Abstract
Holprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by varying degrees of abnormal union of the cerebral hemispheres. These defects are typically co-associated with midline craniofacial anomalies. The combination of forebrain and craniofacial defects that comprise HPE can present along a broad and variable phenotypic spectrum. Both the SHH and NODAL signaling pathways play important roles in the pathogenesis of this disorder. Disruption of these pathways by chromosomal rearrangements, mutations in pathway-related genes and/or biochemical alterations are proposed to contribute to HPE in a large number of patients. Additional factors that are not yet fully delineated are also very likely to be involved in the pathogenesis and phenotypic heterogeneity of the disorder. Genetic loss of GAS1, a cell membrane receptor and positive regulator of SHH, has been demonstrated to contribute to the HPE phenotypic spectrum in animal models. We have evaluated the coding and flanking sequence of GAS1 in 394 patients who have clinical findings within the HPE phenotypic spectrum, and now report five novel missense sequence variants among five unrelated HPE probands. Finally, we tested the effect of these variants (as well as previously reported GAS1 variants) on the ability of GAS1 to bind to SHH. Here, we demonstrate that sequence variants in GAS1 can impair its physical interaction with SHH, suggesting a decrease in the SHH downstream signaling cascade as a pathogenic mechanism of disease.
Similar content being viewed by others
References
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nat Methods 7(4):248–249. doi:10.1038/nmeth0410-248
Allen BL, Tenzen T, McMahon AP (2007) The Hedgehog-binding proteins Gas1 and Cdo cooperate to positively regulate Shh signaling during mouse development. Genes Dev 21(10):1244–1257. doi:10.1101/gad.1543607
Beachy PA, Hymowitz SG, Lazarus RA, Leahy DJ, Siebold C (2010) Interactions between Hedgehog proteins and their binding partners come into view. Genes Dev 24(18):2001–2012. doi:10.1101/gad.1951710
Brown SA, Warburton D, Brown LY, Yu CY, Roeder ER, Stengel-Rutkowski S, Hennekam RC, Muenke M (1998) Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd-paired. Nat Genet 20(2):180–183. doi:10.1038/2484
Cabrera JR, Sanchez-Pulido L, Rojas AM, Valencia A, Manes S, Naranjo JR, Mellstrom B (2006) Gas1 is related to the glial cell-derived neurotrophic factor family receptors alpha and regulates Ret signaling. J Biol Chem 281(20):14330–14339. doi:10.1074/jbc.M509572200
Demyer W, Zeman W, Palmer CG (1964) The Face Predicts the Brain: Diagnostic Significance of Median Facial Anomalies for Holoprosencephaly (Arhinencephaly). Pediatrics 34:256–263
Domené S, Roessler E, El-Jaick KB, Snir M, Brown JL, Velez JI, Bale S, Lacbawan F, Muenke M, Feldman B (2008) Mutations in the human SIX3 gene in holoprosencephaly are loss of function. Hum Mol Genet 17(24):3919–3928. doi:10.1093/hmg/ddn294
Gripp KW, Wotton D, Edwards MC, Roessler E, Ades L, Meinecke P, Richieri-Costa A, Zackai EH, Massague J, Muenke M, Elledge SJ (2000) Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet 25(2):205–208. doi:10.1038/76074
Haas D, Morgenthaler J, Lacbawan F, Long B, Runz H, Garbade SF, Zschocke J, Kelley RI, Okun JG, Hoffmann GF, Muenke M (2007) Abnormal sterol metabolism in holoprosencephaly: studies in cultured lymphoblasts. J Med Genet 44(5):298–305. doi:jmg.2006.047258
Hahn JS, Barnes PD (2010) Neuroimaging advances in holoprosencephaly: Refining the spectrum of the midline malformation. Am J Med Genet C Semin Med Genet 154C(1):120–132. doi:10.1002/ajmg.c.30238
Kang JS, Zhang W, Krauss RS (2007) Hedgehog signaling: cooking with Gas1. Sci STKE 2007 (403):pe50. doi:10.1126/stke.4032007pe50
Lacbawan F, Solomon BD, Roessler E, El-Jaick K, Domene S, Velez JI, Zhou N, Hadley D, Balog JZ, Long R, Fryer A, Smith W, Omar S, McLean SD, Clarkson K, Lichty A, Clegg NJ, Delgado MR, Levey E, Stashinko E, Potocki L, Vanallen MI, Clayton-Smith J, Donnai D, Bianchi DW, Juliusson PB, Njolstad PR, Brunner HG, Carey JC, Hehr U, Musebeck J, Wieacker PF, Postra A, Hennekam RC, van den Boogaard MJ, van Haeringen A, Paulussen A, Herbergs J, Schrander-Stumpel CT, Janecke AR, Chitayat D, Hahn J, McDonald-McGinn DM, Zackai EH, Dobyns WB, Muenke M (2009) Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function. J Med Genet 46(6):389–398. doi:10.1136/jmg.2008.063818
Lee CS, Fan CM (2001) Embryonic expression patterns of the mouse and chick Gas1 genes. Mech Dev 101(1–2):293–297. S0925477301002830[pii]
Lee CS, Buttitta L, Fan CM (2001) Evidence that the WNT-inducible growth arrest-specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite. Proc Natl Acad Sci USA 98(20):11347–11352. doi:10.1073/pnas
Martinelli DC, Fan CM (2007a) Gas1 extends the range of Hedgehog action by facilitating its signaling. Genes Dev 21(10):1231–1243. doi:10.1101/gad.1546307
Martinelli DC, Fan CM (2007b) The role of Gas1 in embryonic development and its implications for human disease. Cell Cycle 6(21):2650–2655. 4877[pii]
Martinelli DC, Fan CM (2009) A sonic hedgehog missense mutation associated with holoprosencephaly causes defective binding to GAS1. J Biol Chem 284(29):19169–19172. doi:10.1074/jbc.C109.011957
Matsunaga E, Shiota K (1977) Holoprosencephaly in human embryos: epidemiologic studies of 150 cases. Teratology 16(3):261–272. doi:10.1002/tera.1420160304
Ming JE, Muenke M (2002) Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J Hum Genet 71(5):1017–1032. doi:10.1086/344412
Ming JE, Kaupas ME, Roessler E, Brunner HG, Golabi M, Tekin M, Stratton RF, Sujansky E, Bale SJ, Muenke M (2002) Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly. Hum Genet 110(4):297–301. doi:10.1007/s00439-002-0695-5
Muenke M, Beachy PA (2000) Genetics of ventral forebrain development and holoprosencephaly. Curr Opin Genet Dev 10(3):262–269. S0959-437X(00)00084-8[pii]
Nanni L, Ming JE, Bocian M, Steinhaus K, Bianchi DW, Die-Smulders C, Giannotti A, Imaizumi K, Jones KL, Campo MD, Martin RA, Meinecke P, Pierpont ME, Robin NH, Young ID, Roessler E, Muenke M (1999) The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly. Hum Mol Genet 8(13):2479–2488. ddc285[pii]
Pineda-Alvarez DE, Dubourg C, David V, Roessler E, Muenke M (2010) Current recommendations for the molecular evaluation of newly diagnosed holoprosencephaly patients. Am J Med Genet C Semin Med Genet 154C(1):93–101. doi:10.1002/ajmg.c.30253
Ribeiro LA, Quiezi RG, Nascimento A, Bertolacini CP, Richieri-Costa A (2010) Holoprosencephaly and holoprosencephaly-like phenotype and GAS1 DNA sequence changes: Report of four Brazilian patients. Am J Med Genet A 152A(7):1688–1694. doi:10.1002/ajmg.a.33466
Roessler E, Muenke M (2003) How a Hedgehog might see holoprosencephaly. Hum Mol Genet 12(Spec No 1):R15–R25
Roessler E, Muenke M (2010) The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet 154C(1):52–61. doi:10.1002/ajmg.c.30236
Roessler E, Belloni E, Gaudenz K, Jay P, Berta P, Scherer SW, Tsui LC, Muenke M (1996) Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat Genet 14(3):357–360. doi:10.1038/ng1196-357
Roessler E, Du YZ, Mullor JL, Casas E, Allen WP, Gillessen-Kaesbach G, Roeder ER, Ming JE, Ruiz i Altaba A, Muenke M (2003) Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features. Proc Natl Acad Sci USA 100(23):13424–13429. doi:10.1073/pnas
Roessler E, Ermilov AN, Grange DK, Wang A, Grachtchouk M, Dlugosz AA, Muenke M (2005) A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and disease-associated human GLI2. Hum Mol Genet 14(15):2181–2188. doi:10.1093/hmg/ddi222
Roessler E, El-Jaick KB, Dubourg C, Velez JI, Solomon BD, Pineda-Alvarez DE, Lacbawan F, Zhou N, Ouspenskaia M, Paulussen A, Smeets HJ, Hehr U, Bendavid C, Bale S, Odent S, David V, Muenke M (2009a) The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis. Hum Mutat 30(10):E921–E935. doi:10.1002/humu.21090
Roessler E, Lacbawan F, Dubourg C, Paulussen A, Herbergs J, Hehr U, Bendavid C, Zhou N, Ouspenskaia M, Bale S, Odent S, David V, Muenke M (2009b) The full spectrum of holoprosencephaly-associated mutations within the ZIC2 gene in humans predicts loss-of-function as the predominant disease mechanism. Hum Mutat 30(4):E541–E554. doi:10.1002/humu.20982
Roessler E, Pei W, Ouspenskaia MV, Karkera JD, Velez JI, Banerjee-Basu S, Gibney G, Lupo PJ, Mitchell LE, Towbin JA, Bowers P, Belmont JW, Goldmuntz E, Baxevanis AD, Feldman B, Muenke M (2009c) Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly. Mol Genet Metab 98(1–2):225–234. doi:10.1016/j.ymgme.2009.05.005
Seppala M, Depew MJ, Martinelli DC, Fan CM, Sharpe PT, Cobourne MT (2007) Gas1 is a modifier for holoprosencephaly and genetically interacts with sonic hedgehog. J Clin Invest 117(6):1575–1584. doi:10.1172/JCI32032
Solomon BD, Lacbawan F, Jain M, Domene S, Roessler E, Moore C, Dobyns WB, Muenke M (2009) A novel SIX3 mutation segregates with holoprosencephaly in a large family. Am J Med Genet A 149A(5):919–925. doi:10.1002/ajmg.a.32813
Solomon BD, Lacbawan F, Mercier S, Clegg NJ, Delgado MR, Rosenbaum K, Dubourg C, David V, Olney AH, Wehner LE, Hehr U, Bale S, Paulussen A, Smeets HJ, Hardisty E, Tylki-Szymanska A, Pronicka E, Clemens M, McPherson E, Hennekam RC, Hahn J, Stashinko E, Levey E, Wieczorek D, Roeder E, Schell-Apacik CC, Booth CW, Thomas RL, Kenwrick S, Cummings DA, Bous SM, Keaton A, Balog JZ, Hadley D, Zhou N, Long R, Velez JI, Pineda-Alvarez DE, Odent S, Roessler E, Muenke M (2010a) Mutations in ZIC2 in human holoprosencephaly: description of a Novel ZIC2 specific phenotype and comprehensive analysis of 157 individuals. J Med Genet 47(8):513–524. doi:10.1136/jmg.2009.073049
Solomon BD, Mercier S, Velez JI, Pineda-Alvarez DE, Wyllie A, Zhou N, Dubourg C, David V, Odent S, Roessler E, Muenke M (2010b) Analysis of genotype-phenotype correlations in human holoprosencephaly. Am J Med Genet C Semin Med Genet 154C(1):133–141. doi:10.1002/ajmg.c.30240
Stebel M, Vatta P, Ruaro ME, Del Sal G, Parton RG, Schneider C (2000) The growth suppressing gas1 product is a GPI-linked protein. FEBS Lett 481(2):152–158. S0014-5793(00)02004-4[pii]
Tokhunts R, Singh S, Chu T, D’Angelo G, Baubet V, Goetz JA, Huang Z, Yuan Z, Ascano M, Zavros Y, Therond PP, Kunes S, Dahmane N, Robbins DJ (2010) The full-length unprocessed hedgehog protein is an active signaling molecule. J Biol Chem 285(4):2562–2568. doi:10.1074/jbc.M109.078626
Wallis DE, Roessler E, Hehr U, Nanni L, Wiltshire T, Richieri-Costa A, Gillessen-Kaesbach G, Zackai EH, Rommens J, Muenke M (1999) Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nat Genet 22(2):196–198. doi:10.1038/9718
Zheng X, Mann RK, Sever N, Beachy PA (2010) Genetic and biochemical definition of the Hedgehog receptor. Genes Dev 24(1):57–71. doi:10.1101/gad.1870310
Acknowledgments
We thank the families who participated in these research studies, and the National Institute of Neurological Disorders and Stroke’s (NINDS) DNA Sequencing Facility for their technical support with DNA sequencing. This work was supported in part by the Division of Intramural Research (DIR) of the National Human Genome Research Institute (MM) and RO1 DK084963 (C-M F).
Author information
Authors and Affiliations
Corresponding author
Additional information
D. E. Pineda-Alvarez and E. Roessler contributed equally to this research work.
Rights and permissions
About this article
Cite this article
Pineda-Alvarez, D.E., Roessler, E., Hu, P. et al. Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand, SHH. Hum Genet 131, 301–310 (2012). https://doi.org/10.1007/s00439-011-1078-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00439-011-1078-6