Skip to main content
PMC home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2017 Jan 25;13(3):267–273. doi: 10.1007/s12519-017-0002-0

Identifying key genes associated with Hirschsprung’s disease based on bioinformatics analysis of RNA-sequencing data

Wei-Kang Pan 1, Ya-Fei Zhang 2, Hui Yu 1, Ya Gao 1,✉, Bai-Jun Zheng 1, Peng Li 1, Chong Xie 1, Xin Ge 1
PMCID: PMC7091079  PMID: 28120235

Abstract

Background

Hirschsprung’s disease (HSCR) is a type of megacolon induced by deficiency or dysfunction of ganglion cells in the distal intestine and is associated with developmental disorders of the enteric nervous system. To explore the mechanisms of HSCR, we analyzed the RNA-sequencing data of the expansion and the narrow segments of colon tissues separated from children with HSCR.

Methods

RNA-sequencing of the expansion segments and the narrow segments of colon tissues isolated from children with HSCR was performed. After differentially expressed genes (DEGs) were identified using the edgeR package in R, functional and pathway enrichment analyses of DEGs were carried out using DAVID software. To further screen the key genes, protein-protein interaction (PPI) network and module analyses were conducted separately using Cytoscape software.

Results

A total of 117 DEGs were identified in the expansion segment samples, including 47 up-regulated and 70 down-regulated genes. Functional enrichment analysis suggested that FOS and DUSP1 were implicated in response to endogenous stimulus. In the PPI network analysis, FOS (degree=20), EGR1 (degree=16), ATF3 (degree=9), NOS1 (degree=8), CCL5 (degree=8), DUSP1 (degree=7), CXCL3 (degree=6), VIP (degree=6), FOSB (degree=5), and NOS2 (degree=4) had higher degrees, which could interact with other genes. In addition, two significant modules (module 1 and module 2) were identified from the PPI network.

Conclusions

Several genes (including FOS, EGR1, ATF3, NOS1, CCL5, DUSP1, CXCL3, VIP, FOSB, and NOS2) might be involved in the development of HSCR through their effect on the nervous system.

Keywords: differentially expressed genes, functional and pathway enrichment analysis, Hirschsprung’s disease, protein-protein interaction network

References

  • 1.Puri P, Shinkai T. Pathogenesis of Hirschsprung’s disease and its variants: recent progress. Semin Pediatr Surg. 2004;13:18–24. doi: 10.1053/j.sempedsurg.2003.09.004. [DOI] [PubMed] [Google Scholar]
  • 2.De LF, Boeckxstaens GE, Benninga MA. Symptomatology, pathophysiology, diagnostic work-up, and treatment of Hirschsprung disease in infancy and childhood. Curr Gastroenterol Rep. 2007;9:245–253. doi: 10.1007/s11894-007-0026-z. [DOI] [PubMed] [Google Scholar]
  • 3.Butler Tjaden NE, Trainor PA. The developmental etiology and pathogenesis of Hirschsprung disease. Transl Res. 2013;162:1–15. doi: 10.1016/j.trsl.2013.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Suita S, Taguchi T, Ieiri S, Nakatsuji T. Hirschsprung’s disease in Japan: analysis of 3852 patients based on a nationwide survey in 30 years. J Pediatr Surg. 2005;40:197–202. doi: 10.1016/j.jpedsurg.2004.09.052. [DOI] [PubMed] [Google Scholar]
  • 5.Okamura Y, Saga Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development. 2008;135:3555–3565. doi: 10.1242/dev.022319. [DOI] [PubMed] [Google Scholar]
  • 6.de Pontual L, Pelet A, Trochet D, Jaubert F, Espinosa-Parrilla Y, Munnich A, et al. Mutations of the RET gene in isolated and syndromic Hirschsprung’s disease in human disclose major and modifier alleles at a single locus. J Med Genet. 2006;43:419–423. doi: 10.1136/jmg.2005.040113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Garcia-Barcelo MM, Tang CS, Ngan ES, Lui VC, Chen Y, So MT, et al. Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung’s disease. Proc Natl Acad Sci U S A. 2009;106:2694–2699. doi: 10.1073/pnas.0809630105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Phusantisampan T, Sangkhathat S, Phongdara A, Chiengkriwate P, Patrapinyokul S, Mahasirimongkol S. Association of genetic polymorphisms in the RET-protooncogene and NRG1 with Hirschsprung disease in Thai patients. J Hum Genet. 2012;57:286–293. doi: 10.1038/jhg.2012.18. [DOI] [PubMed] [Google Scholar]
  • 9.Uesaka T, Jain S, Yonemura S, Uchiyama Y, Milbrandt J, Enomoto H. Conditional ablation of GFRa1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung’s disease phenotype. Development. 2007;134:2171–2181. doi: 10.1242/dev.001388. [DOI] [PubMed] [Google Scholar]
  • 10.Fernández RM, Sánchez-Mejías A, Mena M, Ruiz-Ferrer M, López-Alonso M, Antiñolo G, et al. A novel point variant in NTRK3, R645C, suggests a role of this gene in the pathogenesis of Hirschsprung disease. Ann Hum Genet. 2009;73:19–25. doi: 10.1111/j.1469-1809.2008.00479.x. [DOI] [PubMed] [Google Scholar]
  • 11.Ruiz-Ferrer M, Fernandez RM, Antiñolo G, Lopez-Alonso M, Borrego S. NTF-3, a gene involved in the enteric nervous system development, as a candidate gene for Hirschsprung disease. J Pediatr Surg. 2008;43:1308–1311. doi: 10.1016/j.jpedsurg.2008.02.076. [DOI] [PubMed] [Google Scholar]
  • 12.Duan X-L, Zhang X-S, Li G-W. Clinical relationship between EDN-3 gene, EDNRB gene and Hirschsprung’s disease. World J Gastroenterol. 2003;9:2839. doi: 10.3748/wjg.v9.i12.2839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cantrell VA, Owens SE, Chandler RL, Airey DC, Bradley KM, Smith JR, et al. Interactions between Sox10 and EdnrB modulate penetrance and severity of aganglionosis in the Sox10Dom mouse model of Hirschsprung disease. Hum Mol Genet. 2004;13:2289–2301. doi: 10.1093/hmg/ddh243. [DOI] [PubMed] [Google Scholar]
  • 14.Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011;12:87–98. doi: 10.1038/nrg2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Saeed A, Barreto L, Neogii SG, Loos A, Mcfarlane I, Aslam A. Identification of novel genes in Hirschsprung disease pathway using whole genome expression study. J Pediatr Surg. 2012;47:303–307. doi: 10.1016/j.jpedsurg.2011.11.017. [DOI] [PubMed] [Google Scholar]
  • 16.Afsari B, Geman D, Fertig EJ. Cancer Inform. 2014. Learning dysregulated pathways in cancers from differential variability analysis; p. 61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Patel RK, Jain M. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One. 2012;7:e30619. doi: 10.1371/journal.pone.0030619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome biol. 2013;14:R36. doi: 10.1186/gb-2013-14-4-r36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu Y, et al. The UCSC genome browser database. Nucleic Acids Res. 2003;31:51–54. doi: 10.1093/nar/gkg129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–578. doi: 10.1038/nprot.2012.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Smyth GK. Limma: linear models for microarray data. In: Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S, editors. Bioinformatics and computational biology solutions using R and Bioconductor. New York: Springer; 2005. pp. 397–420. [Google Scholar]
  • 22.Consortium GO. The Gene Ontology in 2010: extensions and refinements. Nucleic acids research. 2010;38:D331–D335. doi: 10.1093/nar/gkp1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008;36:D480–D484. doi: 10.1093/nar/gkm882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43:D447–D52. doi: 10.1093/nar/gku1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Saito R, Smoot ME, Ono K, Ruscheinski J, Wang P-L, Lotia S, et al. A travel guide to Cytoscape plugins. Nat Methods. 2012;9:1069–1076. doi: 10.1038/nmeth.2212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bader GD, Hogue CW. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003;4:2. doi: 10.1186/1471-2105-4-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Furness JB. The enteric nervous system: normal functions and enteric neuropathies. Neurogastroenterol Motil. 2008;20:32–38. doi: 10.1111/j.1365-2982.2008.01094.x. [DOI] [PubMed] [Google Scholar]
  • 28.Gershon MD. Developmental determinants of the independence and complexity of the enteric nervous system. Trends Neurosci. 2010;33:446–456. doi: 10.1016/j.tins.2010.06.002. [DOI] [PubMed] [Google Scholar]
  • 29.McKeown SJ, Stamp L, Hao MM, Young HM. Hirschsprung disease: a developmental disorder of the enteric nervous system. Wiley Interdiscip Rev Dev Biol. 2013;2:113–129. doi: 10.1002/wdev.57. [DOI] [PubMed] [Google Scholar]
  • 30.Oshitari T, Dezawa M, Okada S, Takano M, Negishi H, Horie H, et al. The role of c-fos in cell death and regeneration of retinal ganglion cells. Invest Ophthalmol Vis Sci. 2002;43:2442–2449. [PubMed] [Google Scholar]
  • 31.Howe BM, Bruno SB, Higgs KA, Stigers RL, Cunningham JT. FosB expression in the central nervous system following isotonic volume expansion in unanesthetized rats. Exp Neurol. 2004;187:190–198. doi: 10.1016/j.expneurol.2004.01.020. [DOI] [PubMed] [Google Scholar]
  • 32.Horita H, Wada K, Rivas MV, Hara E, Jarvis ED. The dusp1 immediate early gene is regulated by natural stimuli predominantly in sensory input neurons. J Comp Neurol. 2010;518:2873–2901. doi: 10.1002/cne.22370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Glass WG, Hickey MJ, Hardison JL, Liu MT, Manning JE, Lane TE. Antibody targeting of the CC chemokine ligand 5 results in diminished leukocyte infiltration into the central nervous system and reduced neurologic disease in a viral model of multiple sclerosis. J Immunol. 2004;172:4018–4025. doi: 10.4049/jimmunol.172.7.4018. [DOI] [PubMed] [Google Scholar]
  • 34.Glass WG, Liu MT, Kuziel WA, Lane TE. Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus. Virology. 2001;288:8–17. doi: 10.1006/viro.2001.1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Glass WG, Lane TE. Functional expression of chemokine receptor CCR5 on CD4+ T cells during virus-induced central nervous system disease. J Virol. 2003;77:191–198. doi: 10.1128/JVI.77.1.191-198.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Glass WG, Lane TE. Functional analysis of the CC chemokine receptor 5 (CCR5) on virus-specific CD8+ T cells following coronavirus infection of the central nervous system. Virology. 2003;312:407–414. doi: 10.1016/S0042-6822(03)00237-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Farioli-Vecchioli S, Cinà I, Ceccarelli M, Micheli L, Leonardi L, Ciotti MT, et al. Tis 21 knock-out enhances the frequency of medulloblastoma in Patched1 heterozygous mice by inhibiting the Cxcl3-dependent migration of cerebellar neurons. J Neurosci. 2012;32:15547–15564. doi: 10.1523/JNEUROSCI.0412-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, et al. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: A novel neuronal marker of nerve injury. Mol Cell Neurosci. 2000;15:170–182. doi: 10.1006/mcne.1999.0814. [DOI] [PubMed] [Google Scholar]
  • 39.Seijffers R, Mills CD, Woolf CJ. ATF3 increases the intrinsic growth state of DRG neurons to enhance peripheral nerve regeneration. J Neurosci. 2007;27:7911–7920. doi: 10.1523/JNEUROSCI.5313-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hunt D, Raivich G, Anderson PN. Activating transcription factor 3 and the nervous system. Front Mol Neurosci. 2012;5:7. doi: 10.3389/fnmol.2012.00007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Seijffers R, Allchorne AJ, Woolf CJ. The transcription factor ATF-3 promotes neurite outgrowth. Mol Cell Neurosci. 2006;32:143–154. doi: 10.1016/j.mcn.2006.03.005. [DOI] [PubMed] [Google Scholar]
  • 42.Pearson AG, Gray CW, Pearson JF, Greenwood JM, During MJ, Dragunow M. ATF3 enhances c-Jun-mediated neurite sprouting. Mol Brain Res. 2003;120:38–45. doi: 10.1016/j.molbrainres.2003.09.014. [DOI] [PubMed] [Google Scholar]
  • 43.Waldman D, Gardner J, Zfass A, Makhlouf G. Effects of vasoactive intestinal peptide, secretin, and related peptides on rat colonic transport and adenylate cyclase activity. Gastroenterology. 1977;73:518–523. [PubMed] [Google Scholar]
  • 44.Mourad FH, Barada KA, Rached NAB, Khoury CI, Saadé NE, Nassar CF. Inhibitory effect of experimental colitis on fluid absorption in rat jejunum: role of the enteric nervous system, VIP, and nitric oxide. Am J Physiol-Gastrl. 2006;290:G262–G268. doi: 10.1152/ajpcell.00070.2005. [DOI] [PubMed] [Google Scholar]
  • 45.Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res. 1999;43:521–531. doi: 10.1016/S0008-6363(99)00115-7. [DOI] [PubMed] [Google Scholar]
  • 46.Savidge TC. Importance of NO and its related compounds in enteric nervous system regulation of gut homeostasis and disease susceptibility. Curr Opin Pharmacol. 2014;19:54–60. doi: 10.1016/j.coph.2014.07.009. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from World Journal of Pediatrics are provided here courtesy of Nature Publishing Group

RESOURCES