Advertisement

Genome-Wide Association Analysis Identifies Dcc as an Essential Factor in the Innervation of the Peripheral Vestibular System in Inbred Mice

  • Pezhman Salehi
  • Anthony Myint
  • Young J. Kim
  • Marshall X. Ge
  • Joel Lavinsky
  • Maria K. Ho
  • Amanda L. Crow
  • Charlene Cruz
  • Maya Monges-Hernadez
  • Juemei Wang
  • Jaana Hartiala
  • Li I. Zhang
  • Hooman Allayee
  • Aldons J. Lusis
  • Takahiro OhyamaEmail author
  • Rick A. FriedmanEmail author
Research Article

Abstract

This study aimed to investigate the genetic causes of vestibular dysfunction. We used vestibular sensory-evoked potentials (VsEPs) to characterize the vestibular function of 35 inbred mouse strains selected from the Hybrid Mouse Diversity Panel and demonstrated strain-dependent phenotypic variation in vestibular function. Using these phenotypic data, we performed the first genome-wide association study controlling for population structure that has revealed two highly suggestive loci, one of which lies within a haplotype block containing five genes (Stard6, 4930503L19Rik, Poli, Mbd2, Dcc) on Chr. 18 (peak SNP rs29632020), one gene, deleted in colorectal carcinoma (Dcc) has a well-established role in nervous system development. An in-depth analysis of Dcc-deficient mice demonstrated elevation in mean VsEP threshold for Dcc +/− mice (−11.86 dB) compared to wild-type (−9.68 dB) littermates. Synaptic ribbon studies revealed Dcc −/− (P0) and Dcc +/− (6-week-old) mice showed lower density of the presynaptic marker (CtBP2) as compared to wild-type controls. Vestibular ganglion cell counts of Dcc −/− (P0) was lower than controls. Whole-mount preparations showed abnormal innervation of the utricle, saccule, and crista ampullaris at E14.5, E16.5, and E18.5. Postnatal studies were limited by the perinatal lethality in Dcc −/− mice. Expression analyses using in situ hybridization and immunohistochemistry showed Dcc expression in the mouse vestibular ganglion (E15.5), and utricle and crista ampullaris (6-week-old), respectively. In summary, we report the first GWAS for vestibular functional variation in inbred mice and provide evidence for the role of Dcc in the normal innervation of the peripheral vestibular system.

Keywords

genome-wide association study vestibular system Hybrid Mouse Diversity Panel deleted in colorectal carcinoma (Dccaxonal migration vestibular sensory evoked potential utricle crista vestibular ganglia 

Notes

Acknowledgments

This research is supported by the National Institutes of Health (NIH) grants R01 DC010856-01 (RAF) and HL28481 (AJL). The authors would like to thank Dr. Neil Segil, Dr. Sherri Jones, Dr. Bernd Fritzsch, and Dr. Radha Kalluri for thoughtful discussion and advice. We would also like to thank Litao Tao, Juan Llamas, Toru Miwa, Mete Civelek, Calvin Pan, Maria Gómez-Casati, Sarath Vijayakumar, Christopher Ventura, Nicole Grepo, and Olav Olsen for technical advice. We would like to thank Roman Wunderlich for providing the Dcc in situ probe and Dr. Marc Tessier-Lavigne for providing the Dcc knock-out mice.

Contribution of Authors

R.A.F., J.L., T.O., and A.J.L. designed and supervised the experiments. P.S performed VsEP for Dcc +/−, CTBP2 count for adult mice, immunostaining of VG, prepared images for in situ hybridization, Dcc expression in adult mice, qPCR, Fig. 8D–F, I–O, organized all figures and tables, and completed manuscript writing. A.M. performed VG cell count, P0 CTBP2 count, validated the variation of the HMDP strains for VsEP, and participated in manuscript writing. Y.J.K. and L.I.Z. designed vestibular system neuron projection pattern at embryonic stage and prepared Fig. 8A–C, G, H. Manhattan and regional plots were prepared by A.M.C., J.H., E.E, and H.A following FaST-LMM. M.K.H. set up the VsEP device and VsEP protocol. J.L. performed and analyzed VsEP for HMDP strains. C.C and T.O. preformed in situ hybridization. M.G. contributed in genotyping and confocal imaging.

References

  1. Abbas L, Whitfield TT (2009) Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva. Development 136:2837–2848CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bennett BJ, Farber CR, Orozco L, Kang HM, Ghazalpour A, Siemers N, Neubauer M, Neuhaus I, Yordanova R, Guan B, et al. (2010) A high-resolution association mapping panel for the dissection of complex traits in mice. Genome Res 20:281–290CrossRefPubMedPubMedCentralGoogle Scholar
  3. Berndt SI, Sampson J, Yeager M, et al. (2011) Large-scale fine mapping of the HNF1B locus and prostate cancer risk. Hum Mol Genet 16:3322–3329CrossRefGoogle Scholar
  4. Bush WS, Moore JH (2012) Chapter 11: Genome-wide association studies. PLoS Comput Biol 8:e1002822CrossRefPubMedPubMedCentralGoogle Scholar
  5. Civelek M, Lusis AJ (2014) Systems genetics approaches to understand complex traits. Nat Rev Genet 15:34–48CrossRefPubMedGoogle Scholar
  6. Davis RC, van Nas A, Bennett B, Orozco L, Pan C, Rau CD, Eskin E, Lusis AJ (2013) Genome-wide association mapping of blood cell traits in mice. Mamm Genome 24:105–118CrossRefPubMedPubMedCentralGoogle Scholar
  7. Delpire E, Lu J, England R, Dull C, Thorne T (1999) Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter. Nat Genet 22:192–195CrossRefPubMedGoogle Scholar
  8. Eppsteiner RW, Smith RJ (2011) Genetic disorders of the vestibular system. Curr Opin Otolaryngol Head Neck Surg 19:397–402CrossRefPubMedPubMedCentralGoogle Scholar
  9. Farber CR, Bennett BJ, Orozco L, Zou W, Lira A, Kostem E, Kang HM, Furlotte N, Berberyan A, Ghazalpour A, et al. (2011) Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet 7:e1002038CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fazeli A, Dickinson SL, Hermiston ML, Tighe RV, Steen RG, Small CG, Stoeckli ET, Keino-Masu K, Masu M, Rayburn H, et al. (1997) Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature 386:796–804CrossRefPubMedGoogle Scholar
  11. Ghazalpour A, Rau CD, Farber CR, Bennett BJ, Orozco LD, van Nas A, Pan C, Allayee H, Beaven SW, Civelek M, et al. (2012) Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits. Mamm Genome 23:680–692CrossRefPubMedPubMedCentralGoogle Scholar
  12. Goodyear RJ, Jones SM, Sharifi L, Forge A, Richardson GP (2012) Hair bundle defects and loss of function in the vestibular end organs of mice lacking the receptor-like inositol lipid phosphatase PTPRQ. J Neurosci 32:2762–2772CrossRefPubMedPubMedCentralGoogle Scholar
  13. Ghoussaini M, Fletcher O, Michailidou K, et al. (2012) Genome-wide association analysis identifies three new breast cancer susceptibility loci. Nat Genet 3:312–318CrossRefGoogle Scholar
  14. Guryev V, Koudijs MJ, et al. (2006) Genetic variation in the zebrafish. Genome Res 4:491–497CrossRefGoogle Scholar
  15. Haddick PC, Tom I, Luis E, Quinones G, Wranik BJ, Ramani SR, Stephan JP, Tessier-Lavigne M, Gonzalez LC (2014) Defining the ligand specificity of the deleted in colorectal cancer (DCC) receptor. PLoS One 9:e84823CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hein R, Maranian M, Hopper JL, et al. (2012) Comparison of 6q25 breast cancer hits from Asian and European Genome Wide Association Studies in the Breast Cancer Association Consortium (BCAC). PLoS One 8,e42380Google Scholar
  17. Horn KE, Glasgow SD, Gobert D, et al. (2013) DCC expression by neurons regulates synaptic plasticity in the adult brain. Cell Rep 1:173–185CrossRefGoogle Scholar
  18. Huang L, Kuo YM, Gitschier J (1999) The pallid gene encodes a novel, syntaxin 13-interacting protein involved in platelet storage pool deficiency. Nat Genet 23:329–332CrossRefPubMedGoogle Scholar
  19. Hui ST, Parks BW, Org E, Norheim F, Che N, Pan C, Castellani LW, Charugundla S, Dirks DL, Psychogios N, et al. (2015) The genetic architecture of NAFLD among inbred strains of mice. Elife 4:e05607CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hurle B, Ignatova E, Massironi SM, Mashimo T, Rios X, Thalmann I, Thalmann R, Ornitz DM (2003) Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1. Hum Mol Genet 12:777–789CrossRefPubMedGoogle Scholar
  21. Isosomppi J, Vastinsalo H, Geller SF, Heon E, Flannery JG, Sankila EM (2009) Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane. Mol Vis 15:1806–1818PubMedPubMedCentralGoogle Scholar
  22. Jen JC (2008) Recent advances in the genetics of recurrent vertigo and vestibulopathy. Curr Opin Neurol 21:3–7CrossRefPubMedGoogle Scholar
  23. Jones SM, Johnson KR, Yu H, Erway LC, Alagramam KN, Pollak N, Jones TA (2005) A quantitative survey of gravity receptor function in mutant mouse strains. JARO 6:297–310CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jones SM, Jones TA (2014) Genetics of peripheral vestibular dysfunction: lessons from mutant mouse strains. J Am Acad Audiol 25:289–301CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jones SM, Jones TA, Johnson KR, Yu H, Erway LC, Zheng QY (2006) A comparison of vestibular and auditory phenotypes in inbred mouse strains. Brain Res 1091:40–46CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jones SM, Robertson NG, Given S, Giersch AB, Liberman MC, Morton CC (2011) Hearing and vestibular deficits in the Coch(−/−) null mouse model: comparison to the Coch (G88E/G88E) mouse and to DFNA9 hearing and balance disorder. Hear Res 272:42–48CrossRefPubMedGoogle Scholar
  27. Jones TA, Jones SM (1999) Short latency compound action potentials from mammalian gravity receptor organs. Hear Res 136:75–85CrossRefPubMedGoogle Scholar
  28. Keino-Masu K, Masu M, Hinck L, Leonardo ED, Chan SS, Culotti JG, Tessier-Lavigne M (1996) Deleted in colorectal cancer (DCC) encodes a netrin receptor. Cell 2:175–185CrossRefGoogle Scholar
  29. Kerber KA, Meurer WJ, West BT, Fendrick AM (2008) Dizziness presentations in U.S. emergency departments, 1995-2004. Acad Emerg Med 15:744–750CrossRefPubMedGoogle Scholar
  30. Krimpenfort P, Song JY, Proost N, Zevenhoven J, Jonkers J, Berns A (2012) Deleted in colorectal carcinoma suppresses metastasis in p53-deficient mammary tumours. Nature 482:538–541CrossRefPubMedGoogle Scholar
  31. Lavinsky J, Crow AL, Pan C, Wang J, Aaron KA, Ho MK, Li Q, Salehide P, Myint A, Monges-Hernadez M, et al. (2015) Genome-wide association study identifies nox3 as a critical gene for susceptibility to noise-induced hearing loss. PLoS Genet 11:e1005094CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lee SI, Conrad T, Jones SM, Lagziel A, Starost MF, Belyantseva IA, Friedman TB, Morell RJ (2013) A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction. Hear Res 300:1–9CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lippert C, Listgarten J, Liu Y, Kadie CM, Davidson RI, Heckerman D (2011) FaST linear mixed models for genome-wide association studies. Nat Methods 8:833–835CrossRefPubMedGoogle Scholar
  34. Matilainen T, Haugas M, Kreidberg JA, Salminen M (2007) Analysis of Netrin 1 receptors during inner ear development. Int J Dev Biol 51:409–413CrossRefPubMedGoogle Scholar
  35. Mbiene JP, Favre D, Sans A (1988) Early innervation and differentiation of hair cells in the vestibular epithelia of mouse embryos: SEM and TEM study. Anat Embryol 4:331–340CrossRefGoogle Scholar
  36. Mehlen P, Mazelin L (2003) The dependence receptors DCC and UNC5H as a link between neuronal guidance and survival. Biol Cell 7:425–436CrossRefGoogle Scholar
  37. Mehlen P, Delloye-Bourgeois C, Chedotal A (2011) Novel roles for Slits and netrins: axon guidance cues as anticancer targets? Nat Rev Cancer 11:188–197CrossRefPubMedGoogle Scholar
  38. Nakano Y, Longo-Guess CM, Bergstrom DE, Nauseef WM, Jones SM, Banfi B (2008) Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice. J Clin Invest 118:1176–1185PubMedPubMedCentralGoogle Scholar
  39. Nagel AN, Marshak S, Manitt C, Santos RA, Piercy MA, Mortero SD, Shirkey-Son NJ, Cohen-Cory S (2015) Netrin-1 directs dendritic growth and connectivity of vertebrate central neurons in vivo. Neural Dev. doi: 10.1186/s13064-015-0041-y PubMedPubMedCentralGoogle Scholar
  40. Neuhauser HK, von Brevern M, Radtke A, Lezius F, Feldmann M, Ziese T, Lempert T (2005) Epidemiology of vestibular vertigo: a neurotologic survey of the general population. Neurology 65:898–904CrossRefPubMedGoogle Scholar
  41. Ohmen J, Kang EY, Li X, Joo JW, Hormozdiari F, Zheng QY, Davis RC, Lusis AJ, Eskin E, Friedman RA (2014) Genome-wide association study for age-related hearing loss (AHL) in the mouse: a meta-analysis. JARO 15:335–352CrossRefPubMedPubMedCentralGoogle Scholar
  42. Ohyama T, Basch ML, Mishina Y, Lyons KM, Segil N, Groves AK (2010) BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea. J Neurosci 30:15044–15051CrossRefPubMedPubMedCentralGoogle Scholar
  43. Paffenholz R, Bergstrom RA, Pasutto F, Wabnitz P, Munroe RJ, Jagla W, Heinzmann U, Marquardt A, Bareiss A, Laufs J, et al. (2004) Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase. Genes Dev 18:486–491CrossRefPubMedPubMedCentralGoogle Scholar
  44. Park CC, Gale GD, de Jong S, Ghazalpour A, Bennett BJ, Farber CR, Langfelder P, Lin A, Khan AH, Eskin E, et al. (2011) Gene networks associated with conditional fear in mice identified using a systems genetics approach. BMC Syst Biol 5:43CrossRefPubMedPubMedCentralGoogle Scholar
  45. Rau CD, Parks B, Wang Y, Eskin E, Simecek P, Churchill GA, Lusis AJ (2015) High-density genotypes of inbred mouse strains: improved power and precision of association mapping. G3 (Bethesda) 5:2021–2026CrossRefGoogle Scholar
  46. Sanna S, Li B, Mulas A, Sidore C, et al. (2011) Fine mapping of five loci associated with low-density lipoprotein cholesterol detects variants that double the explained heritability. PLoS Genet 7, e1002198Google Scholar
  47. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  48. Shi M, Zheng MH, Liu ZR, ZL H, Huang Y, Chen JY, Zhao G, Han H, Ding YQ (2010) DCC is specifically required for the survival of retinal ganglion and displaced amacrine cells in the developing mouse retina. Dev Biol 1:87–96CrossRefGoogle Scholar
  49. Smolock EM, Ilyushkina IA, Ghazalpour A, Gerloff J, Murashev AN, Lusis AJ, Korshunov VA (2012) Genetic locus on mouse chromosome 7 controls elevated heart rate. Physiol Genomics 44:689–698CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sul JH, Eskin E (2013) Mixed models can correct for population structure for genomic regions under selection. Nat Rev Genet 4:300CrossRefGoogle Scholar
  51. Vincent PF, Bouleau Y, Safieddine S, Petit C, Dulon D (2014) Exocytotic machineries of vestibular type I and cochlear ribbon synapses display similar intrinsic otoferlin-dependent Ca2+ sensitivity but a different coupling to Ca2+ channels. J Neurosci 34:10853–10869CrossRefPubMedGoogle Scholar
  52. Yang H, Ding Y, Hutchins LN, Szatkiewicz J, Bell TA, Paigen BJ, Graber JH, de Villena FP, Churchill GA (2009) A customized and versatile high-density genotyping array for the mouse. Nat Methods 6:663–666CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhou X, Crow AL, Hartiala J, Spindler TJ, Ghazalpour A, Barsky LW, Bennett BJ, Parks BW, Eskin E, Jain R, et al. (2015) The genetic landscape of hematopoietic stem cell frequency in mice. Stem Cell Rep 5:125–138CrossRefGoogle Scholar

Copyright information

© Association for Research in Otolaryngology 2016

Authors and Affiliations

  • Pezhman Salehi
    • 1
  • Anthony Myint
    • 1
  • Young J. Kim
    • 2
  • Marshall X. Ge
    • 1
  • Joel Lavinsky
    • 1
    • 3
  • Maria K. Ho
    • 1
    • 4
  • Amanda L. Crow
    • 5
  • Charlene Cruz
    • 1
  • Maya Monges-Hernadez
    • 1
  • Juemei Wang
    • 1
  • Jaana Hartiala
    • 5
  • Li I. Zhang
    • 2
  • Hooman Allayee
    • 5
  • Aldons J. Lusis
    • 5
    • 6
  • Takahiro Ohyama
    • 1
    Email author
  • Rick A. Friedman
    • 1
    • 7
    Email author
  1. 1.USC Tina and Rick Caruso Department of Otolaryngology-Head & Neck Surgery, Zilkha Neurogenetic Institute, Keck Medicine of USCUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, USC Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA
  3. 3.Graduate Program in Surgical SciencesFederal University of Rio Grande do SulPorto AlegreBrazil
  4. 4.Department of Internal Medicine, Charles E. Schmidt College of MedicineFlorida Atlantic UniversityBoca RatonUSA
  5. 5.Department of Preventive Medicine and Institute for Genetic Medicine, USC Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA
  6. 6.Department of Microbiology, Immunology, and Molecular GeneticsUniversity of California, Los AngelesLos AngelesUSA
  7. 7.Department of Otolaryngology, Zilkha Neurogenetic Institute, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA

Personalised recommendations