Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema


Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Pedigrees of four lymphoedema families linked to chromosome 5q.
Figure 2: Missense mutations in FLT4 alleles of the lymphoedema patients abolish the receptor tyrosyl phosphorylation.
Figure 3: Analysis of VEGFR-3 autophosphorylation in a ligand-dependent system.
Figure 4: The turnover times of mutant and wild-type VEGFR-3 analysed by pulse-chase labelling analysis.
Figure 5: Ability of wild-type and mutant VEGFR-3 to activate downstream gene expression.
Figure 6: Model of the VEGFR-3 protein fold showing the positions of lymphoedema-associated mutations.

Accession codes




  1. 1

    Risau, W. Mechanisms of angiogenesis. Nature 386, 671–674 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Korpelainen, E.I. & Alitalo, K. Signaling angiogenesis and lymphangiogenesis. Curr. Opin. Cell Biol. 10, 159–164 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Ferrara, N. Molecular and biological properties of vascular endothelial growth factor . J. Mol. Med. 77, 527– 543 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Veikkola, T., Karkkainen, M.J., Claesson-Welsh, L. & Alitalo, K. Regulation of angiogenesis via vascular endothelial growth factor receptors . Cancer Res. 60, 203–212 (2000).

    CAS  PubMed  Google Scholar 

  5. 5

    Pajusola, K. et al. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res. 52, 5738–5743 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Galland, F. et al. The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor. Oncogene 8, 1233–1240 ( 1993).

    CAS  Google Scholar 

  7. 7

    Joukov, V. et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 15, 290–298 ( 1996).

    CAS  Article  Google Scholar 

  8. 8

    Lee, J. et al. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc. Natl Acad. Sci. USA 93, 1988–1992 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Achen, M.G. et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4) . Proc. Natl Acad. Sci. USA 95, 548– 553 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Kaipainen, A. et al. Expression of the fms-like tyrosine kinase FLT4 gene becomes restricted to endothelium of lymphatic vessels during development . Proc. Natl Acad. Sci. USA 92, 3566– 3570 (1995).

    CAS  Article  Google Scholar 

  11. 11

    Dumont, D.J. et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282, 946– 949 (1998).

    CAS  Article  Google Scholar 

  12. 12

    Kukk, E. et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggest a role in lymphatic vascular development. Development 122, 3829–3837 (1996).

    CAS  Google Scholar 

  13. 13

    Jeltsch, M. et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice . Science 276, 1423–1425 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Oh, S.J. et al. VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96–109 ( 1997).

    CAS  Article  Google Scholar 

  15. 15

    Milroy, W.F. An undescribed variety of hereditary oedema. NY Med. J. 56, 505–508 (1892).

    Google Scholar 

  16. 16

    Meige, H. Dystophie oedematoeuse hereditaire. Presse Med. 6, 341–343 (1898).

    Google Scholar 

  17. 17

    Leu, J.J. & Lie, J.T. Diseases of the veins and lymphatic vessels, including angiodysplasias. in Vascular Pathology (eds Stehbens, W.E. & Lie, J.T.) 489–516 (Chapman & Hall, London, 1995).

    Google Scholar 

  18. 18

    Ferrell, R.E. et al. Hereditary lymphedema: evidence for linkage and genetic heterogeneity . Hum. Mol. Genet. 7, 2073– 2078 (1998).

    CAS  Article  Google Scholar 

  19. 19

    Witte, M.H. et al. Phenotypic and genotypic heterogeneity in familial Milroy lymphedema. Lymphology 31, 145– 155 (1998).

    CAS  PubMed  Google Scholar 

  20. 20

    Evans, A.L. et al. Mapping of primary congenital lymphedema to the 5q35.3 region . Am. J. Hum. Genet. 64, 547– 555 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Jussila, L. et al. Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res. 58, 1599–1604 (1998).

    CAS  Google Scholar 

  22. 22

    Pajusola, K. et al. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 9, 3545–3555 ( 1994).

    CAS  Google Scholar 

  23. 23

    van der Geer, P., Hunter, T. & Lindberg, R. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10, 251– 337 (1994).

    CAS  Article  Google Scholar 

  24. 24

    McTigue, M.A. et al. Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis. Structure 7, 319–330 ( 1999).

    CAS  Article  Google Scholar 

  25. 25

    Hanks, S.K. & Quinn, A.M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 200, 38–62 (1991).

    CAS  Article  Google Scholar 

  26. 26

    Hubbard, S.R. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581 (1997).

    CAS  Article  Google Scholar 

  27. 27

    Zheng, J. et al. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 32, 2154–2161 ( 1993).

    CAS  Article  Google Scholar 

  28. 28

    The Protein Kinase Facts Book: Protein Tyrosine Kinases (eds Hardie, G. & Hanks, S.) (Academic, San Diego, 1995).

  29. 29

    Felder, S., LaVin, J., Ullrich, A. & Schlessinger, J. Kinetics of binding, endocytosis, and recycling of EGF receptor mutants. J. Cell Biol. 117, 203–212 (1992).

    CAS  Article  Google Scholar 

  30. 30

    Millauer, B., Shawver, L.K., Plate, K.H., Risau, W. & Ullrich, A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 367 , 576–579 (1994).

    CAS  Article  Google Scholar 

  31. 31

    Burke, D., Wilkes, D., Blundell, T.L. & Malcolm, S. Fibroblast growth factor receptors: lessons from the genes. Trends Biochem. Sci. 23, 59–62 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Muenke, M. & Schell, U. Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet. 11, 308–313 (1995).

    CAS  Article  Google Scholar 

  33. 33

    Bardelli, A., Pugliese, L. & Comoglio, P.M. “Invasive-growth” signaling by the Met/HGF receptor: the hereditary renal carcinoma connection. Biochim. Biophys. Acta 1333, M41–51 ( 1997).

    CAS  PubMed  Google Scholar 

  34. 34

    Schmidt, L. et al. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nature Genet. 16, 68–73 ( 1997).

    CAS  Article  Google Scholar 

  35. 35

    Pasini, B., Ceccherini, I. & Romeo, G. RET mutations in human disease. Trends Genet. 12, 138–144 ( 1996).

    CAS  Article  Google Scholar 

  36. 36

    Robertson, K., Mason, I. & Hall, S. Hirschsprung's disease: genetic mutations in mice and men. Gut 41, 436–441 ( 1997).

    CAS  Article  Google Scholar 

  37. 37

    Spritz, R.A. Molecular basis of human piebaldism. J. Invest. Dermatol. 103, 137S–140S (1994).

    Article  Google Scholar 

  38. 38

    Besmer, P. et al. The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev. Suppl. 125–137 (1993).

  39. 39

    Schinzel, A. Catalog of Unbalanced Chromosome Aberrations in Man (Walter de Gruyter, Berlin, 1983).

    Google Scholar 

  40. 40

    Groen, S.E., Drewes, J.G., de Boer, E.G., Hoovers, J.M.N. & Hennekam, R.C.M. Repeated unbalanced offspring due to a familial translocation involving chromosomes 5 and 6. Am. J. Med. Genet. 80, 448–453 (1998).

    CAS  Article  Google Scholar 

  41. 41

    Barber, J.C. et al. Unbalanced translocation in a mother and her son in one of two 5;10 translocation families. Am. J. Med. Genet. 62, 84–90 (1996).

    CAS  Article  Google Scholar 

  42. 42

    Mowat, D., Jauch, A., Robson, L. & Smith, A. Duplication within chromosome 5q characterized by fluorescence in situ hybridization. Am. J. Med. Genet. 83, 361–364 (1999).

    CAS  Article  Google Scholar 

  43. 43

    Mangion, J. et al. A gene for lymphedema-distichiasis maps to 16q24.3. Am. J. Hum. Genet. 65, 427–432 (1999).

    CAS  Article  Google Scholar 

  44. 44

    Korpelainen, E.I., Karkkainen, M., Gunji, Y., Vikkula, M. & Alitalo, K. Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2 causing venous malformations signals a distinct STAT activation response. Oncogene 18, 1–8 (1999).

    CAS  Article  Google Scholar 

  45. 45

    Pajusola, K., Aprelikova, O., Armstrong, E., Morris, S. & Alitalo, K. Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxyterminal tails are produced by alternative processing of primary transcripts. Oncogene 8, 2931–2937 (1993).

    CAS  PubMed  Google Scholar 

  46. 46

    Joukov, V. et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16, 3898– 3911 (1997).

    CAS  Article  Google Scholar 

  47. 47

    Brünger, A.T. X-PLOR (Version 3.1) Manual (The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 1992).

    Google Scholar 

Download references


We thank J. Esman for clinical evaluation of the families; the family members for participation; and S. Karttunen, T. Tainola, A. Parsons, P. Ylikantola and M. Helantera for technical assistance. This study was supported by grants from the Finnish Cancer Organization, Finnish Cultural Foundation, Emil Aaltonen Foundation, Ida Montini Foundation, the Finnish Academy and the European Union (Biomed grant no. PL 963380), N.I.H. grant no. HD35174 and a grant from the D.T. Watson Rehabilitation Hospital.

Author information



Corresponding author

Correspondence to Robert E. Ferrell.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Karkkainen, M., Ferrell, R., Lawrence, E. et al. Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema . Nat Genet 25, 153–159 (2000). https://doi.org/10.1038/75997

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing