Biology, University of Melbourne, Melbourne 3010, VIC, Australia
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Chalazonitis A, Gershon MD, Greene LA. Cell death and the developing enteric nervous system. Neurochem Int 2012; 61:839-47. [PMID: 22342822 DOI: 10.1016/j.neuint.2012.01.028] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2012] [Revised: 01/25/2012] [Accepted: 01/27/2012] [Indexed: 12/16/2022]
Abstract
This review discusses current knowledge about cell death in the developing enteric nervous system (ENS). It also includes findings about the molecular mechanisms by which such death is mediated. Additional consideration is given to trophic factors that contribute to survival of the precursors and neurons and glia of the ENS, as well to genes that, when mutated or deleted, trigger their death. Although further confirmation is needed, present observations support the view that enteric neural crest-derived precursor cells en route to the gut undergo substantial levels of apoptotic death, but that once these cells colonize the gut, there is relatively little death of precursor cells or of neurons and glia during the fetal period. There are also indications that normal neuron loss occurs in the ENS, but at times beyond the perinatal stage. Taken together, these findings suggest that ENS development is similar is some ways, but different in others from extra-enteric areas of the vertebrate central and peripheral nervous systems, in which large-scale apoptotic death of precursor neurons and glia occurs during the fetal and perinatal periods. Potential reasons for these differences are discussed such as a fetal enteric microenvironment that is especially rich in trophic support. In addition to the cell death that occurs during normal ENS development, this review discusses mechanisms of experimentally-induced ENS cell death, such as those that are associated with defective glial cell-line derived neurotrophic factor/Ret signaling, which are an animal model of human congenital megacolon (aganglionosis; Hirschsprung's disease). Such considerations underscore the importance of understanding cell death in the developing ENS, not just from a curiosity-driven point of view, but also because the pathophysiology behind many disorders of human gastrointestinal function may originate in abnormalities of the mechanisms that govern cell survival and death during ENS development.
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Affiliation(s)
- Alcmène Chalazonitis
- Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.
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49
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Wallace AS, Anderson RB. Genetic interactions and modifier genes in Hirschsprung's disease. World J Gastroenterol 2011; 17:4937-44. [PMID: 22174542 PMCID: PMC3236992 DOI: 10.3748/wjg.v17.i45.4937] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2011] [Revised: 06/09/2011] [Accepted: 06/16/2011] [Indexed: 02/06/2023] Open
Abstract
Hirschsprung’s disease is a congenital disorder that occurs in 1:5000 live births. It is characterised by an absence of enteric neurons along a variable region of the gastrointestinal tract. Hirschsprung’s disease is classified as a multigenic disorder, because the same phenotype is associated with mutations in multiple distinct genes. Furthermore, the genetics of Hirschsprung’s disease are highly complex and not strictly Mendelian. The phenotypic variability and incomplete penetrance observed in Hirschsprung’s disease also suggests the involvement of modifier genes. Here, we summarise the current knowledge of the genetics underlying Hirschsprung’s disease based on human and animal studies, focusing on the principal causative genes, their interactions, and the role of modifier genes.
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50
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Dang R, Torigoe D, Sasaki N, Agui T. QTL analysis identifies a modifier locus of aganglionosis in the rat model of Hirschsprung disease carrying Ednrb(sl) mutations. PLoS One 2011; 6:e27902. [PMID: 22132166 PMCID: PMC3222640 DOI: 10.1371/journal.pone.0027902] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2011] [Accepted: 10/27/2011] [Indexed: 01/31/2023] Open
Abstract
Hirschsprung disease (HSCR) exhibits complex genetics with incomplete penetrance and variable severity thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. As reported previously, when the same null mutation of the Ednrb gene, Ednrbsl, was introgressed into the F344 strain, almost 60% of F344-Ednrbsl/sl pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile. These findings strongly suggested that the severity of HSCR was affected by strain-specific genetic factor (s). In this study, the genetic basis of such large strain differences in the severity of aganglionosis in the rat model was studied by whole-genome scanning for quantitative trait loci (QTLs) using an intercross of (AGH-Ednrbsl×F344-Ednrbsl) F1 with the varying severity of aganglionosis. Genome linkage analysis identified one significant QTL on chromosome 2 for the severity of aganglionosis. Our QTL analyses using rat models of HSCR revealed that multiple genetic factors regulated the severity of aganglionosis. Moreover, a known HSCR susceptibility gene, Gdnf, was found in QTL that suggested a novel non-coding sequence mutation in GDNF that modifies the penetrance and severity of the aganglionosis phenotype in EDNRB-deficient rats. A further identification and analysis of responsible genes located on the identified QTL could lead to the richer understanding of the genetic basis of HSCR development.
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Affiliation(s)
- Ruihua Dang
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Daisuke Torigoe
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Nobuya Sasaki
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Takashi Agui
- Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
- * E-mail:
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