CLINICAL AND GENETIC STUDY OF 46 ITALIAN PATIENTS WITH PRIMARY LYMPHEDEMA

S. Michelini, D. Degiorgio, M. Cestari, D. Corda, M. Ricci, M. Cardone,
A. Mander, L. Famoso, E. Contini, R. Serrani, L. Pinelli, S. Cecchin, M. Bertelli

Department of Vascular Rehabilitation (SM,MC), San Giovanni Battista Hospital, Rome, Italy; Laboratory of Molecular Genetics (DD,LP,SC,MB), International Association of Medical Genetics -MAGI-onlus-; Pianeta Linfedema Study Center (MC,LF); Centro Medico e Riabilitativo per la Ricerca, la Diagnosi e la Cura del Linfedema (DC,EC); Medicina Riabilitativa (MR, RS), Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona; Rehabilitative Centre Vaclav Vojta (AM), Roma, Italy.

ABSTRACT
Primary lymphedema is characterized by altered morphological development of lymphatic vessels causing fluid accumulation in interstitial spaces. In familial forms, it is primarily transmitted as a dominant Mendelian trait with heterozygous mutations in genes involved in lymphangiogenesis. We used PCR and direct sequencing to analyze the region of the fms-related tyrosine kinase 4 (FLT4) gene encoding the "tyrosine-kinase domain" and the single exon of the forkhead box C2 (FOXC2) gene in 46 Italian probands with primary lymphedema, 42 of whom had familial forms. We identified 12 mutations in 12 patients (12/46, 26%), six in the FLT4 gene and six in the FOXC2 gene. Most of the mutations (9/12, 75%) were new, and none were identified in 100 healthy subjects or listed in the NCBI dbSNP. A clear relation emerged between genotype and phenotype because 4/5 (80%) probands with onset at birth showed FLT4 mutations and 4/5 (80%) probands without distichiasis and with FOXC2 mutations had an amino-acid substitution outside the forkhead domain. Besides the allelic heterogeneity shown by unique mutations in each proband, the absence of mutations in almost 75% of  familial cases of primary lymphedema also suggests genetic heterogeneity.

REFERENCES
1. Connell, F, G Brice, P Mortimer: Phenotypic characterization of primary lymphedema. Ann. N. Y. Acad. Sci. 1131 (2008), 140-146.
2. Fang, J, LD Susan, RP Erickson, et al: Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am. J. Hum. Genet. 67 (2000), 1382-1388.
3. Holberg, CJ, RP Erickson, MJ Bernas, et al: Segregation analyses and a genome-wide linkage search confirm genetic heterogeneity and suggest oligogenic inheritance in some Milroy congenital primary lymphedema families. Am. J. Med. Genet. 98 (2001), 303-312.
4. Ferrell, RE, KL Levinson, JH Esman, et al: Hereditary lymphedema: Evidence for linkage and genetic heterogeneity. Hum. Mol. Genet. 7 (1998), 2073-2078.
5. Neufeld, G, T Cohen, S Gengrinovitch, et al: Vascular endothelial growth factor (VEGF) and its receptors. FASEB. J. 13 (1999), 9-22.
6. Bell, R, G Brice, AH Child, et al:  Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene. Hum. Genet. 108 (2001), 546-551.
7. Szuba, A, M Skobe, MJ Karkkainen, et al: Therapeutic lymphangiogenesis with human recombinant VEGF-C. FASEB J. 16 (2002), 1985-1987.
8. Kriederman BM, TL Myloyde, MH Witte, et al.  FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum. Mol. Genet. 12 (2003), 1179-1185.
9. Finegold, DN, V Schacht, MA Kimak, et al: HGF and MET mutations in primary and secondary lymphedema. Lymphat. Res. Biol. 6 (2008), 65-68.
10. Ferrell, RE, MA Kimak, EC Lawrence, et al: Candidate gene analysis in primary lymphedema. Lymphat. Res. Biol. 6 (2008), 69-76.
11. Ferrell, RE, CJ Baty, MA Kimak, et al: GJC2 missense mutations cause human lymphedema. Am. J. Hum. Genet. 86 (2010), 943-948.
12. Irrthum, A, D Koenraad, D Chitayat et al: Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema- telangiectasia. Am. J. Hum. Genet. 72 (2003), 1470-1478.
13. Ostergaard, P, MA Simpson, FC Connell, et al: Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome). Nat. Genet. 43 (2011), 929-931.
14. Alders, M, BM Hogan, E Gjini, et al: Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans. Nat. Genet. 41 (2009), 1272-1274.
15. Connell, FC, P Ostergaard, C Carver, et al: Analysis of the coding regions of VEGFR3 and VEGFC in Milroy disease and other primary lymphoedemas. Hum. Genet. 124 (2009), 625-631.
16. Brice, G, AH Child, A Evans, et al: Milroy disease and the VEGFR-3 mutation phenotype. J. Med. Genet. 42 (2005), 98-102.
17. Mangion, J, N Rahman, S Mansour, et al: A gene for lymphedema-distichiasis maps to 16q24.3. Am. J. Hum. Genet. 65 (1999), 427-432.
18. Finegold, DN, MA Kimak, EC Lawrence, et al: Truncating mutations in FOXC2 cause multiple lymphedema syndromes. Hum. Mol. Genet. 10 (2001), 1185-1189.
19. Brice, G, S Mansour, R Bell, et al: Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J. Med. Genet. 39 (2002), 478-483.
20. Bahuau, M, C Houdayer, M Tredano, et al: FOXC2 truncating mutation in distichiasis, lymphedema, and cleft palate. Clin. Genet. 62 (2002), 470-473.
21. Fabretto, A, A Shardlow, F Faletra, et al: A case of lymphedema-distichiasis syndrome carrying a new de novo frameshift FOXC2 mutation. Ophthalmic Genet. 31 (2010), 98-100.
22. Mellor, RH, G Brice, AW Stanton, et al: Mutations in FOXC2 are strongly associated with primary valve failure in veins of the lower limb. Circulation. 115 (2007), 1912-1920.
23. van Steensel, MA, RJ Damstra, MV Heitink, et al: Novel missense mutations in the FOXC2 gene alter transcriptional activity. Hum. Mutat. 30 (2009), 1002-1009.
24. International Society of Lymphology: 2009 Consensus Document of the International Society of Lymphology: the diagnosis and treatment of peripheral lymphedema. Lymphology 42 (2009), 51-60.
25. Betts, MJ, RB Russell: Amino acid properties and consequences of substitutions. In: Bioinformatics for Geneticists. Barnes, MR, IC Gray (Eds.), Wiley, 2003.
26. Berry, FB, Y Tamimi, MV Carle, et al: The establishment of a predictive mutational model of the forkhead domain through the analyses of FOXC2 missense mutations identified in patients with hereditary lymphedema with distichiasis. Hum. Mol. Genet. 14 (2005), 2619-2627.
27. Evans, AL, R Bell, G Brice, et al: Identification of eight novel VEGFR-3 mutations in families with primary congenital lymphoedema. J. Med. Genet. 40 (2003), 697-703.
28. Ghalamkarpour, A, S Morlot, A Raas-Rothschild, et al: Hereditary lymphedema type I associated with VEGFR3 mutation: The first de novo case and atypical presentations. Clin. Genet. 70 (2006), 330-335.
29. Sholto-Douglas-Vernon, C, R Bell, G Brice, et al: Lymphoedema-distichiasis and FOXC2: unreported mutations, de novo mutation estimate, families without coding mutations. Hum. Genet. 117 (2005), 238-242.
30. Spiegel, R, A Ghalamkarpour, E Daniel-Spiegel, et al: Wide clinical spectrum in a family with hereditary lymphedema type I due to a novel missense mutation in VEGFR3. J. Hum. Genet. 51 (2006), 846-850.
31. Cheung, L, J Han, A Beilhack, et al: An experimental model for the study of lymphedema and its response to therapeutic lymphangiogenesis. BioDrugs. 20 (2006), 363-370.