Human Genetics

, Volume 133, Issue 8, pp 997–1009 | Cite as

CHD7, the gene mutated in CHARGE syndrome, regulates genes involved in neural crest cell guidance

  • Yvonne Schulz
  • Peter Wehner
  • Lennart Opitz
  • Gabriela Salinas-Riester
  • Ernie M. H. F. Bongers
  • Conny M. A. van Ravenswaaij-Arts
  • Josephine Wincent
  • Jacqueline Schoumans
  • Jürgen Kohlhase
  • Annette Borchers
  • Silke Pauli
Original Investigation


Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7Whi/+ and Chd7Whi/Whi) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7Whi/Whi embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.

Supplementary material

439_2014_1444_MOESM1_ESM.pptx (791 kb)
Supplementary material 1 (PPTX 790 kb) Figure S1. Chd7 loss of function results in a downregulation of Sema3a expression in Xenopus laevis. Embryos were injected in one blastomere at the two-cell stage with different Morpholino Oligonucleotides (MO) in combination with lacZ RNA as a lineage tracer. Sema3a expression was detected by whole mount in situ hybridization at neurula stages (stage 20-22). (A, B) Embryos injected with 20 ng of a Control Morpholino (Co MO) show Sema3a expression in the midbrain–hindbrain boundary (mh) (A) as well as in the somites (s) (B). (C–F) Different concentrations of a Chd7 MO were injected. (C, D) Embryos injected with 10 ng Chd7 MO show a reduction in Sema3a expression at the midbrain–hindbrain boundary and in the somites. (E, F) Injection of 20 ng Chd7 MO strongly reduced Sema3a expression. (G) The graph summarizes three independent experiments. Numbers indicate the number of injected embryos. Reduction of Sema3a expression or patterning defects are indicated as mild defects, whereas loss of Sema3a expression is scored as severe defect. Standard error of the means is shown
439_2014_1444_MOESM2_ESM.pptx (3.2 mb)
Supplementary material 2 (PPTX 3229 kb) Figure S2. Chd7 loss of function inhibits Sema3a expression at tailbud stages. Embryos were injected with 10 ng or 20 ng MO in combination with lacZ RNA as a lineage tracer in one blastomere at the 2-cell stage. At tailbud stage 27, the Sema3a expression was analyzed by whole mount in situ hybridization. (A-C) Injected side is shown on the right. (A) Embryo injected with 20 ng control MO (Co MO) shows normal Sema3a expression in the splanchnic mesoderm (sm), the somites (s) and the midbrain–hindbrain boundary (mh). (B) Embryo injected with 10 ng Chd7 MO showing a reduction in Sema3a expression on the injected site. (C) Embryos injected with 20 ng Chd7 MO exhibiting a severe reduction in Sema3a expression. (D) Graph summarizing the percentage of mild and severe defects in Sema3a expression of one representative experiment. Numbers indicate the number of injected embryos
439_2014_1444_MOESM3_ESM.pptx (513 kb)
Supplementary material 3 (PPTX 513 kb) Figure S3. The Chd7 MO phenotype can be rescued by injection of human CHD7 RNA, which is not recognized by the Chd7 MO. Embryos were co-injected with 10 ng MO and 1 ng human CHD7 RNA (hCHD7 RNA) in one blastomere at the 2-cell stage and Sema3a expression was analyzed at neurula stages (20-22). One representative rescue experiment is shown. (A,C,E,G) Anterior view of the embryos. (B,D,F,H) Dorsal view of the embryos, posterior is up. (A-H) shows embryos injected as indicated; controls are uninjected embryos. (I) Graph summarizing the percentage of mild and severe defects in Sema3a expression of one representative rescue experiment. Numbers indicate the number of injected embryos
439_2014_1444_MOESM4_ESM.pptx (261 kb)
Supplementary material 4 (PPTX 261 kb) Figure S4. Repetition of the experiment shown in Figure S3 using 1.5 ng hCHD7 RNA


  1. Aramaki M, Udaka T, Kosaki R, Makita Y, Okamoto N, Yoshihashi H, Oki H, Nanao K, Moriyama N, Oku S, Hasegawa T, Takahashi T, Fukushima Y, Kawame H, Kosaki K (2006) Phenotypic spectrum of CHARGE syndrome with CHD7 mutations. J Pediatr 148:410–414. doi:10.1016/j.jpeds.2005.10.044 PubMedCrossRefGoogle Scholar
  2. Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Y, Helms J, Chang CP, Zhao Y, Swigut T, Wysocka J (2010) CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463:958–962. doi:10.1038/nature08733 PubMedCentralPubMedCrossRefGoogle Scholar
  3. Bergman JE, Janssen N, Hoefsloot LH, Jongmans MC, Hofstra RM, van Ravenswaaij-Arts CM (2011) CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype. J Med Genet 48:334–342. doi:10.1136/jmg.2010.087106 PubMedCrossRefGoogle Scholar
  4. Borchers A, David R, Wedlich D (2001) Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification. Development 128:3049–3060PubMedGoogle Scholar
  5. Bosman EA, Penn AC, Ambrose JC, Kettleborough R, Stemple DL, Steel KP (2005) Multiple mutations in mouse Chd7 provide models for CHARGE syndrome. Hum Mol Genet 14:3463–3476. doi:10.1093/hmg/ddi375 PubMedCrossRefGoogle Scholar
  6. Chalouhi C, Faulcon P, Le Bihan C, Hertz-Pannier L, Bonfils P, Abadie V (2005) Olfactory evaluation in children: application to the CHARGE syndrome. Pediatrics 116:e81–e88. doi:10.1542/peds.2004-1970 PubMedCrossRefGoogle Scholar
  7. da Huang W, Sherman BT, Lempicki RA (2009a) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13. doi:10.1093/nar/gkn923 PubMedCentralCrossRefGoogle Scholar
  8. da Huang W, Sherman BT, Lempicki RA (2009b) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. doi:10.1038/nprot.2008.211 CrossRefGoogle Scholar
  9. Dode C, Hardelin JP (2009) Kallmann syndrome. Eur J Hum Genet 17:139–146. doi:10.1038/ejhg.2008.206 PubMedCentralPubMedCrossRefGoogle Scholar
  10. Gammill LS, Bronner-Fraser M (2003) Neural crest specification: migrating into genomics. Nat Rev Neurosci 4:795–805. doi:10.1038/nrn1219 PubMedCrossRefGoogle Scholar
  11. Hall BK (2000) The neural crest as a fourth germ layer and vertebrates as quadroblastic not triploblastic. Evol Dev 2:3–5PubMedCrossRefGoogle Scholar
  12. Hanchate NK, Giacobini P, Lhuillier P, Parkash J, Espy C, Fouveaut C, Leroy C, Baron S, Campagne C, Vanacker C, Collier F, Cruaud C, Meyer V, Garcia-Pinero A, Dewailly D, Cortet-Rudelli C, Gersak K, Metz C, Chabrier G, Pugeat M, Young J, Hardelin JP, Prevot V, Dode C (2012) SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome. PLoS Genet 8:e1002896. doi:10.1371/journal.pgen.1002896 PubMedCentralPubMedCrossRefGoogle Scholar
  13. Harland RM (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36:685–695PubMedCrossRefGoogle Scholar
  14. Hawker K, Fuchs H, Angelis MH, Steel KP (2005) Two new mouse mutants with vestibular defects that map to the highly mutable locus on chromosome 4. Int J Audiol 44:171–177PubMedCrossRefGoogle Scholar
  15. Hrabe de Angelis MH, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, Marschall S, Heffner S, Pargent W, Wuensch K, Jung M, Reis A, Richter T, Alessandrini F, Jakob T, Fuchs E, Kolb H, Kremmer E, Schaeble K, Rollinski B, Roscher A, Peters C, Meitinger T, Strom T, Steckler T, Holsboer F, Klopstock T, Gekeler F, Schindewolf C, Jung T, Avraham K, Behrendt H, Ring J, Zimmer A, Schughart K, Pfeffer K, Wolf E, Balling R (2000) Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 25:444–447. doi:10.1038/78146 PubMedCrossRefGoogle Scholar
  16. Janssen N, Bergman JE, Swertz MA, Tranebjaerg L, Lodahl M, Schoots J, Hofstra RM, van Ravenswaaij-Arts CM, Hoefsloot LH (2012) Mutation update on the CHD7 gene involved in CHARGE syndrome. Hum Mutat 33:1149–1160. doi:10.1002/humu.22086 PubMedCrossRefGoogle Scholar
  17. Jongmans MC, Admiraal RJ, van der Donk KP, Vissers LE, Baas AF, Kapusta L, van Hagen JM, Donnai D, de Ravel TJ, Veltman JA, Geurts van Kessel A, De Vries BB, Brunner HG, Hoefsloot LH, van Ravenswaaij CM (2006) CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene. J Med Genet 43:306–314. doi:10.1136/jmg.2005.036061 PubMedCentralPubMedCrossRefGoogle Scholar
  18. Jongmans MC, van Ravenswaaij-Arts CM, Pitteloud N, Ogata T, Sato N, Claahsen-van der Grinten HL, van der Donk K, Seminara S, Bergman JE, Brunner HG, Crowley WF Jr, Hoefsloot LH (2009) CHD7 mutations in patients initially diagnosed with Kallmann syndrome: the clinical overlap with CHARGE syndrome. Clin Genet 75:65–71. doi:10.1111/j.1399-0004.2008.01107.x PubMedCentralPubMedCrossRefGoogle Scholar
  19. Kim HG, Kurth I, Lan F, Meliciani I, Wenzel W, Eom SH, Kang GB, Rosenberger G, Tekin M, Ozata M, Bick DP, Sherins RJ, Walker SL, Shi Y, Gusella JF, Layman LC (2008) Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome. Am J Hum Genet 83:511–519. doi:10.1016/j.ajhg.2008.09.005 PubMedCentralPubMedCrossRefGoogle Scholar
  20. Kirby ML, Hutson MR (2010) Factors controlling cardiac neural crest cell migration. Cell Adh Migr 4:609–621PubMedCentralPubMedCrossRefGoogle Scholar
  21. Koestner U, Shnitsar I, Linnemannstons K, Hufton AL, Borchers A (2008) Semaphorin and neuropilin expression during early morphogenesis of Xenopus laevis. Dev Dyn 237:3853–3863. doi:10.1002/dvdy.21785 PubMedCrossRefGoogle Scholar
  22. Lalani SR, Safiullah AM, Fernbach SD, Harutyunyan KG, Thaller C, Peterson LE, McPherson JD, Gibbs RA, White LD, Hefner M, Davenport SL, Graham JM, Bacino CA, Glass NL, Towbin JA, Craigen WJ, Neish SR, Lin AE, Belmont JW (2006) Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation. Am J Hum Genet 78:303–314. doi:10.1086/500273 PubMedCentralPubMedCrossRefGoogle Scholar
  23. Morgan D, Bailey M, Phelps P, Bellman S, Grace A, Wyse R (1993) Ear-nose-throat abnormalities in the CHARGE association. Arch Otolaryngol Head Neck Surg 119:49–54PubMedCrossRefGoogle Scholar
  24. Ogata T, Fujiwara I, Ogawa E, Sato N, Udaka T, Kosaki K (2006) Kallmann syndrome phenotype in a female patient with CHARGE syndrome and CHD7 mutation. Endocr J 53:741–743PubMedCrossRefGoogle Scholar
  25. Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beissbarth T, Gaedcke J (2010) Impact of RNA degradation on gene expression profiling. BMC Med Genomics 3:36. doi:10.1186/1755-8794-3-36 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Pinto G, Abadie V, Mesnage R, Blustajn J, Cabrol S, Amiel J, Hertz-Pannier L, Bertrand AM, Lyonnet S, Rappaport R, Netchine I (2005) CHARGE syndrome includes hypogonadotropic hypogonadism and abnormal olfactory bulb development. J Clin Endocrinol Metab 90:5621–5626. doi:10.1210/jc.2004-2474 PubMedCrossRefGoogle Scholar
  27. Randall V, McCue K, Roberts C, Kyriakopoulou V, Beddow S, Barrett AN, Vitelli F, Prescott K, Shaw-Smith C, Devriendt K, Bosman E, Steffes G, Steel KP, Simrick S, Basson MA, Illingworth E, Scambler PJ (2009) Great vessel development requires biallelic expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice. J Clin Invest 119:3301–3310. doi:10.1172/JCI37561 PubMedCentralPubMedGoogle Scholar
  28. Sanlaville D, Verloes A (2007) CHARGE syndrome: an update. Eur J Hum Genet 15:389–399. doi:10.1038/sj.ejhg.5201778 PubMedCrossRefGoogle Scholar
  29. Sanlaville D, Etchevers HC, Gonzales M, Martinovic J, Clement-Ziza M, Delezoide AL, Aubry MC, Pelet A, Chemouny S, Cruaud C, Audollent S, Esculpavit C, Goudefroye G, Ozilou C, Fredouille C, Joye N, Morichon-Delvallez N, Dumez Y, Weissenbach J, Munnich A, Amiel J, Encha-Razavi F, Lyonnet S, Vekemans M, Attie-Bitach T (2006) Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development. J Med Genet 43:211–217. doi:10.1136/jmg.2005.036160 PubMedCentralPubMedCrossRefGoogle Scholar
  30. Schwanzel-Fukuda M, Pfaff DW (1989) Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161–164. doi:10.1038/338161a0 PubMedCrossRefGoogle Scholar
  31. Siebert JR, Graham JM Jr, MacDonald C (1985) Pathologic features of the CHARGE association: support for involvement of the neural crest. Teratology 31:331–336. doi:10.1002/tera.1420310303 PubMedCrossRefGoogle Scholar
  32. Smith WC, Harland RM (1991) Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell 67:753–765PubMedCrossRefGoogle Scholar
  33. Teixeira L, Guimiot F, Dode C, Fallet-Bianco C, Millar RP, Delezoide AL, Hardelin JP (2010) Defective migration of neuroendocrine GnRH cells in human arrhinencephalic conditions. J Clin Invest 120:3668–3672. doi:10.1172/JCI43699 PubMedCentralPubMedCrossRefGoogle Scholar
  34. Van Meter TD, Weaver DD (1996) Oculo-auriculo-vertebral spectrum and the CHARGE association: clinical evidence for a common pathogenetic mechanism. Clin Dysmorphol 5:187–196PubMedGoogle Scholar
  35. Vissers LE, van Ravenswaaij CM, Admiraal R, Hurst JA, de Vries BB, Janssen IM, van der Vliet WA, Huys EH, de Jong PJ, Hamel BC, Schoenmakers EF, Brunner HG, Veltman JA, van Kessel AG (2004) Mutations in a new member of the chromodomain gene family cause CHARGE syndrome. Nat Genet 36:955–957. doi:10.1038/ng1407 PubMedCrossRefGoogle Scholar
  36. Williams MS (2005) Speculations on the pathogenesis of CHARGE syndrome. Am J Med Genet A 133A:318–325. doi:10.1002/ajmg.a.30561 PubMedCrossRefGoogle Scholar
  37. Wincent J, Holmberg E, Stromland K, Soller M, Mirzaei L, Djureinovic T, Robinson K, Anderlid B, Schoumans J (2008) CHD7 mutation spectrum in 28 Swedish patients diagnosed with CHARGE syndrome. Clin Genet 74:31–38. doi:10.1111/j.1399-0004.2008.01014.x PubMedCrossRefGoogle Scholar
  38. Yazdani U, Terman JR (2006) The semaphorins. Genome Biol 7:211. doi:10.1186/gb-2006-7-3-211 PubMedCentralPubMedCrossRefGoogle Scholar
  39. Young J, Metay C, Bouligand J, Tou B, Francou B, Maione L, Tosca L, Sarfati J, Brioude F, Esteva B, Briand-Suleau A, Brisset S, Goossens M, Tachdjian G, Guiochon-Mantel A (2012) SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development. Hum Reprod 27:1460–1465. doi:10.1093/humrep/des022 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yvonne Schulz
    • 1
  • Peter Wehner
    • 2
  • Lennart Opitz
    • 4
  • Gabriela Salinas-Riester
    • 4
  • Ernie M. H. F. Bongers
    • 5
  • Conny M. A. van Ravenswaaij-Arts
    • 6
  • Josephine Wincent
    • 7
  • Jacqueline Schoumans
    • 7
    • 8
  • Jürgen Kohlhase
    • 9
  • Annette Borchers
    • 2
    • 3
  • Silke Pauli
    • 1
  1. 1.Institute of Human GeneticsUniversity Medical Center GöttingenGöttingenGermany
  2. 2.Department of Developmental Biochemistry, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), GZMBUniversity of GöttingenGöttingenGermany
  3. 3.Department of Biology, Molecular EmbryologyPhilipps-University MarburgMarburgGermany
  4. 4.Department of Developmental BiochemistryUniversity Medical Center GöttingenGöttingenGermany
  5. 5.Department of Human GeneticsRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
  6. 6.Department of GeneticsUniversity Medical Centre Groningen, University of GroningenGroningenThe Netherlands
  7. 7.Department of Molecular Medicine and Surgery and Center for Molecular MedicineKarolinska University HospitalStockholmSweden
  8. 8.Cancer Cytogenetic Unit, Department of Medical GeneticsUniversity Hospital of LausanneLausanneSwitzerland
  9. 9.Center for Human GeneticsFreiburgGermany

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