Skip to main content
PMC home page
American Journal of Human Genetics logoLink to American Journal of Human Genetics
. 1999 Feb;64(2):446–461. doi: 10.1086/302245

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome.

M Oldridge 1, E H Zackai 1, D M McDonald-McGinn 1, S Iseki 1, G M Morriss-Kay 1, S R Twigg 1, D Johnson 1, S A Wall 1, W Jiang 1, C Theda 1, E W Jabs 1, A O Wilkie 1
PMCID: PMC1377754  PMID: 9973282

Abstract

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.

Full Text

The Full Text of this article is available as a PDF (886.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abdelhak S., Kalatzis V., Heilig R., Compain S., Samson D., Vincent C., Levi-Acobas F., Cruaud C., Le Merrer M., Mathieu M. Clustering of mutations responsible for branchio-oto-renal (BOR) syndrome in the eyes absent homologous region (eyaHR) of EYA1. Hum Mol Genet. 1997 Dec;6(13):2247–2255. doi: 10.1093/hmg/6.13.2247. [DOI] [PubMed] [Google Scholar]
  2. Anderson J., Burns H. D., Enriquez-Harris P., Wilkie A. O., Heath J. K. Apert syndrome mutations in fibroblast growth factor receptor 2 exhibit increased affinity for FGF ligand. Hum Mol Genet. 1998 Sep;7(9):1475–1483. doi: 10.1093/hmg/7.9.1475. [DOI] [PubMed] [Google Scholar]
  3. Anderson P. J., Hall C. M., Evans R. D., Jones B. M., Hayward R. D. The feet in Pfeiffer's syndrome. J Craniofac Surg. 1998 Jan;9(1):83–87. doi: 10.1097/00001665-199801000-00018. [DOI] [PubMed] [Google Scholar]
  4. Batzer M. A., Deininger P. L., Hellmann-Blumberg U., Jurka J., Labuda D., Rubin C. M., Schmid C. W., Zietkiewicz E., Zuckerkandl E. Standardized nomenclature for Alu repeats. J Mol Evol. 1996 Jan;42(1):3–6. doi: 10.1007/BF00163204. [DOI] [PubMed] [Google Scholar]
  5. Batzer M. A., Rubin C. M., Hellmann-Blumberg U., Alegria-Hartman M., Leeflang E. P., Stern J. D., Bazan H. A., Shaikh T. H., Deininger P. L., Schmid C. W. Dispersion and insertion polymorphism in two small subfamilies of recently amplified human Alu repeats. J Mol Biol. 1995 Mar 31;247(3):418–427. doi: 10.1006/jmbi.1994.0150. [DOI] [PubMed] [Google Scholar]
  6. Bellus G. A., Gaudenz K., Zackai E. H., Clarke L. A., Szabo J., Francomano C. A., Muenke M. Identical mutations in three different fibroblast growth factor receptor genes in autosomal dominant craniosynostosis syndromes. Nat Genet. 1996 Oct;14(2):174–176. doi: 10.1038/ng1096-174. [DOI] [PubMed] [Google Scholar]
  7. Boeke J. D. LINEs and Alus--the polyA connection. Nat Genet. 1997 May;16(1):6–7. doi: 10.1038/ng0597-6. [DOI] [PubMed] [Google Scholar]
  8. Carstens R. P., McKeehan W. L., Garcia-Blanco M. A. An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing. Mol Cell Biol. 1998 Apr;18(4):2205–2217. doi: 10.1128/mcb.18.4.2205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Del Gatto F., Plet A., Gesnel M. C., Fort C., Breathnach R. Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon. Mol Cell Biol. 1997 Sep;17(9):5106–5116. doi: 10.1128/mcb.17.9.5106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dell K. R., Williams L. T. A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity. J Biol Chem. 1992 Oct 15;267(29):21225–21229. [PubMed] [Google Scholar]
  12. Dib C., Fauré S., Fizames C., Samson D., Drouot N., Vignal A., Millasseau P., Marc S., Hazan J., Seboun E. A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature. 1996 Mar 14;380(6570):152–154. doi: 10.1038/380152a0. [DOI] [PubMed] [Google Scholar]
  13. Dionne C. A., Crumley G., Bellot F., Kaplow J. M., Searfoss G., Ruta M., Burgess W. H., Jaye M., Schlessinger J. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors. EMBO J. 1990 Sep;9(9):2685–2692. doi: 10.1002/j.1460-2075.1990.tb07454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Escobar V., Bixler D. On the classification of the acrocephalosyndactyly syndromes. Clin Genet. 1977 Sep;12(3):169–178. doi: 10.1111/j.1399-0004.1977.tb00920.x. [DOI] [PubMed] [Google Scholar]
  15. Finch P. W., Cunha G. R., Rubin J. S., Wong J., Ron D. Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions. Dev Dyn. 1995 Jun;203(2):223–240. doi: 10.1002/aja.1002030210. [DOI] [PubMed] [Google Scholar]
  16. Gilbert E., Del Gatto F., Champion-Arnaud P., Gesnel M. C., Breathnach R. Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA. Mol Cell Biol. 1993 Sep;13(9):5461–5468. doi: 10.1128/mcb.13.9.5461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hu M. C., Qiu W. R., Wang Y. P., Hill D., Ring B. D., Scully S., Bolon B., DeRose M., Luethy R., Simonet W. S. FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation. Mol Cell Biol. 1998 Oct;18(10):6063–6074. doi: 10.1128/mcb.18.10.6063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iseki S., Wilkie A. O., Heath J. K., Ishimaru T., Eto K., Morriss-Kay G. M. Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development. 1997 Sep;124(17):3375–3384. doi: 10.1242/dev.124.17.3375. [DOI] [PubMed] [Google Scholar]
  19. Janicic N., Pausova Z., Cole D. E., Hendy G. N. Insertion of an Alu sequence in the Ca(2+)-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am J Hum Genet. 1995 Apr;56(4):880–886. [PMC free article] [PubMed] [Google Scholar]
  20. Johnson D. E., Williams L. T. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res. 1993;60:1–41. doi: 10.1016/s0065-230x(08)60821-0. [DOI] [PubMed] [Google Scholar]
  21. Johnston C. L., Cox H. C., Gomm J. J., Coombes R. C. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem. 1995 Dec 22;270(51):30643–30650. doi: 10.1074/jbc.270.51.30643. [DOI] [PubMed] [Google Scholar]
  22. Jurka J. Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1872–1877. doi: 10.1073/pnas.94.5.1872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katoh M., Hattori Y., Sasaki H., Tanaka M., Sugano K., Yazaki Y., Sugimura T., Terada M. K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2960–2964. doi: 10.1073/pnas.89.7.2960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lajeunie E., Ma H. W., Bonaventure J., Munnich A., Le Merrer M., Renier D. FGFR2 mutations in Pfeiffer syndrome. Nat Genet. 1995 Feb;9(2):108–108. doi: 10.1038/ng0295-108. [DOI] [PubMed] [Google Scholar]
  25. Makałowski W., Mitchell G. A., Labuda D. Alu sequences in the coding regions of mRNA: a source of protein variability. Trends Genet. 1994 Jun;10(6):188–193. doi: 10.1016/0168-9525(94)90254-2. [DOI] [PubMed] [Google Scholar]
  26. Martin G. R. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 1998 Jun 1;12(11):1571–1586. doi: 10.1101/gad.12.11.1571. [DOI] [PubMed] [Google Scholar]
  27. Mason I. J., Fuller-Pace F., Smith R., Dickson C. FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech Dev. 1994 Jan;45(1):15–30. doi: 10.1016/0925-4773(94)90050-7. [DOI] [PubMed] [Google Scholar]
  28. Miki T., Bottaro D. P., Fleming T. P., Smith C. L., Burgess W. H., Chan A. M., Aaronson S. A. Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):246–250. doi: 10.1073/pnas.89.1.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miki Y., Katagiri T., Kasumi F., Yoshimoto T., Nakamura Y. Mutation analysis in the BRCA2 gene in primary breast cancers. Nat Genet. 1996 Jun;13(2):245–247. doi: 10.1038/ng0696-245. [DOI] [PubMed] [Google Scholar]
  30. Moloney D. M., Slaney S. F., Oldridge M., Wall S. A., Sahlin P., Stenman G., Wilkie A. O. Exclusive paternal origin of new mutations in Apert syndrome. Nat Genet. 1996 May;13(1):48–53. doi: 10.1038/ng0596-48. [DOI] [PubMed] [Google Scholar]
  31. Muenke M., Schell U., Hehr A., Robin N. H., Losken H. W., Schinzel A., Pulleyn L. J., Rutland P., Reardon W., Malcolm S. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat Genet. 1994 Nov;8(3):269–274. doi: 10.1038/ng1194-269. [DOI] [PubMed] [Google Scholar]
  32. Muratani K., Hada T., Yamamoto Y., Kaneko T., Shigeto Y., Ohue T., Furuyama J., Higashino K. Inactivation of the cholinesterase gene by Alu insertion: possible mechanism for human gene transposition. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11315–11319. doi: 10.1073/pnas.88.24.11315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neilson K. M., Friesel R. Ligand-independent activation of fibroblast growth factor receptors by point mutations in the extracellular, transmembrane, and kinase domains. J Biol Chem. 1996 Oct 4;271(40):25049–25057. doi: 10.1074/jbc.271.40.25049. [DOI] [PubMed] [Google Scholar]
  34. Ohbayashi N., Hoshikawa M., Kimura S., Yamasaki M., Fukui S., Itoh N. Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18. J Biol Chem. 1998 Jul 17;273(29):18161–18164. doi: 10.1074/jbc.273.29.18161. [DOI] [PubMed] [Google Scholar]
  35. Ohuchi H., Nakagawa T., Yamamoto A., Araga A., Ohata T., Ishimaru Y., Yoshioka H., Kuwana T., Nohno T., Yamasaki M. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development. 1997 Jun;124(11):2235–2244. doi: 10.1242/dev.124.11.2235. [DOI] [PubMed] [Google Scholar]
  36. Oldridge M., Lunt P. W., Zackai E. H., McDonald-McGinn D. M., Muenke M., Moloney D. M., Twigg S. R., Heath J. K., Howard T. D., Hoganson G. Genotype-phenotype correlation for nucleotide substitutions in the IgII-IgIII linker of FGFR2. Hum Mol Genet. 1997 Jan;6(1):137–143. doi: 10.1093/hmg/6.1.137. [DOI] [PubMed] [Google Scholar]
  37. Ornitz D. M., Xu J., Colvin J. S., McEwen D. G., MacArthur C. A., Coulier F., Gao G., Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem. 1996 Jun 21;271(25):15292–15297. doi: 10.1074/jbc.271.25.15292. [DOI] [PubMed] [Google Scholar]
  38. Orr-Urtreger A., Bedford M. T., Burakova T., Arman E., Zimmer Y., Yayon A., Givol D., Lonai P. Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol. 1993 Aug;158(2):475–486. doi: 10.1006/dbio.1993.1205. [DOI] [PubMed] [Google Scholar]
  39. Park W. J., Theda C., Maestri N. E., Meyers G. A., Fryburg J. S., Dufresne C., Cohen M. M., Jr, Jabs E. W. Analysis of phenotypic features and FGFR2 mutations in Apert syndrome. Am J Hum Genet. 1995 Aug;57(2):321–328. [PMC free article] [PubMed] [Google Scholar]
  40. Passos-Bueno M. R., Richieri-Costa A., Sertié A. L., Kneppers A. Presence of the Apert canonical S252W FGFR2 mutation in a patient without severe syndactyly. J Med Genet. 1998 Aug;35(8):677–679. doi: 10.1136/jmg.35.8.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Passos-Bueno M. R., Sertié A. L., Zatz M., Richieri-Costa A. Pfeiffer mutation in an Apert patient: how wide is the spectrum of variability due to mutations in the FGFR2 gene? Am J Med Genet. 1997 Aug 8;71(2):243–245. [PubMed] [Google Scholar]
  42. Przylepa K. A., Paznekas W., Zhang M., Golabi M., Bias W., Bamshad M. J., Carey J. C., Hall B. D., Stevenson R., Orlow S. Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome. Nat Genet. 1996 Aug;13(4):492–494. doi: 10.1038/ng0896-492. [DOI] [PubMed] [Google Scholar]
  43. Robertson S. C., Meyer A. N., Hart K. C., Galvin B. D., Webster M. K., Donoghue D. J. Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4567–4572. doi: 10.1073/pnas.95.8.4567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schell U., Hehr A., Feldman G. J., Robin N. H., Zackai E. H., de Die-Smulders C., Viskochil D. H., Stewart J. M., Wolff G., Ohashi H. Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome. Hum Mol Genet. 1995 Mar;4(3):323–328. doi: 10.1093/hmg/4.3.323. [DOI] [PubMed] [Google Scholar]
  45. Schmid C. W. Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog Nucleic Acid Res Mol Biol. 1996;53:283–319. doi: 10.1016/s0079-6603(08)60148-8. [DOI] [PubMed] [Google Scholar]
  46. Slaney S. F., Oldridge M., Hurst J. A., Moriss-Kay G. M., Hall C. M., Poole M. D., Wilkie A. O. Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome. Am J Hum Genet. 1996 May;58(5):923–932. [PMC free article] [PubMed] [Google Scholar]
  47. Spivak-Kroizman T., Lemmon M. A., Dikic I., Ladbury J. E., Pinchasi D., Huang J., Jaye M., Crumley G., Schlessinger J., Lax I. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994 Dec 16;79(6):1015–1024. doi: 10.1016/0092-8674(94)90032-9. [DOI] [PubMed] [Google Scholar]
  48. Twigg S. R., Burns H. D., Oldridge M., Heath J. K., Wilkie A. O. Conserved use of a non-canonical 5' splice site (/GA) in alternative splicing by fibroblast growth factor receptors 1, 2 and 3. Hum Mol Genet. 1998 Apr;7(4):685–691. doi: 10.1093/hmg/7.4.685. [DOI] [PubMed] [Google Scholar]
  49. Upton J. Apert syndrome. Classification and pathologic anatomy of limb anomalies. Clin Plast Surg. 1991 Apr;18(2):321–355. [PubMed] [Google Scholar]
  50. Vidaud D., Vidaud M., Bahnak B. R., Siguret V., Gispert Sanchez S., Laurian Y., Meyer D., Goossens M., Lavergne J. M. Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene. Eur J Hum Genet. 1993;1(1):30–36. doi: 10.1159/000472385. [DOI] [PubMed] [Google Scholar]
  51. Wallace M. R., Andersen L. B., Saulino A. M., Gregory P. E., Glover T. W., Collins F. S. A de novo Alu insertion results in neurofibromatosis type 1. Nature. 1991 Oct 31;353(6347):864–866. doi: 10.1038/353864a0. [DOI] [PubMed] [Google Scholar]
  52. Wilkie A. O. Craniosynostosis: genes and mechanisms. Hum Mol Genet. 1997;6(10):1647–1656. doi: 10.1093/hmg/6.10.1647. [DOI] [PubMed] [Google Scholar]
  53. Wilkie A. O., Slaney S. F., Oldridge M., Poole M. D., Ashworth G. J., Hockley A. D., Hayward R. D., David D. J., Pulleyn L. J., Rutland P. Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet. 1995 Feb;9(2):165–172. doi: 10.1038/ng0295-165. [DOI] [PubMed] [Google Scholar]
  54. Xu X., Weinstein M., Li C., Naski M., Cohen R. I., Ornitz D. M., Leder P., Deng C. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development. 1998 Feb;125(4):753–765. doi: 10.1242/dev.125.4.753. [DOI] [PubMed] [Google Scholar]

Articles from American Journal of Human Genetics are provided here courtesy of American Society of Human Genetics

RESOURCES