Advertisement

World Journal of Pediatrics

, Volume 13, Issue 3, pp 267–273 | Cite as

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

  • Wei-Kang Pan
  • Ya-Fei Zhang
  • Hui Yu
  • Ya Gao
  • Bai-Jun Zheng
  • Peng Li
  • Chong Xie
  • Xin Ge
Original Article

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 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Puri P, Shinkai T. Pathogenesis of Hirschsprung’s disease and its variants: recent progress. Semin Pediatr Surg 2004;13:18–24.CrossRefPubMedGoogle Scholar
  2. 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.CrossRefGoogle Scholar
  3. 3.
    Butler Tjaden NE, Trainor PA. The developmental etiology and pathogenesis of Hirschsprung disease. Transl Res 2013;162:1–15.CrossRefPubMedGoogle Scholar
  4. 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.CrossRefPubMedGoogle Scholar
  5. 5.
    Okamura Y, Saga Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development 2008;135:3555–3565.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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.CrossRefPubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 14.
    Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 2011;12:87–98.CrossRefPubMedGoogle Scholar
  15. 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.CrossRefPubMedGoogle Scholar
  16. 16.
    Afsari B, Geman D, Fertig EJ. Learning dysregulated pathways in cancers from differential variability analysis. Cancer Inform 2014:61.Google Scholar
  17. 17.
    Patel RK, Jain M. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 2012;7:e30619.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Smyth GK. Limma: linear models for microarray data. In: Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S, eds. Bioinformatics and computational biology solutions using R and Bioconductor. New York: Springer, 2005: 397–420.CrossRefGoogle Scholar
  22. 22.
    Consortium GO. The Gene Ontology in 2010: extensions and refinements. Nucleic acids research 2010;38:D331–D335.CrossRefGoogle Scholar
  23. 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.CrossRefPubMedGoogle Scholar
  24. 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.CrossRefPubMedGoogle Scholar
  25. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Bader GD, Hogue CW. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics 2003;4:2.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Furness JB. The enteric nervous system: normal functions and enteric neuropathies. Neurogastroenterol Motil 2008;20 Suppl 1:32–38.CrossRefPubMedGoogle Scholar
  28. 28.
    Gershon MD. Developmental determinants of the independence and complexity of the enteric nervous system. Trends Neurosci 2010;33:446–456.CrossRefPubMedGoogle Scholar
  29. 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.CrossRefPubMedGoogle Scholar
  30. 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.PubMedGoogle Scholar
  31. 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.CrossRefPubMedGoogle Scholar
  32. 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.PubMedPubMedCentralGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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.CrossRefPubMedGoogle Scholar
  37. 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.CrossRefPubMedGoogle Scholar
  38. 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.CrossRefPubMedGoogle Scholar
  39. 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.CrossRefPubMedGoogle Scholar
  40. 40.
    Hunt D, Raivich G, Anderson PN. Activating transcription factor 3 and the nervous system. Front Mol Neurosci 2012;5:7.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Seijffers R, Allchorne AJ, Woolf CJ. The transcription factor ATF-3 promotes neurite outgrowth. Mol Cell Neurosci 2006;32:143–154.CrossRefPubMedGoogle Scholar
  42. 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.CrossRefPubMedGoogle Scholar
  43. 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.PubMedGoogle Scholar
  44. 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.CrossRefGoogle Scholar
  45. 45.
    Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res 1999;43:521–531.CrossRefPubMedGoogle Scholar
  46. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Children's Hospital, Zhejiang University School of Medicine and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • 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
  1. 1.Department of Pediatric SurgeryThe Second Affiliated Hospital of Xi’an Jiaotong UniversityXi’anChina
  2. 2.Department of EndoscopyShaanxi Nuclear Industry 215 HospitalXianyangChina

Personalised recommendations