CA2283470A1 - Screening and therapy for lymphatic disorders involving the flt4 receptor tyrosine kinase (vegfr-3) - Google Patents

Abstract

The present invention provides materials and methods for screening for and treating hereditary lymphedema in human subjects.

Description

SCREENING AND THERAPY FOR
LYMPHATIC DISORDERS INVOLVING THE
FLT4 RECEPTOR TYROSINE KINASE (VEGFR-3) This application is a Continuation-in-Part of International Patent Application No. PCT/LTS99/06133, filed March 26, 1999, incorporated herein by reference in its entirety.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with United States and Finnish government support, including support under contract R03-HD35174, awarded by the U.S.
National Institutes ofHealth. The U.S. Government has certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular biology and medicine; more particularly to the areas of genetic screening and the identification and treatment of hereditary disorders; and more particularly to identification and treatment of hereditary lymphedema.
DESCRIPTION OF RELATED ART
The lymphatic system is a complex structure organized in parallel fashion to the circulatory system. In contrast to the circulatory system, which utilizes the heart to pump blood throughout the body, the lymphatic system pumps lymph fluid using the inherent contractility of the lymphatic vessels. The lymphatic vessels are not interconnected in the same manner as the blood vessels, but rather form a set of coordinated structures including the initial lymphatic sinuses [Jeltsch et al." Science, 276:1423-1425 (1997); and Castenholz, A., in Olszewski, W.L. (ed.), Lymph Stasis:
Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton, Florida (1991), pp.15-42] which drain into the lymphatic capillaries and subsequently to the collecting lymphatics which drain into the lymphatic trunks and the thoracic duct which ultimately drains into the venous circulation. The composition of the channels through which lymph passes is varied [Olszewski, W.L., in Olszewski, W.L. (ed), Lymph Stasis: Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton, Florida (1991), pp. 235-258; and Kinmonth, J.B., in Kinmonth, J.B. (ed), The Lymphatics:
Diseases, Lymphography and Surgery. Edward Arnold Publishers: London, England (1972), pp. 82-86], including the single endothelial layers of the initial lymphatics, the multiple layers of the collecting lymphatics including endothelium, muscular and adventitial layers, and the complex organization of the lymph node. The various organs of the body such as skin, lung, and GI tract have components of the lymphatics with various unique features. [See Ohkuma, M., in Olszewski (1991), supra, at pp.
157-190; Uhley, H. and Leeds, S., in Olszewski (1991), supra, at pp. 191-210;
and Barrowman, J.A., in Olszewski (1991), at pp. 221-234).]
Molecular biology has identified at least a few genes and proteins postulated to have roles mediating the growth and/or embryonic development of the lymphatic system. One such gene/protein is the receptor tyrosine kinase designated Flt4 (fms-1_ike Iyrosine kinase 4), cloned from human erythroleukaemia cell and placental cDNA libraries. [See U.S. Patent No. 5,776,755; Aprelikova et al., Cancer Res., 52: 746-748 (1992); Galland et al., Genomics, 13: 475-478 (1992);
Galland et al., Oncogene, 8: 1233-1240 (1993); and Pajusola et al., Cancer Res., 52: 5738-(1992), all incorporated herein by reference.] Studies showed that, in mouse embryos, a targeted disruption of the Flt4 gene leads to a failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [Dumont et al., Science, 282: 946-949 (1998)]. These studies suggested that Flt4 has an essential role in the development of the embryonic vasculature, before the emergence of the lymphatic vessels. However, additional studies indicated that, during further development, the expression of Flt4 becomes restricted mainly to lymphatic vessels [Kaipainen, et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)].
In humans, there are two isoforms of the Flt4 protein, designated as Flt4s (short, Genbank Accession No. X68203) and F1t41 (long, Genbank Accession Nos. X68203 and 566407, SEQ ID NO: 1). The sequence of these isoforms is largely identical, except for divergence that occurs at the carboxyl terminus of the receptor as a result of alternative mRNA splicing at the 3' end. The C-terminus of the long form contains three tyrosyl residues, and one of them (Y1337 (SEQ ID NO: 2)) serves as an autophosphorylation site in the receptor [Fournier et al., Oncogene, ll: 921-(1995); and Pajusola, et al., Oncogene, 8: 2931-2937 (1993)]. Only the long form is detected in human erythroleukaemia (HEL) and in a megakaryoblastic cell line (the DAMI cells), and the mouse Flt4 gene (Genbank Accession No. L07296) only produces one mRNA transcript, corresponding to F1t41 [Galland et al., Oncogene, 8:
1233-1240 (1993); and Pajusola et al., Cancer Res., 52: 5738-5743 (1992)].
These findings suggest that the long form of Flt4 may be responsible for most of the biological properties of this receptor. The Flt4 protein is glycosylated and proteolytically processed in transfected cells [Pajusola et al., Oncogene, 9:

(1994)]. During this process, the 175 kD form of the receptor matures to a 195 kD
form, which is subsequently cleaved into a 125 kD C-terminal fragment, and a 75 kD
extracellular domain-containing fragment, which are linked by disulphide bonding in the mature receptor.
Two growth factors, named vascular endothelial growth factors C and D (VEGF-C and VEGF-D) due to amino acid sequence similarity to earlier-discovered vascular endothelial growth factor, have been shown to bind and activate the tyrosine phosphorylation of Flt4. [Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-(1998); Joukov et al, EMBOJ., 16: 3898-3911; and Joukov et al., EMBO J., I5:
290-298 (1996)]. Because of Flt4's growth factor binding properties and the fact that Flt4 possesses amino acid sequence similarity to two previously identified VEGF
receptors (Fltl/VEGFR-1 and KDR/VEGFR-2), Flt4 has also been designated VEGFR-3, and these terms are used interchangeably herein.
When VEGF-C was intentionally over-expressed under a basal keratin promoter in transgeruc mice, a hyperplastic lymphatic vessel network in the skin was observed. [Jeltsch et al., Science, 276:1423-1425 (1997).] The results of this study, when combined with the expression pattern of VEGFR-3 in the lymphatic vasculature, suggest that lymphatic growth may be induced by VEGF-C and mediated via VEGFR-3. Notwithstanding the foregoing insights involving one cell surface receptor and the two apparent ligands therefor, little is known about the developmental regulation of the lymphatic system.
Hereditary or primary lymphedema, first described by Milroy in 1892 [Milroy, N. Y. Med. J., 56:505-508 (1892)], is a developmental disorder ofthe lymphatic system which leads to a disabling and disfiguring swelling of the extremities.
Hereditary lymphedema generally shows an autosomal dominant pattern of inheritance with reduced penetrance, variable expression, and variable age-at-onset [Greenlee et al., Lymphology, 26:156-168 (1993)]. Swelling may appear in one or all limbs, varying in degree and distribution. If untreated, such swelling worsens over time. In rare instances, angiosarcoma may develop in affected tissues [Offori et al., Clin. Exp.
Dermatol., 18:174-177 (1993)]. Despite having been described over a century ago, little progress has been made in understanding the mechanisms causing lymphedema.
A long-felt need exists for the identification of the presumed genetic variations that underlie hereditary lymphedema, to permit better informed genetic counseling in affected families, earlier diagnosis and treatment, and the development of more targeted and effective lymphedema therapeutic regimens. In addition, identification of genetic markers and high risk members of lymphedema families facilitates the identification and management of environmental factors that influence the expression and severity of a lymphedema phenotype.
SUMMARY OF THE INVENTION
The present invention provides materials and methods that address one or more of the long-felt needs identified above by identifying a genetic marker that correlates and is posited to have a causative role in the development of hereditary lymphedema. The invention is based in part on the discovery that, in several families with members afflicted with hereditary lymphedema, the lymphedema phenotype correlates with genetic markers localized to chromosome Sq34-q35; and that in at least some such families, a missense mutation in the VEGFR-3 gene (which maps to chromosome Sq34-q35) exists that appears to behave in a loss-of function dominant negative manner to decrease tyrosine kinase signaling of the receptor. In view of the fact that VEGFR-3 acts as a high affinity receptor for vascular endothelial growth factor C (VEGF-C); a growth factor whose effects include modulation of the growth of the lymphatic vascular network, these linkage and biochemical studies provide an important marker for determining a genetic predisposition for lymphedema in healthy individuals; and for diagnosing hereditary lymphedema in symptomatic individuals.
Materials and methods for performing such genetic analyses are considered aspects of the present invention.
Thus, the invention provides genetic screening procedures that entail analyzing a person's genome -- in particular their VEGFR-3 alleles -- to determine whether the individual possesses a genetic characteristic found in other individuals that are considered to be afflicted with, or at risk for, developing hereditary lymphedema.

-S-For example, in one embodiment, the invention provides a method for determining a hereditary lymphedema development potential in a human subject comprising the steps of analyzing the coding sequence of the VEGFR-3 genes from the human subject; and determining hereditary lymphedema development potential in said human subject from the analyzing step.
In another embodiment, the invention provides a method of screening a human subject for an increased risk of developing a lymphatic disorder, comprising the steps of (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the encoded VEGFR-3 amino acid sequence or expression of at least one VEGFR-3 allele; and (b) screening for an increased risk of developing a lymphatic disorder from the presence or absence of said mutation.
By "human subject" is meant any human being, human embryo, or human fetus. It will be apparent that methods of the present invention will be of particular interest to individuals that have themselves been diagnosed with lymphedema or have relatives that have been diagnosed with lymphedema.
By "screening for an increased risk" is meant determination of whether a genetic variation exists in the human subject that correlates with a greater likelihood of developing lymphedema than exists for the human population as a whole, or for a relevant racial or ethnic human sub-population to which the individual belongs. Both positive and negative determinations (i.e., determinations that a genetic predisposition marker is present or is absent) are intended to fall within the scope of screening methods of the invention. In preferred embodiments, the presence of a mutation altering the sequence or expression of at least one Flt4 receptor tyrosine kinase allele in the nucleic acid is correlated with an increased risk of developing a lymphatic disorder, whereas the absence of such a mutation is reported as a negative determination.
By "lymphatic disorder" is meant any clinical condition affecting the lymphatic system, including but not limited to lymphedemas, lymphangiomas, lymphangiosarcomas, lymphangiomatosis, lymphangiectasis, and cystic hygroma.
Preferred embodiments are methods of screening a human subject for an increased risk of developing a lymphedema disorder, i.e., any disorder that physicians would diagnose as lymphedema and that is characterized by swelling associated with lymph accumulation, other than lymphedemas for which non-genetic causes (e.g., parasites, surgery) are known. By way of example, lymphedema disorders include Milroy-Nonne (OMIM 153100) syndrome-early onset lymphedema [Milroy, N. Y. Med. J., 56:505-508 (1892); and Dale, J. Med. Genet., 22: 274-278 (1985)] and lymphedema praecox (Meige syndrome, OMIM 153200)-late onset lymphedema [Lewis et al., J. Ped., 104:641-648 (1984); Holmes et al., Pediatrics 61: 575-579 (1978); and Wheeler et al., Plastic Reconstructive Surg., 67:362-364 (1981)] which generally are described as separate entities, both characterized by dominant inheritance. However, there is confusion in the literature about the separation of these disorders. In Milroy's syndrome, the presence of edema, which is usually more severe in the lower extremities, is seen from birth. Lymphedema praecox presents in a similar fashion but the onset of swelling is usually around puberty. Some cases have been reported to develop in the post-pubertal period. In the particular analyses described herein, the lymphedema families showing linkage to 5q34-q35 show an early onset for most affected individuals, but individuals in these pedigrees have presented during or after puberty.
The "assaying" step of the invention may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well-known techniques such as single-strand conformation polymorphism analysis (SSCP) [Orita et al., Proc Natl. Acad. Sci. USA, 86: 2766-2770 (1989)];
heteroduplex analysis [White et al., Genomics, 12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci.
USA, 80:
1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)];
DNA
sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)];
chemical cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995);
and Roberts et al., Nucl. Acids Res., 25: 3377-3378 (1997)]; restriction fragment length polymorphism analysis; single nucleotide primer extension analysis [Shumaker et al., Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614 (1997)]; 5' nuclease assays [Pease et al., Proc. Natl. Acad. Sci. USA, 91:5022-(1994)]; DNA Microchip analysis [Ramsay, G., Nature Biotechnology, 16: 40-48 (1999); and Chee et al., U.S. Patent No. 5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Patent No. 5,521,065]. [See generally, Schafer and Hawkins, Nature Biotechnology, 16: 33-39 (1998).] All of the foregoing documents are hereby incorporated by reference in their entirety.

_7_ In one preferred embodiment, the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique. [See, e.g., Sanger et al., Proc. Natl. Acad Sci. (USA), 74: 5463-(1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998); U.S. Patent No. 5,202,231; and Science, 260: 1649-1652 (1993) (sequencing by hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992) (sequencing by primer walking); (Douglas et al., Biotechniques, 14: 824-828 (1993) (Direct sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241 (1994);
Maxam and Gilbert, Meth. Enrymol., 65: 499-560 (1977) (chemical termination sequencing), all incorporated herein by reference.] The analysis may entail sequencing of the entire VEGFR-3 gene genomic DNA sequence, or portions thereof; or sequencing of the entire VEGFR-3 coding sequence or portions thereof. In some circumstances, the analysis may involve a determination of whether an individual possesses a particular VEGFR-3 allelic variant, in which case sequencing of only a small portion of nucleic acid -- enough to determine the sequence of a particular codon characterizing the allelic variant -- is su~cient. This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and correlated with heritable lymphedema. More generally, the sequencing may be focused on those portions of the VEGFR-3 sequence that encode a VEGFR-3 kinase domain, since several different and apparently causative mutations in affected individuals that have been identified correspond to residues within an intracellular VEGFR-3 kinase domain. Referring to SEQ ID NOs: 1 and 2, the two kinase domains of human wild type VEGFR-3 correspond to nucleotides to 2848 and 3044 to 3514 of SEQ ID NO: 1, which encode residues 843 to 943 and 1009 to 1165 of SEQ ID NO: 2. Such kinase domains are localized to exons 17-20 and 22-26 in the VEGFR-3 gene, so the sequencing/analysis may be focused on those exons in particular. Molecular modeling suggests that, within these domains, residues 6852, 6854, 6857, K879, E896, H1035, D1037, N1042, D1055, F1056, 61057, E 1084, D 1096, and Rl 159 are of particular importance in comprising or shaping the catalytic pocket of the VEGFR-3 kinase domains, so the sequencing may focus on _$_ these residues (in addition to residues described herein for which mutations have already been identified).
In a related embodiment, the invention provides PCR primers useful for amplifying particular exon sequences of human VEGFR-3 genomic DNA. The Examples below identify preferred primers for amplifying Exon 17, Exon 22, and Exon 24 sequences, where specific missense mutations described herein map. In addition, the Examples below describe the Exon-Intron junctions of human VEGFR-3, which, in combination with the VEGFR-3 cDNA sequence provided herein, permit the manufacture of appropriate oligonucleotide primers for other exons. Any such primers of, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides that are identical or exactly complementary to a human VEGFR-genomic sequence and that includes or is within 50 nucleotides of a VEGFR-3 exon-intron splice site is intended to be within the scope of the invention.
In another embodiment, the assaying step comprises performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences. In a preferred embodiment, the hybridization involves a determination of whether nucleic acid derived from the human subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that correspond identically to a portion of the VEGFR-3 gene sequence, preferably the VEGFR-3 coding sequence set forth in SEQ ID NO: 1, or that correspond identically except for one mismatch.
The hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases. Such hybridization experiments thereby can provide single nucleotide polymorphism sequence information about the nucleic acid from the human subject, by virtue of knowing the sequences of the oligonucleotides used in the experiments.
Several of the techniques outlined above involve an analysis wherein one performs a polynucleotide migration assay, e.g., on a polyacrylamide electrophoresis gel, under denaturing or non-denaturing conditions. Nucleic acid derived from the human subject is subjected to gel electrophoresis, usually adjacent to one or more reference nucleic acids, such as reference VEGFR-3 sequences having a coding sequence identical to all or a portion of SEQ ID NO: 1, or identical except for one known polymorphism. The nucleic acid from the human subject and the reference sequences) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the polynucleotides will show a differential migration pattern, unless they contain identical sequences. [See generally Ausubel et al. (eds.), Current Protocols in Molecular Biology, New York: John Wiley &
Sons, Inc. (1987-1999); and Sambrook et al., (eds.), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989), both incorporated herein by reference in their entirety.]
In the context of assaying, the term "nucleic acid of a human subject" is intended to include nucleic acid obtained directly from the human subject (e.g., DNA
or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample); and also nucleic acid derived from nucleic acid obtained directly from the human subject. By way of non-limiting examples, well known procedures exist for creating cDNA that is complementary to RNA derived from a biological sample from a human subject, and for amplifying (e.g., via polymerase chain reaction (PCR)) DNA or RNA derived from a biological sample obtained from a human subject. Any such derived polynucleotide which retains relevant nucleotide sequence information of the human subject's own DNA/RNA is intended to fall within the definition of "nucleic acid of a human subject" for the purposes of the present invention.
In the context of assaying, the term "mutation" includes addition, deletion, and/or substitution of one or more nucleotides in the VEGFR-3 gene sequence. The invention is demonstrated by way of non-limiting examples set forth below that identify several mutations in VEGFR-3, including single nucleotide polymorphisms that introduce missense mutations into the YEGFR-3 coding sequence (as compared to the VEGFR-3 cDNA sequence set forth in SEQ ID NO: 1) and other polymorphisms that occur in introns and that are identifiable via sequencing, restriction fragment length polymorphism, or other techniques. Example 2 provides an assay to determine whether a VEGFR-3 mutation inhibits VEGFR-3 signaling. Additional assays to study both ligand binding and signaling activities of VEGFR-3 are disclosed, e.g., in U.S. Patent No. 5,776,755 and International Patent Publication No. WO
98/33917, published 06 August 1998, both ofwhich are incorporated herein by reference in their entirety. Evidence that a VEGFR-3 mutation inhibits VEGFR-3 signaling is evidence that the mutation may have a causative role in lymphedema phenotype. However, even mutations that have no apparent causative role may serve as useful markers for heritable lymphedema, provided that the appearance of the mutation correlates reliably with the appearance of lymphedema.
In a related embodiment, the invention provides a method of screening for a VEGFR-3 hereditary lymphedema genotype in a human subject, comprising the steps of (a) providing a biological sample comprising nucleic acid from a human subject; (b) analyzing the nucleic acid for the presence of a mutation or mutations in a VEGFR-3 allele in the nucleic acid of the human subject; (c) determining a genotype from said analyzing step; and (d) correlating the presence of a mutation in a VEGFR-3 allele with a hereditary lymphedema genotype. In a preferred embodiment, the biological sample is a cell sample containing human cells that contain genomic DNA of the human subject.
Although more time consuming and expensive than methods involving nucleic acid analysis, the invention also may be practiced by assaying protein of a human subject to determine the presence or absence of an amino acid sequence variation in VEGFR-3 protein from the human subject. Such protein analyses may be performed, e.g., by fragmenting VEGFR-3 protein via chemical or enzymatic methods and sequencing the resultant peptides; or by Western analyses using an antibody having specificity for a particular allelic variant of VEGFR-3.
The invention also provides materials that are useful for performing methods of the invention. For example, the present invention provides oligonucleotides useful as probes in the many analyzing techniques described above.
In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that is identical, or exactly complementary, to a portion of a human VEGFR-3 gene sequence, or that is identical or exactly complementary except for one nucleotide substitution. In a preferred embodiment, the oligonucleotides have a sequence that corresponds in the foregoing manner to a human VEGFR-3 coding sequence, and in particular, the VEGFR-3 coding sequence set forth in SEQ B7 NO: 1. In one variation, an oligonucleotide probe of the invention is purified and isolated.
In another variation, the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore, or fluorophore. In yet another variation, the probe is covalently attached to a solid support. [See generally Ausubel et al. And Sambrook et al., supra.]
In preferred embodiments, the invention comprises an oligonucleotide probe useful for detecting one or more of several mutations that have been characterized herein in affected individuals, including:
(1) a missense mutation at nucleotide 3360 of SEQ 117 NO: 1, causing a proline to leucine change at residue 1114 in SEQ ID NO: 2;
(2) a missense mutation at nucleotide 2588 of SEQ ID NO: 1, causing a glycine to arginine change at residue 857 in SEQ ID NO: 2;
(3) a missense mutation at nucleotide 3141 of SEQ ID NO: 1, causing an arginine to proline change at residue 1041 in SEQ ID NO: 2;
(4) a missense mutation at nucleotide 3150 in SEQ ID NO: 1, causing a leucine to proline change at residue 1044 in SEQ ID NO: 2; and (5) a missense mutation at nucleotide 3164 of SEQ 117 NO: 1, causing an aspartic acid to asparagine change at residue 1049 in SEQ ID NO: 2.
For example, the invention provides oligonucleotides comprising anywhere from 6 to 50 nucleotides that have a sequence that is identical to, or exactly complementary to, a portion of the human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, except for a nucleotide substitution corresponding to nucleotide 3360 of SEQ 117 NO:
1.
Such oligonucleotides may be generically described by the formula X"YZm or its complement; where n and m are integers from 0 to 49; where 5 <_ (n + m) <_ 49;
where X" is a stretch of n nucleotides identical to a first portion of SEQ ID NO: 1 and Zm is a stretch of m nucleotides identical to a second portion of SEQ ID NO: 1, wherein the first and second portions are separated in SEQ ID NO: 1 by one nucleotide; and wherein Y represents a nucleotide other than the nucleotide that separates the first and second portions of SEQ 117 NO: 1. For example, where XI, represents 0 to 49 nucleotides immediately upstream (S') of nucleotide 3360 of SEQ B7 NO: 1 and Zm represents 0 to 49 nucleotides immediately downstream (3') of nucleotide 3360 of SEQ ID NO: 1, Y represents a nucleotide other than cytosine, since a cytosine nucleotide is found at position 3360 of SEQ ID NO: 1. In a preferred embodiment, Y
is a thymine nucleotide. Similar examples are contemplated for the other specific mutations identified immediately above.

In a related embodiment, the invention provides a kit comprising at least two such oligonucleotide probes. Preferably, the two or more probes are provided in separate containers, or attached to separate solid supports, or attached separately to the same solid support, e.g., on a DNA microchip.
In still another related embodiment, the invention provides an array of oligonucleotide probes immobilized on a solid support, the array having at least 4 probes, preferably at least 100 probes, and preferably up to 100,000, 10,000, or 1000 probes, wherein each probe occupies a separate known site in the array. In a preferred embodiment, the array includes probe sets comprising two to four probes, wherein one probe is exactly identical or exactly complementary to a human VEGFR-3 coding sequence, and the other one to three members of the set are exactly identical to the first member , but for at least one different nucleotide, which different nucleotide is located in the same position in each of the one to three additional set members. In one preferred embodiment, the array comprises several such sets of probes, wherein the sets correspond to different segments of the human VEGFR-3 gene sequence. In a highly preferred embodiment, the array comprises enough sets of oligonucleotides of length N to correspond to every particular N-mer sequence of the VEGFR-3 gene, where N is preferably 6 to 25 and more preferably 9 to 20. Materials and methods for making such probes are known in the art and are described, for example, in U.S. Patent Nos. 5,837,832, 5,202,231, 5,002,867, and 5,143,854.
Moreover, the discoveries which underlie the present invention identify a target for therapeutic intervention in cases of hereditary lymphedema. The causative mutations in the families that have been studied in greatest detail are mutations that appear to result in VEGFR-3 signaling that is reduced in heterozygous affected individuals, but not completely eliminated. This data supports a therapeutic indication for administration of agents, such as VEGFR-3 ligand polypeptides, that will induce VEGFR-3 signaling in the lymphatic endothelia of affected individuals to effect improvement in the structure and function of the lymphatic vasculature of such individuals. In addition, therapeutic gene therapy, to replace defective VEGFR-alleles or increase production of VEGFR-3 ligand polypeptides in vivo, is envisioned as an aspect of the invention.
Thus, in yet another aspect, the invention provides a therapeutic or prophylactic method of treatment for lymphedema, comprising the step of administering to a mammalian subject in need of therapeutic or prophylactic treatment for lymphedema a composition comprising a compound effective to induce intracellular signaling of VEGFR-3 in lymphatic endothelial cells that express said receptor. In a preferred embodiment, the compound comprises a polypeptide ligand for VEGFR-3, or a polynucleotide encoding such a ligand, wherein the polynucleotide is administered in a form that results in transcription and translation of the polynucleotide in the mammalian subject to produce the ligand in vivo. In another preferred embodiment, the compound comprises any small molecule that is capable of binding to the VEGFR-3 receptor extracellular or intracellular domain and inducing intracellular signaling.
For example, the invention provides a therapeutic or prophylactic method of treatment for lymphedema, comprising the step of administering to a mammalian subject in need of therapeutic or prophylactic treatment for lymphedema a composition comprising a polynucleotide, the polynucleotide comprising a nucleotide sequence that encodes a vascular endothelial growth factor C (VEGF-C) polypeptide.
In a preferred embodiment, the subject is a human subject.
While it is contemplated that the VEGF-C polynucleotide could be administered purely as a prophylactic treatment to prevent lymphedema in subjects at risk for developing lymphedema, it is contemplated in a preferred embodiment that the polynucleotide be administered to subjects afflicted with lymphedema, for the purpose of ameliorating its symptoms (e.g., swelling due to the accumulation of lymph). The polynucleotide is included in the composition in an amount and in a form effective to promote expression of a VEGF-C polypeptide in or near the lymphatic endothelia of the mammalian subject, to stimulate VEGFR-3 signaling in the lymphatic endothelia of the subject.
In a preferred embodiment, the mammalian subject is a human subject.
Practice of methods of the invention in other mammalian subjects, especially mammals that are conventionally used as models for demonstrating therapeutic efFlcacy in humans (e.g., primate, porcine, canine, equine, murine, or rabbit animals), also is contemplated. Several potential animal models for hereditary lymphedema have been described in the literature. [See, e.g., Lyon et al., Mouse News Lett. 71: 26 (1984), Mouse News Lett. 74: 96 (1986), and Genetic variants and strains of the laboratory mouse, 2nd ed., New York: Oxford University Press (1989), p. 70 (Chylous ascites mouse); Dumont et al., Science, 282: 946-949 (1998) (heterozygous VEGFR-3 knockout mouse); Patterson et al., "Hereditary Lymphedema," Comparative Pathology Bulletin, 3: 2 (1971) (canine hereditary lymphedema model); van der Putte, "Congenital Hereditary Lymphedema in the Pig," Lympho, ll: 1-9 (1978); and Campbell-Beggs et al., "Chyloabdomen in a neonatal foal," Veterinary Record, 137:
96-98 (1995).] Those models which are determined to have analogous mutations to the VEGFR-3 gene, such as the Chylous ascetei (Chy) mouse, are preferred. The present inventors have analyzed the VEGFR-3 genes of the Chy mouse and determined that affected mice contain a missense mutation that results in a phenylalanine (rather than an isoleucine) in the VEGFR-3 sequence at a position corresponding to the isoleucine at position 1053 of SEQ 1D NO: 2. This mutation maps to the catalytic pocket region of the tyrosine kinase domain of the VEGFR-3 protein, and may represent a viable model for identical mutations in human (if discovered) or other mutations in humans that similarly affect the tyrosine kinase catalytic domain. The Chy mouse has peripheral swelling (oedema) after birth and chyle ascites. In another embodiment, "knock in" homologous recombination genetic engineering strategies are used to create an animal model (e.g., a mouse model) having a VEGFR-3 allelic variation analogous to the human variations described herein. [See, e.g., Partanen et al., Genes & Development, 12: 2332-2344 (1998) (gene targeting to introduce mutations into a receptor protein (FGFR-1) in mice).] Such mice can also be bread to the heterozygous VEGFR-3 knockout mice or Chy mice described above to further modify the phenotypic severity of the lymphedema disease.
For the practice of methods of the invention, the term "VEGF-C
polypeptide" is intended to include any polypeptide that has a VEGF-C or VEGF-C
analog amino acid sequence (as defined elsewhere herein in greater detail) and that is able to bind the VEGFR-3 extracellular domain and stimulate VEGFR-3 signaling in vivo. The term "VEGF-C polynucleotide" is intended to include any polynucleotide (e.g., DNA or RNA, single- or double-stranded) comprising a nucleotide sequence that encodes a VEGF-C polypeptide. Due to the well-known degeneracy of the genetic code, multiple VEGF-C polynucleotide sequences exist that encode any selected VEGF-C polypeptide. Preferred VEGF-C polynucleotides, polypeptides, and VEGF-C variants and analogs for use in this invention are disclosed in International Patent Application No. PCTlUS98/01973, published as WO 98/33917, incorporated herein by reference in its entirety.

For treatment of humans, VEGF-C polypeptides with an amino acid sequence of a human VEGF-C are highly preferred, and polynucleotides comprising a nucleotide sequence of a human VEGF-C cDNA are highly preferred. By "human VEGF-C" is meant a polypeptide corresponding to a naturally occurring protein (prepro-protein, partially-processed protein, or fully-processed mature protein) encoded by any allele of the human VEGF-C gene, or a polypeptide comprising a biologically active fragment of a naturally-occurring mature protein. By way of example, a human VEGF-C comprises a continuous portion of the amino acid sequence set forth in SEQ ID NO: 4 sufficient to permit the polypeptide to bind and stimulate VEGFR-3 phosphorylation in cells that express such receptors. A
polypeptide comprising amino acids 131-211 of SEQ ID NO: 4 is specifically contemplated. For example, polypeptides having an amino acid sequence comprising a continuous portion of SEQ ID NO: 4, the continuous portion having, as its amino terminus, an amino acid selected from the group consisting of positions 30-131 of SEQ
ID NO: 4, and having, as its carboxyl terminus, an amino acid selected from the group consisting of positions 211-419 of SEQ ID NO: 4 are contemplated. An amino terminus selected from the group consisting of positions 102-131 of SEQ ID NO:
4 is preferred, and an amino terminus selected from the group consisting of positions 103-113 of SEQ ID NO: 4 is highly preferred. Likewise, a carboxyl terminus selected from the group consisting of positions 211-227 of SEQ ID NO: 4 is preferred.
As stated above, the term "human VEGF-C" also is intended to encompass polypeptides encoded by allelic variants of the human VEGF-C characterized by the sequences set forth in SEQ ID NOs: 3 & 4.
Moreover, since the therapeutic VEGF-C is to be administered as recombinant VEGF-C or indirectly via somatic gene therapy, it is within the skill in the art to make and use analogs of human VEGF-C (and polynucleotides that encode such analogs) wherein one or more amino acids have been added, deleted, or replaced with other amino acids, especially with conservative replacements, and wherein the VEGFR-3-stimulatory biological activity has been retained. Analogs that retain VEGFR-3-stimulatory VEGF-C biological activity are contemplated as VEGF-C
polypeptides for use in the present invention. In a preferred embodiment, analogs having l, 2, 3, 4, S, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 such modifications and that retain VEGFR-3-stimulatory VEGF-C biological activity are contemplated as VEGF-C polypeptides for use in the present invention.
Analogs having a deletion of or substitution for the cysteine residue at position 156 of SEQ ID NO: 4 and that retain VEGFR-3 stimulatory activity but have reduced activity toward the receptor VEGFR-2, which is expressed in blood vessels, are specifically contemplated. See WO 98/33917. Polynucleotides encoding such analogs are generated using conventional PCR, site-directed mutagenesis, and chemical synthesis techniques.
Also contemplated as VEGF-C polypeptides are non-human mammalian or avian VEGF-C polypeptides and polynucleotides. By "mammalian VEGF-C" is meant a polypeptide corresponding to a naturally occurring protein (prepro-protein, partially-processed protein, or fully-processed mature protein) encoded by any allele of a VEGF-C gene of any mammal, or a polypeptide comprising a biologically active fragment of a mature protein. The term "mammalian VEGF-C polypeptide" is intended to include analogs of mammalian VEGF-C's that possess the in vivo VEGFR-3-stimulatory effects of the mammalian VEGF-C.
Irrespective of which encoded VEGF-C polypeptide is chosen, any VEGF-C polynucleotide gene therapy pharmaceutical encoding it preferably comprises a nucleotide sequence encoding a secretory signal peptide fused in-frame with the VEGF-C polypeptide sequence. The secretory signal peptide directs secretion of the VEGF-C polypeptide by the cells that express the polynucleotide, and is cleaved by the cell from the secreted VEGF-C polypeptide. For example, the VEGF-C
polynucleotide could encode the complete prepro-VEGF-C sequence set forth in SEQ
117 NO: 4; or could encode the VEGF-C signal peptide fused in-frame to a sequence encoding a fully-processed VEGF-C (e.g., amino acids 103-227 of SEQ ID NO: 4) or VEGF-C analog. Moreover, there is no requirement that the signal peptide be derived from VEGF-C. The signal peptide sequence can be that of another secreted protein, or can be a completely synthetic signal sequence effective to direct secretion in cells of the mammalian subject.
In one embodiment, the VEGF-C polynucleotide of the invention comprises a nucleotide sequence that will hybridize to a polynucleotide that is complementary to the human VEGF-C cDNA sequence specified in SEQ ID NO: 3 under the following exemplary stringent hybridization conditions:
hybridization at 42°C in 50% formamide, SX SSC, 20 mM Na~P04, pH 6.8; and washing in 1X
SSC at SS°C for 30 minutes; and wherein the nucleotide sequence encodes a polypeptide that binds and stimulates human VEGFR-3. It is understood that variation in these exemplary conditions occur based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for S determining appropriate hybridization conditions. [See Sambrook et al., Molecular Cloning: A Laboratory Manual (Second ed., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1989) ~~ 9.47-9.51.]
In preferred embodiments, the VEGF-C polynucleotide further comprises additional sequences to facilitate the VEGF-C gene therapy. In one embodiment, a "naked" VEGF-C transgene (i.e., a transgene without a viral, liposomal, or other vector to facilitate transfection) is employed for gene therapy. In this embodiment, the VEGF-C polynucleotide preferably comprises a suitable promoter and/or enhancer sequence (e.g., cytomegalovirus promoter/enhancer [Lehner et al., J.
Clirz Microbiol., 29:2494-2502 (1991); Boshart et al., Cell, 41: 521-530 (1985)];
Rous sarcoma virus promoter [Davis et al., Hum. Gene Ther., 4:151 (1993)]; Tie promoter [Korhonen et al., Blood, 8G(S): 1828-1835 (1995)]; or simian virus 40 promoter) for expression in the target mammalian cells, the promoter being operatively linked upstream (i.e., S') of the VEGF-C coding sequence. The VEGF-C
polynucleotide also preferably further includes a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (i.e., 3') of the VEGF-C coding sequence. The polynucleotide may further optionally comprise sequences whose only intended function is to facilitate large-scale production of the vector, e.g., in bacteria, such as a bacterial origin of replication and a sequence encoding a selectable marker. However, in a preferred embodiment, such extraneous sequences are at least partially cleaved off prior to administration to humans according to methods of the invention. One can manufacture and administer such polynucleotides to achieve successful gene therapy using procedures that have been described in the literature for other transgenes.
See, e.g., Isner et al., Circulation, 91: 2687-2692 (1995); and Isner et al., Human Gene Therapy, 7: 989-1011 (1996); incorporated herein by reference in the entirety.
Any suitable vector may be used to introduce the VEGF-C transgene into the host. Exemplary vectors that have been described in the literature include replication-deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al., .I. Virol., 72(I): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46.]; adeno-associated viral vectors [Gnatenko et al., J.
Investig. Med., 45: 87-98 (1997)]; adenoviral vectors [See, e.g., U.S. Patent No.
5,792,453; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992);
Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630 (1992); and Rosenfeld et al., Cell, 68: 143-155 (1992)]; Lipofectin-mediated gene transfer (BRL); liposomal vectors [See, e.g., U.S. Patent No. 5,631,237 (Liposomes comprising Sendai virus proteins)] ;
and combinations thereof. All of the foregoing documents are incorporated herein by reference in the entirety. Replication-deficient adenoviral vectors constitute a preferred embodiment.
In embodiments employing a viral vector, preferred polynucleotides still include a suitable promoter and polyadenylation sequence as described above.
Moreover, it will be readily apparent that, in these embodiments, the polynucleotide further includes vector polynucleotide sequences (e.g., adenoviral polynucleotide sequences) operably connected to the sequence encoding a VEGF-C polypeptide.
Thus, in one embodiment the composition to be administered comprises a vector, wherein the vector comprises the VEGF-C polynucleotide. In a preferred embodiment, the vector is an adenovirus vector. In a highly preferred embodiment, the adenovirus vector is replication-deficient, i.e., it cannot replicate in the mammalian subject due to deletion of essential viral-replication sequences from the adenoviral genome. For example, the inventors contemplate a method wherein the vector comprises a replication-deficient adenovirus, the adenovirus comprising the VEGF-C
polynucleotide operably connected to a promoter and flanked on either end by adenoviral polynucleotide sequences.
The composition to be administered according to methods of the invention preferably comprises (in addition to the polynucleotide or vector) a pharmaceutically-acceptable carrier solution such as water, saline, phosphate-bui~ered saline, glucose, or other Garners conventionally used to deliver therapeutics intravascularly. Multi-gene therapy is also contemplated, in which case the composition optionally comprises both the VEGF-C polynucleotide/vector and another polynucleotide/vector. As described in greater detail below, a VEGF-D
transgene is a preferred candidate for co-administration with the VEGF-C transgene.

The "administering" that is performed according to the present method may be performed using any medically-accepted means for introducing a therapeutic directly or indirectly into a mammalian subject to reach the lymph or the lymphatic system, including but not limited to injections; oral ingestion; intranasal or topical administration; and the like. In a preferred embodiment, administration of the composition comprising the VEGF-C polynucleotide is performed intravascularly, such as by intravenous or intra-arterial injection, or by subcutaneous injection or local depot administration. In a highly preferred embodiment, the composition is administered locally, e.g., to the site of swelling.
In still another variation, endothelial cells or endothelial progenitor cells are transfected ex vivo with a wild type VEGFR-3 transgene, and the transfected cells are administered to the mammalian subject.
In another aspect, the invention provides a therapeutic or prophylactic method of treating for lymphedema, comprising the step of administering to a mammalian subject in need of treatment for lymphedema a composition comprising a VEGF-C polypeptide, in an amount effective to treat or prevent swelling associated with lymphedema. Administration via one or more intravenous or subcutaneous injections is contemplated. Co-administration of VEGF-C polynucleotides and VEGF-C polypeptides is also contemplated.
In yet another embodiment, the invention provides the use of a VEGF-C polynucleotide or VEGF-C polypeptide for the manufacture of a medicament for the treatment or prevention of lymphedema.
In still another embodiment, the invention provides a therapeutic or prophylactic method of treatment for lymphedema, comprising the step of administering to a mammalian subject in need of therapeutic or prophylactic treatment of lymphedema a composition comprising a polynucleotide, the polynucleotide comprising a nucleotide sequence that encodes a vascular endothelial growth factor D
(VEGF-D) polypeptide. Such methods are practiced essentially as described herein with respect to VEGF-C-encoding polynucleotides, except that polynucleotides encoding VEGF-D are employed. A detailed description of the human VEGF-D gene and protein are provided in Achen, et al., Proc. Nat'l Acad. Sci. U.S.A., 95(2): 548-553 (1998); International Patent Publication No. WO 98/07832, published 26 February 1998; and in Genbank Accession No. AJ000185, all incorporated herein by reference.

A cDNA and deduced amino acid sequence for prepro-VEGF-D is set forth herein in SEQ ID NOs: 5 and 6. Of course, due to the well-known degeneracy of the genetic code, multiple VEGF-D encoding polynucleotide sequence exist, any of which may be employed according to the methods taught herein.
As described herein in detail with respect to VEGF-C, the use of polynucleotides that encode VEGF-D fragments, VEGF-D analogs, VEGF-D allelic and interspecies variants, and the like which possess in vivo stimulatory effects of human VEGF-D are all contemplated as being encompassed by the present invention.
In yet another embodiment, the invention provides a therapeutic or prophylactic method of treatment for lymphedema, comprising the step of administering to a mammalian subject in need of treatment for lymphedema a composition comprising a VEGF-D polypeptide, in an amount effective to treat or prevent swelling associated with lymphedema. Administration via one or more intravenous or subcutaneous injections is contemplated.
The VEGFR-3 allelic variant polynucleotides and polypeptides described herein that were discovered and characterized by the present inventors are themselves considered aspects of the invention. Such polynucleotides and polypeptides are useful, for example, in screening assays (e.g., cell-based assays or assays involving transgenic mice that express the polynucleotide in lieu of a native VEGF-3 allele) to study the biological activities of VEGFR-3 variant alleles and identify compounds that are capable of modulating that activity, e.g., to identify therapeutic candidates for treatment of lymphedema. Such screening assays are also considered aspects of the invention.
The polypeptides of the invention are intended to include complete VEGFR-3 polypeptides with signal peptide (e.g., approximately residues 1 to 20 of SEQ ID NO: 2), mature VEGFR-3 polypeptides lacking any signal peptide, and recombinant variants wherein a foreign or synthetic signal peptide has been fused to the mature VEGFR-3 polypeptide. Polynucleotides of the invention include all polynucleotides that encode all such polypeptides. It will be understood that for essentially any polypeptide, many polynucleotides can be constructed that encode the polypeptide by virtue of the well known degeneracy of the genetic code. All such polynucleotides are intended as aspects of the invention.

Thus, in yet another aspect, the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a human VEGFR-3 protein variant, wherein said polynucleotide is capable of hybridizing to the complement of SEQ m NO: 1 under stringent hybridization conditions, and wherein the encoded VEGFR-3 protein variant has an amino acid sequence that differs at position 1114, 857, 1041, 1044 or 1049 from the amino acid sequence set forth in SEQ ID NO:
1.
Exemplary conditions are as follows: hybridization at 42°C in 50%
formamide, SX
SSC, 20 mM Na~P04, pH 6.8; and washing in 0.2X SSC at 55 °C. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining appropriate hybridization conditions. [See Sambrook et al. ( 1989), supra, ~ ~ 9.47-9. S 1. ]
In a related embodiment, the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a VEGFR-3 protein of a human that is affected with heritable lymphedema or other lymphatic disorder;
wherein the polynucleotide is capable of hybridizing to the complement of SEQ 117 NO:

under stringent hybridization conditions, and wherein the encoded polynucleotide has an amino acid sequence that differs from SEQ ID NO: 1 at at least one codon.
It will be understood that conventional recombinant techniques can be used to isolate such polynucleotides from individuals affected with heritable lymphedema or their relatives.
The wildtype VEGFR-3 cDNA sequence set forth in SEQ ID NO: 1 (or its complement, or fragments thereof) is used as a probe to identify and isolate sequences from nucleic acid derived from the individuals. Alternatively, PCR
amplification primers based on the wildtype VEGFR-3 sequence are generated and used to amplify either VEGFR-3 genomic DNA or VEGFR-3 mRNA from the human subject. The resultant amplified genomic DNA or cDNA is sequenced to determine the variations that characterize the VEGFR-3 lymphedema allele of the individual.
Preferred VEGFR-3 lymphedema alleles include, but are not limited to the P1114L, G857R, R1041P, L1044P and D1049N alleles described in detail herein.
In addition, the invention provides vectors that comprise the polynucleotides of the invention. Such vectors are useful for amplifying and expressing the VEGFR-3 proteins encoded by the polynucleotides, and for creating recombinant host cells and/or transgenic animals that express the polynucleotides. The invention further provides a host cell transformed or transfected with polynucleotides (including vectors) of the invention. In a preferred embodiment, the host cell expresses the encoded VEGFR-3 protein on its surface. Such host cells are useful in cell-based screening assays for identifying modulators that stimulate or inhibit signaling of the encoded VEGFR-3. Modulators that stimulate VEGFR-3 signaling have utility as therapeutics to treat lymphedemas, whereas modulators that are inhibitory have utility for treating hyperplastic lymphatic conditions mediated by the allelic variant VEGFR-3.
In a preferred embodiment, host cells of the invention are co-transfected with both a wildtype and an allelic variant VEGFR-3 polynucleotide, such that the cells express both receptor types on their surface. Such host cells are preferred for simulating a heterozygous VEGFR-3 genotype of many individuals afl;'ected with lymphedema.
In yet another aspect, the invention provides a transgenic mammal, e.g., mouse, characterized by a non-native VEGFR-3 allele that has been introduced into the mouse, and the transgenic progeny thereof. Preferred allelic variants include allelic variants that correlate with hereditary lymphedema in human subjects, such as an allelic variant wherein a P1114L, G857R, R1041P, L1044P or D1049N missense mutation has been introduced into the murine VEGFR-3 gene, or wherein the human P
1114L, G857R, R1041P, L1044P or D1049N allelic variant has been substituted for a murine VEGFR-3 allele. Such mice are produced using standard methods. [See, e.g., Hogan et al. (eds.), Manipulating the Mouse Embryo, Cold Spring Harbor, New York:
Cold Spring Harbor Laboratory (1986).] The introduction of the human-like mutations into non-human sequences is readily achieved with standard techniques, such as site-directed mutagenesis. The determination of which residues in a non-human sequence to alter to mimic the foregoing human mutations is routine since the foregoing mutations all occur in regions of the VEGFR-3 sequence that contain residues that are highly conserved between species. See Figs. 3A-3B.
In yet another aspect, the invention provides assays for identifying modulators of VEGFR-3 signaling, particularly modulators of the signaling of allelic variants of VEGFR-3 that correlate with lymphatic disorders such as heritable lymphedema. For example, the invention provides a method for identifying a modulator of intracellular VEGFR-3 signaling, comprising the steps o~
contacting a cell expressing at least one mutant mammalian VEGFR-3 polypeptide in the presence and in the absence of a putative modulator compound; b) detecting VEGFR-3 signaling in the cell; and c) identifying a putative modulator compound in view of decreased or increased signaling in the presence of the putative modulator, as compared to signaling in the absence of the putative modulator.
By "mutant mammalian VEGFR-3 polypeptide" is meant a VEGFR-3 polypeptide that varies from a wildtype mammalian VEGFR-3 polypeptide (e.g., by virtue of one or more amino acid additions, deletions, or substitutions), wherein the variation is reflective of a naturally occurring variation that has been correlated with a lymphatic disorder, such as lymphedema. By way of example, the previously described substitution variations of human VEGFR-3, such as P1114L, have been correlated with heritable lymphedema. Any of the human allelic variants described above, or analogous human allelic variants having a different substitution at the indicated amino acid positions, or a non-human VEGFR-3 into which a mutation at the position corresponding to any of the described positions has been introduced are all examples of mutant mammalian VEGFR-3 polypeptides.
The detecting step can entail the detection of any parameter indicative of VEGFR-3 signaling. For example, the detecting step can entail a measurement of VEGFR-3 autophosphorylation, or a measurement of VEGFR-3-mediated cell growth, or a measurement of any step in the VEGFR-3 signaling cascade between VEGFR-3 autophosphorylation and cell growth.
In a preferred embodiment, the method is practiced with a cell that expresses the mutant mammalian VEGFR-3 polypeptide and a wildtype mammalian VEGFR-3 polypeptide. Such cells are thought to better mimic the conditions in heterozygous individuals suffering from a VEGFR-3-mediated lymphatic disorder.
In a highly preferred embodiment, the mutant and wildtype VEGFR-3 polypeptides are human. In the preferred embodiments, the mutant VEGFR-3 polypeptide comprises a leucine amino acid at the position corresponding to position 1114 of SEQ ID
NO: 2;
an arginine at the position corresponding to position 857 of SEQ ID NO: 2; a proline amino acid at the position corresponding to position 1041 of SEQ 117 NO: 2; a proline amino acid at the position corresponding to position 1044 of SEQ m NO: 2; or an asparagine at the position corresponding to position 1049 of SEQ ID NO: 2.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the drawing and detailed description, and all such features are intended as aspects of the invention.

Likewise, features of the invention described herein can be re-combined into additional embodiments that are also intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although the applicants) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicants) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-1F depict pedigrees of six hereditary lymphedema families (Families 101, 106, 111, 135, 105, and 127, respectively) informative for linkage.
Filled symbols represent individuals with clinically documented lymphedema.
Crossed symbols represent individuals with an ambiguous phenotype. An ambiguous phenotype is defined as self reported swelling of the limbs with no known cause, without a clinical diagnosis of lymphedema. Individuals of ambiguous phenotype were coded as disease status unknown for the linkage analysis. The proband in each family is indicated by an arrow.
Figure 2 is a graph summarizing VITESSE analysis of lymphedema families with markers localized to chromosome Sq34-q35. In the graph, filled circles represent analyses for Families 101, 105, 106, and 111; open boxes represent analyses for Families 101, 106, and 111; open circles represent the VEGFR-3 gene; and open triangles represent Family 135. The one LOD confidence interval lies completely within the interval flanked by markers DSS1353 and DSS408 and overlaps the most likely location of Flt4 (VEGFR-3). Linkage is excluded for the entire region for family 135.
Figure 3A-3B depict an alignment of portions of the human (top line, SEQ ID NO: 2) and murine (bottom line, GenBank Acc. No. P35917, SEQ ID NO:
19) VEGFR-3 amino acid sequences to demonstrate similarity. Identical residues are marked with a line, and highly conserved and less conserved differences are marked with two dots or a single dot, respectively. The location of various mutations that have been observed to correlate with a heritable lymphedema phenotype are indicated immediately beneath the aligned sequences.
DETAILED DESCRIPTION OF THE INVENTION
Certain therapeutic aspects of the present invention involve the administration of Vascular Endothelial Growth Factor C or D polynucleotides and polypeptides. The growth factor VEGF-C, as well as native human, non-human mammalian, and avian polynucleotide sequences encoding VEGF-C, and VEGF-C
variants and analogs, have been described in detail in International Patent Application Number PCT/LTS98/01973, filed 02 February 1998 and published on 06 August 1998 as International Publication Number WO 98/33917; in Joukov et al., J. Biol.
Chem., 273(12): 6599-6602 (1998); and in Joukov et al., EMBO J., 16(13): 3898-3911 (1997), all of which are incorporated herein by reference in the entirety. As explained therein in detail, human VEGF-C is initially produced in human cells as a prepro-VEGF-C polypeptide of 419 amino acids. A cDNA and deduced amino acid sequence for human prepro-VEGF-C are set forth in SEQ 117 NOs: 3 and 4, respectively, and a cDNA encoding human VEGF-C has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 (USA), pursuant to the provisions of the Budapest Treaty (Deposit date of 24 July 1995 and ATCC Accession Number 97231). VEGF-C sequences from other species have also been reported. See Genbank Accession Nos. MMU73620 (Mus musculus); and CCY15837 (Coturnix coturnix) for example, incorporated herein by reference.
The prepro-VEGF-C polypeptide is processed in multiple stages to produce a mature and most active VEGF-C polypeptide of about 21-23 kD (as assessed by SDS-PAGE under reducing conditions). Such processing includes cleavage of a signal peptide (SEQ 117 NO: 4, residues 1-31); cleavage of a carboxyl-terminal peptide (corresponding approximately to amino acids 228-419 of SEQ ID
NO: 4 and having a pattern of spaced cysteine residues reminiscent of a Balbiani ring 3 protein (BR3P) sequence [Dignam et al., Gene, 88:133-40 (1990); Paulsson et al., J.
Mol. Biol., 211:331-49 (1990)]) to produce a partially-processed form of about kD; and cleavage (apparently extracellularly) of an amino-terminal peptide (corresponding approximately to amino acids 32-103 of SEQ 1D NO: 4) to produce a fully-processed mature form of about 21-23 kD. Experimental evidence demonstrates that partially-processed forms of VEGF-C (e.g., the 29 kD form) are able to bind the VEGFR-3 receptor, whereas high affinity binding to VEGFR-2 occurs only with the fully processed forms of VEGF-C.
Moreover, it has been demonstrated that amino acids 103-227 of SEQ
ID NO: 4 are not all critical for maintaining VEGF-C functions. A polypeptide consisting of amino acids 113-213 (and lacking residues 103-112 and 214-227) of SEQ ID NO: 2 retains the ability to bind and stimulate VEGFR-3, and it is expected that a polypeptide spanning from about residue 131 to about residue 211 will retain VEGF-C biological activity. The cysteine residue at position 156 has been shown to be important for VEGFR-2 binding ability. However, VEGF-C ~Cls6 polypeptides (i.e., analogs that lack this cysteine due to deletion or substitution) remain potent activators of VEGFR-3, and are therefore considered to be among the preferred candidates for treatment of lymphedema. (It has been shown that a VEGF-C C

serine substitution analog promotes lymphatic growth when over-expressed in the skin of transgenic mice behind the K14 promotee, in a manner analogous to what was described in Jeltsch et al., Science, 276:1423 (1997), incorporated herein by reference.) The cysteine at position 165 of SEQ B7 NO: 4 is essential for binding to either receptor, whereas analogs lacking the cysteines at positions 83 or 137 compete with native VEGF-C for binding with both receptors and are able to stimulate both receptors.
An alignment of human VEGF-C with VEGF-C from other species (performed using any generally accepted alignment algorithm) suggests additional residues wherein modifications can be introduced (e.g., insertions, substitutions, and/or deletions) without destroying VEGF-C biological activity. Any position at which aligned VEGF-C polypeptides of two or more species have different amino acids, especially different amino acids with side chains of different chemical character, is a likely position susceptible to modification without concomitant elimination of function.
An exemplary alignment of human, murine, and quail VEGF-C is set forth in Figure 5 of PCT/US98/01973.
Apart from the foregoing considerations, it will be understood that innumerable conservative amino acid substitutions can be performed to a wildtype VEGF-C sequence which are likely to result in a polypeptide that retains VEGF-C
biological activities, especially if the number of such substitutions is small. By "conservative amino acid substitution" is meant substitution of an amino acid with an amino acid having a side chain of a similar chemical character. Similar amino acids for making conservative substitutions include those having an acidic side chain (glutamic acid, aspartic acid); a basic side chain (arginine, lysine, histidine); a polar amide side chain (glutamine, asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine, 1 S valine, alanine, glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine); a small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine). Addition or deletion of one or a few internal amino acids without destroying VEGF-C biological activities also is contemplated.
Without intending to be limited to a particular theory, the mechanism behind the ei~icacy of VEGF-C in treating or preventing lymphedema is believed to relate to the ability of VEGF-C to stimulate VEGFR-3 signaling. Administration of VEGF-C in quantities exceeding those usually found in interstitial fluids is expected to stimulate VEGFR-3 in human subjects who, by virtue of a dominant negative heterozygous mutation, have insufficient VEGFR-3 signaling.
The growth factor named Vascular Endothelial Growth Factor D
(VEGF-D), as well as human sequences encoding VEGF-D, and VEGF-D variants and analogs, have been described in detail in International Patent Application Number PCT/LJS97/14696, filed 21 August 1997 and published on 26 February 1998 as International Publication Number WO 98/07832; and in Achen, et al., Proc.
Nat'l Acad. Sci. U.S.A., 95(2): 548-553 (1998), both incorporated herein by reference in the entirety. As explained therein in detail, human VEGF-D is initially produced in human cells as a prepro-VEGF-D polypeptide of 354 amino acids. A cDNA and deduced amino acid sequence for human prepro-VEGF-D are set forth in SEQ m Nos: 5 and 6, respectively. VEGF-D sequences from other species also have been reported. See Genbank Accession Nos. D89628 (Mus musculus); and AF014827 (Rattus norvegicus), for example, incorporated herein by reference.
The prepro-VEGF-D polypeptide has a putative signal peptide of 21 amino acids and is apparently proteolytically processed in a manner analogous to the processing of prepro-VEGF-C. A "recombinantly matured" VEGF-D lacking residues 1-92 and 202-354 of SEQ ID NO: 6 retains the ability to activate receptors and VEGFR-3, and appears to associate as non-covalently linked dimers. Thus, preferred VEGF-D polynucleotides include those polynucleotides that comprise a nucleotide sequence encoding amino acids 93-201 of SEQ ID NO: 6.
The subject matter of the invention is further described and demonstrated with reference to the following examples.

Demonstration that hereditary lymphedema is linked to the VEGFR-3 locus The following experiments, conducted to identify a gene or genes contributing to susceptibility to develop lymphedema, demonstrated that hereditary lymphedema correlates, in at least some families, to the chromosomal locus for the VEGFR-3 gene.
O VERVIEW
Families with inherited lymphedema were identified for the purpose of conducting a linkage and positional candidate gene analysis. Thirteen distinct families from the United States and Canada were identified through referrals from lymphedema treatment centers, lymphedema support groups, and from Internet correspondence (worldwide web site at www.pitt.edu/~genetics/lymph~. The study protocol was approved by the Institutional Review Board of the University of Pittsburgh and participants gave written informed consent. All members of the families were of western European ancestry. Forty members of one family ("Family 101") were examined during a family reunion by a physiatrist experienced in lymphedema treatment. Family members were considered affected with hereditary lymphedema if they exhibited asymmetry or obvious swelling of one or both legs. Members of the other 12 families were scored as affected if they had received a medical diagnosis of lymphedema, or if there were personal and family reports of extremity swelling or asymmetry. Medical records were obtained to verify status whenever possible.
For the purpose of linkage analysis, individuals with very mild or intermittent swelling, heavyset legs, obesity, or a history of leg infections as the only symptom were considered to have indeterminate disease status.
In the 13 families, 105 individuals were classified as affected, with a male:female ratio of 1:2.3. The age of onset of lymphedema symptoms ranged from prenatal (diagnosed by ultrasound) to age SS. When affected by normal matings were analyzed, 76 of 191 children were affected, yielding a penetrance of 80%.
First degree relatives of affected individuals were considered at risk.
Biological samples were obtained from members of the thirteen families to conduct the genetic analyses. DNA was isolated from the EDTA-anticoagulated whole blood by the method of Miller et al., Nucleic Acids Res., 16: 1215 (1998), and from cytobrush specimens using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Analysis of the markers used in the genome scan were performed 1 S by methods recognized in the art. [See Browman et al., Am. J. Hum.
Genetic., 63:861-869 (1998); see also the NHLBI Mammalian Genotyping Service world-wide web sites (www.marshmed.org/genetics/methods/pcr.htm; and www.marshmed.org/genetics/methods/gel.htm).
Two-point linkage analysis was conducted using an autosomal dominant model predicting 80% penetrance in the heterozygous state, 99%
penetrance in the homozygous state, and a 1% phenocopy rate. The frequency of the disease allele was set at 1/10,000. Microsatellite marker allele frequencies were calculated by counting founder alleles, with the addition of counts of non-transmitted alleles.
Multipoint analysis was carried out using distances obtained from the Location Database (LDB-http://cedar.genetics.soton.ac.uk/public html). Multipoint and 2-point analyses were facilitated using the VITESSE (vl. l) program. [O'Connell, J.R.
and Weeks, D.E., (1995), Nature Genet., 11:402-408].
DETAILED DESCRIPTION OF METHODS AND RESULTS
The first family studied, Family 101, was a large, mufti-generational family demonstrating early onset lymphedema. (See Fig. 1.) Forty individuals of this family were examined and DNA sampled. In addition, blood was obtained from another 11 members from mailing kits. Linkage simulation was performed using SLINK [Weeks et al., Am. J. Hum. Genet. 47: A204 (1990)] and linkage was analyzed using MSIM [Ott, J., Proc. Nat. Acad. Sci. USA, 86:4175-4178 (1989)] to estimate the potential power of two point linkage analysis in the family. Marker genotypes were simulated for a marker with heterozygosity of 0.875 under a linked (8=0) and unlinked (8=0.5) model using the 51 available individuals. The simulation showed that the power to detect linkage was greater than 90% for a LOD score threshold of Z(8) 2Ø The false positive rate was less than 5%.
Shortly thereafter, two additional families (designated Families 106 and 111) segregating for autosomal dominant lymphedema were identified. These three families (Figures lA-1C, Families 101, 106 and 111) were genotyped for 366 autosomal markers by the NHLBI Mammalian Genotyping Service (www.marshmed.org/genetics). Genotypes were checked for consistency using Pedcheck [O'Connell, J.R. and Weeks, D.E., Am. J. Hum. Genet., 61:A288 (1997)].
Two point linkage analysis was performed using VITESSE [O'Connell, J.R. and Weeks, D.E., Nature Genet., 11:402-408 (1995)). The model for linkage assumed an autosomal dominant model of inheritance, a disease allele frequency of 0.0001 and a penetrance of 0.80.
The results from the genomic scan can be briefly summarized as follows. A summed LOD score of greater than 4.0 was observed from distal chromosome 5, markers DSS1456, DSS817 and DSS488. The markers on distal chromosome Sq were the only markers having Z>3.0, the criteria established for statistical significance. LOD scores greater than 2.0 (8=0-0.15) were also detected for chromosome 12 (D12S391 Z=2.03, all families), and chromosome 21 (D21SI440 Z=2.62, all families). The largest two-point LOD (Z=4.3; 8=0) was observed for marker DSS408, localized to chromosome Sq34-q35.
This initial chromosomal mapping was further refined by genotyping the three affected families for eight additional markers localized to region Sq34-q35. Six of these were informative for linkage (DSS653, DSS498, DSS408, DSS2006, and DSS1354). Linkage analysis of these markers using VITESSE yielded a 2-point LOD score of 6.1 at B=0 for marker DSS1354 (Table 1) and a maximum multipoint LOD score of 8.8 at marker DSSI354 (Fig. 2). These findings supported the localization of a gene within chromosome band Sq34-q35 that is a predisposing factor in hereditary lymphedema.

LOD scores for individual families estimated over the interval defined by markers DSS498 and DSS2006.
Z(8) Z(8) 0.01Z(6)0.05 Z(8) Z(6) 0.2 0.0 0.1 Locus DSS498 Family 101 -3.18 -2.33 -0.45 0.42 0.88 Family 106 1.08 1.07 1.05 0.99 0.81 Family 111 -0.85 -0.77 -0.53 -0.34 -0.13 Family 105 1.22 1.20 1.11 0.98 0.72 Family 135 -2.48 -1.85 -1.12 -0.75 -0.38 Locus DSSI353 Family 101 -2.99 -2.48 -1.21 -0.63 -0.18 Family 106 0.28 0.29 0.3 5 0. 3 0. 3 8 Family 111 -1.06 -1.02 -0.88 -0.72 -0.42 Family 105 0.72 0.71 0.65 0.56 0.39 Family 135 -8.03 -4.18 -2.09 -1.13 -0.30 Locus DSS1354 Family 101 6.09 6.02 5.69 5.21 4.07 Family 106 1.42 1.40 1.32 1.20 0.96 Family 111 0.21 0.22 0.23 0.24 0.22 Family 105 0.43 0.42 0.40 0.36 0.28 Family 135 -6.88 -4.91 -3.20 -2.16 -1.07 Locus DSS408 Family 101 2.80 2.74 2.50 2.20 1.56 Family 106 0.66 0.68 0.73 0.76 0.71 Family 111 -1.70 -1.40 -0.80 -0.44 -0.10 Family 105 0.42 0.41 0.38 0.35 0.27 Family 135 -5.22 -4.24 -2.58 -1.67 -0.80 Z(8) Z(8) 0.01 Z(8)0.05 Z(8) 0.1 Z(8) 0.2 0.0 Locus DSS2006 Family 101 4.51 4.70 4.85 4.66 3.80 Family 106 1.17 1.16 1.11 1.03 0.83 Family 111 -1.32 -1.18 -0.82 -0.56 -0.25 Family 105 0.43 0.42 0.40 0.36 0.28 Family 135 -3.86 -3.20 -2.11 -1.45 -0.73 During the completion of the genome scan, an additional ten lymphedema families were ascertained. Two of these families (Families 105 and 135, see Figures lE and 1D), were potentially informative for linkage and were genotyped for markers in the linked region. Examination of the two point LOD scores for the five informative families for markers in the linked region (Table 1) shows that four of the families (101, 105, 106 and 111) are consistent with linkage to chromosome 5q while family 135 excluded linkage across the entire region with LOD scores Z =< -2.0 for all markers. Multipoint linkage analysis of Families 101, 105, 106 and 111 (Fig.
2) yielded a peak LOD score of Z = 10 at marker DSS1354. These findings support the existence of at least two loci which predispose to hereditary lymphedema.
The order of markers DSS1353, DSSI354 and DSS408 with respect to each other was uncertain. Multipoint linkage analysis using alternative orders for these markers gave similar results. Marker DSS498 is a framework marker and marker DSS408 is mapped 11.2 centimorgans distal to DSS498, based on the CHIC
chromosome 5 sex averaged, recombination minimized map, version 3 (www.chlc.org). The physical distance between DSS498 and DSS408 is estimated as 1.45 megabases based on the Genetic Location Database (LDB) chromosome 5 summary map (cedar.genetics.soton.ac.uk/public html~.
Database analysis identified sixteen genes within this region. Two of these genes have been identified as having roles in development (MSX2 and VEGFR-3). MSX2 was considered an unlikely candidate gene for lymphedema because of its known involvement in craniofacial development [Jabs et al., Cell, 75: 443-450 (1993)]. VEGFR-3, the gene encoding a receptor for VEGF-C, was selected as a better candidate gene for initial fi.~rther study for the following reasons.

- 33 _ (1) VEGFR-3 is expressed in developing lymphatic endothelium in the mouse [Kukk et al., Development, 122: 3829-3837 (1996); and Kaipainen et al., Proc. Nat. Acad. Sci. USA, 92: 3566-3570 (1995)];
(2) expression of VEGFR-3 is induced in differentiating avian S chorioallantoic membrane [Oh et al., Dev. Biol., 188:96-109 (1997)];
and (3) overexpression of VEGF-C, a ligand of VEGFR-3, leads to hyperplasia of the lymphatic vessels in transgenic mice [Jeltsch et al., Science, 276:
1423-1425 (1997)].
To explore the potential role of VEGFR-3 in lymphedema, probands from the thirteen lymphedema families were screened for variation by direct sequencing of portions of the VEGFR-3 gene. The sequencing strategy used amplification primers generated based upon the VEGFR-3 cDNA sequence (SEQ ID
NO: 1) and information on the genomic organization of the related vascular endothelial growth factor receptor-2 (VEGFR-2/KDRlflk-I) [Yin et al., Mammalian Genome, 9:
408-410 (1998)]. Variable positions (single nucleotide polymorphisms), the unique sequence primers used to amplify sequences flanking each variable site, and the method of detecting each variant are summarized in Table 2.

Location, amplification primer sequences, amplification conditions, and detection mPthndc fnr fve intrauenic cinule nucleotide nolvmornhisms in the human VEGFR-3 gene Position Primer 1 sequencePrimer 2 Ann. [MgClz]Base Detection sequence in VEGFR-3 temp. changeMethod gene Exon 12, tcaccatcgatccaagcagttctgegtgagccgag56 1.o CT sequencing amino C mM

acid 641 (SEQ m NO: (SEQ m NO:

7) 8) Exon 24, caggacggggtgacttgagcccaggectgtctactg56 1.0 CT Sequencing amino C mM

acid 1114 (SEQ m NO: (SEQ m NO:
9) 10) Exon 3, ccagctcctacgtgttcgggcaacagctggatgtca56 1.0 C-T Hhal amino C mM

acid 175 (SEQ m NO:
11 ) (SEQ m NO:
12) 656p 3' ctgtgagggcgtgggagtgtcctttgagccactgga54 1.5 GA Styl to Exon C mM

(SEQ m NO: (SEQ 117 13) NO: 14) SSbp 3' cacacgtcatcgacaccggtgggcaacagctggatgtca56 1.5 CT Apal to Exon C mM

(SEQ m NO: (SEQ m NO:
15) 16) All amplifications were done for 35 cycles with denaturation at 94° for 30 seconds, annealing as above for 30 seconds, and extension at 72° for 30 seconds.
Amplification and sequencing primers were synthesized by the DNA Synthesis Facility, University of Pittsburgh. Amplification primers were tagged at the S' end with the forward or reverse M13 universal sequence to facilitate direct sequencing.
Amplimers were subjected to cycle sequencing using the dRhodamine terminator ready reaction kit or the Dye Primer ready reaction kit for -M13 and M13 Rev primers (Perkin Elmer) and analyzed on the Prism ABI 377 fluorescent sequencer. Sequences were aligned for further analysis using SEQUENCHER 3.0 (Gene Codes).
Genomic sequence from approximately 50% of the VEGFR-3 gene was determined in this manner, and five single nucleotide variants were observed.
Two of the variants occurred in introns, and a third was a silent substitution in predicted exon 3. These intragenic polymorphisms were used to map the VEGFR-3 gene. As shown in Figure 2, VEGFR-3 maps within the region of chromosome Sq linked to the lymphedema phenotype, consistent with it being selected as a candidate gene.
In two families, (Family 127, pedigree not shown, and Family 135), a C~T transition was identified at nucleotide position 1940 of the VEGFR-3 cDNA (SEQ m NO: 1). This nucleotide substitution is predicted to lead to a non-conservative substitution of serine (codon TCC) for proline (codon CCC) at residue 641 (putative exon 12, within the sixth immunoglobulin-like region of the receptor's extracellular domain) of the amino acid sequence of the receptor (SEQ ID NO: 2). However, this sequence change was observed in 2 of 120 randomly selected individuals from the general population (240 alleles). Also, in one of the two families in which this variant was initially detected, family 135, linkage between lymphedema and chromosome 5q markers was excluded (Table 1 and Figure 2). In probands from the other ten families, wild type sequence was observed at nucleotide position 1940. Collectively, these results suggest that this P641 S variant is not causative.
In one nuclear family (Family 127, pedigree shown in Figure 1F) a C~T
transition was observed at nucleotide position 3360 (SEQ ID NO: 1) of the IfEGFR-3 cDNA. This nucleotide substitution is predicted to lead to a non-conservative substitution of leucine (codon CTG) for proline (codon CCG) at residue 1114 of the amino acid sequence of the receptor (SEQ ID NO: 2). This P1114L mutation is predicted to lie in the intracellular tyrosine kinase domain II involved in intracellular signaling [Pajusola et al., Cancer Res., 52:5738- 5743 (1992)]. Direct sequencing of predicted exon 24 of the VEGFR-3 gene alleles from members of this family identified this substitution only in affected and at-risk family members. This sequence change was not observed in 120 randomly selected individuals of mixed European ancestry from the general population (240 alleles). In probands from the other 11 families, wild type sequence was observed at nucleotide position 3360.
Collectively, this data demonstrates that a missense mutation that causes a non-conservative substitution in a kinase domain of the VEGFR-3 protein correlates strongly with a heritable lymphedema in one family, and suggests that other mutations in the same gene may exist that correlate with heritable lymphedema in other families. As explained above, only a portion of the VEGFR-3 gene sequence was analyzed to identify this first mutant of interest. Additional sequencing, using standard techniques and using the known VEGFR-3 gene sequence for guidance, is expected to identify additional mutations of interest that are observed in affected and at-risk members of other families studied.

Demonstration that a C--T missense mutation at position 3360 in the VEGFR-3 coding sequence results in a tyrosine kinase negative mutant The results set forth in Example 1 identified two missense mutations in the VEGFR-3 coding sequence, one of which (C~T at position 3360) appeared to correlate with heritable lymphedema and one of which (C~T transition at position 1940) did not. The following experiments were conducted to determine the biochemical significance of these mutations on VEGFR-3 biological activity.
To analyze how the two single amino acid substitutions affect the VEGFR-3-mediated signaling, the corresponding mutant receptor expression vectors were generated using site-directed mutagenesis procedures and expressed in 293T cells by transient transfection. The long form of human VEGFR-3 cDNA (SEQ 1D NO: 1) was cloned as a Hind III-Bam HI fragment from the LTR-FLT41 plasmid [Pajusola et al., Oncogene 8: 2931-2937 (1993)] into pcDNA3.1/Z(+) (Invitrogen). The P641S
and P 1114L mutants of VEGFR-3 were generated from this construct with the GeneEditor~ in vitro Site-Directed Mutagenesis System (Promega) using the following oligonucleotides (the C~T mutations are indicated with bold letters):
5'-CCTGAGTATCTCCCGCGTCGC-3' (SEQ ID NO: 17) for P641 S
mutation; and 5'-GGTGCCTCCCTGTACCCTGGG-3' (SEQ 117 NO: 18) for P 1114L
mutation.
For the transient expression studies, 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (GIBCO BRL, Life Technologies, Gaithersburg, MD), glutamine, and antibiotics. Cells were transfected with 20 pg of plasmid encoding the wild type or mutant VEGFR-3 forms using the calcium phosphate method, and harvested 36 hours after transfection for immunoprecipitation and Western blotting. Under these conditions, RTK overexpression results in ligand-independent activation, thus allowing the receptor phosphorylation to be studied. An empty vector was used for mock (control) transfections. (It will be appreciated that ligand stimulation assays of VEGFR-3 forms also can be employed, e.g., as described in U. S. Patent No.
5,776,755, incorporated herein by reference, using VEGF-C or VEGF-D ligands.) In order to investigate the effect of the two VEGFR-3 mutants on the tyrosine phosphorylation of the VEGFR-3, Western blotting analysis was performed using anti-phosphotyrosine antibodies. The cell monolayers were washed three times with cold phosphate-buffered saline (PBS, containing 2 mM vanadate and 2 mM
PMSF) and scraped into RIPA buffer (150 mM NaCI, 1 % Nonidet P40, 0.5 deoxycholic acid sodium salt, 0.1 % SDS, 50 mM Tris-HCI, pH 8.0) containing 2 mM
Vanadate, 2 mM PMSF, and 0.07 U/ml Aprotinin.
The cell lysates were sonicated and centrifuged for 10 minutes at 19,000 X g, and the supernatants were incubated for 2 hours on ice with 2 pg/ml of monoclonal anti-VEGFR-3 antibodies (9D9f~) [Jussila ei al., Cancer Res., 58:
1599-604 (1998)]. Thereafter, Protein A sepharose (Pharmacia) beads were added and incubation was continued for 45 minutes with rotation at +4°C. The sepharose beads were then washed three times with ice-cold RIPA buffer and twice with PBS
(both containing 2 mM vanadate, 2 mM PMSF), analyzed by 7.5 % SDS-PAGE and transferred to a nitrocellulose filter (Protran Nitrocellulose, Schleicher &
Schuell, No.
401196) using semi-dry transfer apparatus. After blocking the filter with 5 %
BSA in TBS-T buffer (10 mM Tris, pH 7.5, 150 mM NaCI, 0.05 % Tween 20), the filters were incubated with the phosphotyrosine-specific primary antibodies (Upstate Biotechnology, #OS-321), followed by biotinylated goat-anti-mouse immunoglobulins (Dako, E0433) and Biotin-Streptavidin HRP complex (Amersham, RPN1051). The bands were visualized by the enhanced chemiluminescence (ECL) method.
After analysis for phosphotyrine-containing proteins, the filters were stripped by washing for 30 minutes at +50°C in 100 mM 2-mercaptoethanol, 2 % SDS, 62.5 mM Tris-HCI, pH 6.7, with occasional agitation. The filters were washed with TBS-T, blocked again with BSA as described above, and analyzed for the presence of VEGFR-3 using the 9D9f~ antibodies and HRP-conjugated rabbit-anti-mouse immunoglobulins (Dako, P0161).
The Western analyses revealed that the P641 S mutant receptor was phosphorylated normally, i.e., in a manner similar to the wild type control.
However, the proteolytic processing of the P641 S receptor protein may be affected, as the 175 kD and 125 kD polypeptides seemed to have a higher relative density when compared to the 195 kD form.

In contrast, no phosphorylated P 1114L mutant protein was detected using the phosphotyrosine antibodies. The expression of similar amounts of the VEGFR-3 protein (normal and both mutants) was confirmed using the monoclonal 9D9f~ antibody, which is directed towards the extracellular domain of the VEGFR-3.
Both the P641 S and the P 1114L mutant VEGFR-3 migrated slightly faster than the wild type VEGFR-3 in the gel electrophoresis.
In order to analyze the possible dominant negative effect of the Pl 114L
mutant on the wild-type receptor, a second, similar set of experiments were performed wherein the 293T yells were transfected with an increasing amount of the expression vector in combination with decreasing amounts of the wild type vector.
Wild type to mutant ratios of 1:0, 3:1, 1:1, 1:3 and 0:1 were used. The cells were lysed 48 hours after transfection and the lysates were analyzed by immunoprecipitation and Western blotting as described above. These experiments permitted evaluation of whether the mutant protein interferes with wild type protein phosphorylation and estimation of the minimal amount of the WT protein needed for observable tyrosyl autophosphorylation. Immunoprecipitates from cells transfected with only the WT
plasmid revealed WT protein that was strongly phosphorylated in this experiment (lane 2), whereas immunoprecipitates from cells transfected with only the mutant plasmid were again inactive (unphosphorylated).
Interestingly, when transfection was made using 75% of WT and 25 of mutant plasmid, the phosphorylation of the receptors was decreased by about 90%.
This result strongly suggests that the P1114L mutant receptor forms heterodimers with the WT receptor, but cannot phosphorylate the WT receptor, thus failing to activate it.
Under this theory, the WT receptor monomers in the heterodimers would also remain inactive, causing a disproportionate decrease of the total amount of activated receptor, when co-transfected with the mutant. Wildtype-wildtype homodimers would remain active and be responsible for the observed signaling. When the wild type and mutant receptor expression vectors were transfected at a 1:1 ratio, the VEGFR-3 phosphorylation was about 4% of the wild type alone, whereas at a 1:3 ratio, no tyrosine phosphorylation of VEGFR-3 was observed.
The foregoing results are consistent with the linkage analyses in Example 1: the mutation at position 641 that did not appear to correlate with lymphedema also did not appear to be disfunctional, whereas the mutation at position 1114 appeared to cause a dominant negative mutation that shows no tyrosine phosphorylation alone and that drastically reduces VEGFR-3 signaling in cells expressing both the mutant and wild type VEGFR-3 genes.
Collectively, these data indicate that the P1114L VEGFR-3 mutant is unable to act as a part of the signaling cascade, and also acts in a dominant negative manner, thus possibly interfering partially with the activation of the wild type VEGFR-3. Such effects of the mutation may eventually lead to lymphedema.

Treatment of lymphedema with a VEGFR-3 ligand The data from Examples 1 and 2 collectively indicate a causative role in heritable lymphedema for a mutation in the VEGFR-3 gene that interferes with VEGFR-3 signaling. Such a mutation behaves in an autosomal dominant pattern, due to the apparent necessity for receptor dimerization in the signaling process.
However, the data from Example 2 suggests that some residual signaling may still occur in 1 S heterozygous affected individuals, presumably through pairing of VEGFR-3 proteins expressed from the wild type allele. The following experiments are designed to demonstrate the efficacy of VEGFR-3 ligand treatment in such affected individuals, to raise VEGFR-3 signaling to levels approaching normal and thereby ameliorate/palliate the symptoms of hereditary lymphedema.
Initially, an appropriate animal model is selected. Several potential animal models have been described in the literature. [See, e.g., Lyon et al., Mouse News Lett. 71: 26 (1984), Mouse News Lett. 74: 96 (1986), and Genetic variants and strains of the laboratory mouse, 2nd ed., New York: Oxford University Press (1989), p. 70 (Chylous ascites mouse); Dumont et al., Science, 282: 946-949 (1998) (heterozygous VEGFR-3 knockout mouse); Patterson et al., "Hereditary Lymphedema," Comparative Pathology Bulletin, 3: 2 (1971) (canine hereditary lymphedema model); van der Putte, "Congenital Hereditary Lymphedema in the Pig,"
Lympho, ll: 1-9 (1978); and Campbell-Beggs et al., "Chyloabdomen in a neonatal foal," Veterinary Record, 137: 96-98 (1995).] Those models which are determined to have analogous mutations to the VEGFR-3 gene are preferred. Analogous mutations would include mutations affecting corresponding residues and also mutations affecting different residues but causing similar functional alterations. The Chylous ascites mouse VEGFR-3 gene contains a missense mutation at a position corresponding to residue 1053 of SEQ ID No. 2, which maps to the catalytic pocket region of the tyrosine kinase catalytic domain. Thus, the "Chy" mouse is expected to display similar functional alterations to human mutations affecting tyrosine kinase activity, a prediction which can be confirmed by functional assays such as those described in Example 2. In a preferred embodiment, "knock in" homologous recombination genetic engineering strategies are used to create an animal model (e.g., a mouse model) having a VEGFR-3 allelic variation analogous to the human variations described herein. [See, e.g., Partanen et al., Genes & Development, 12: 2332-2344 (1998) (gene targeting to introduce mutations into another receptor protein (FGFR-1) in mice).] For example, the P 1114L mutation in human VEGFR-3 occurs in a VEGFR-3 region having highly conserved amino acid identity with murine VEGFR-3 (Genbank Accession No.
L07296). Thus, a corresponding P1114L can be introduced into the murine VEGFR-by "knock-in" homologous recombination. Optionally, such mice can be bred to the heterozygous VEGFR-3 knockout mice or Chy mice described above to further modify the phenotypic severity of the lymphedema disease.
The mice as described above are treated with a candidate therapeutic, e.g., a recombinant mature form of VEGF-C, at various dosing schedules, e.g., once daily by intravenous (IV) or intramuscular (IM) injection at a dose of 1-1000 ng/g body weight, preferably 10-100 ng/g, which should result in a peak level saturating VEGFR-3 (Kd about 150 pM) but not VEGFR-2 (Kd around 400 pM). For VEGFR-3-specific forms, such as VEGF-CoClss, even higher dosing is contemplated, to sustain VEGFR-3-saturating physiological concentrations for longer periods. Direct IM
injection at multiple sites in the muscles of affected extremities is a preferred route of administration. The dosing is adjusted according to the efficacy of the treatment and the presence of possible side effects due to the lowering of blood pressure, which has been observed in response to VEGF administration IV. The efficacy of treatment is measured via IVMRI imaging of the water content and volume of swelling of the abdomen and the extremities of the animals. The amount of fluid in the abdominal cavity is estimated and the animals are weighed during the follow-up.
In studies using VEGFR-3 -l+ x Chy mice progeny, the animals will also have the 13-galactosidase marker in their lymphatic endothelium. After a successful treatment, the treated and non-treated experimental animals and -/+ controls are killed and their lymphatic vessels are visualized by f3-gal and antibody staining. The staining patterns of experimental and control animals are compared for vessel diameter, numbers of endothelial cells, density of blood and lymphatic vessels, and nuclear density/section surface area for the estimation of tissue oedema.
S Such experiments are repeated with various candidate therapeutics (e.g., VEGF-C or VEGF-D recombinant polypeptides; VEGF-C and VEGF-D gene therapy vectors; and combinations thereof) at various dosing schedules to determine an optimum treatment regimen.

Chromosomal structure of the human VEGFR-3 gene Sequencing and mapping of human DNA corresponding to the VEGFR-3 locus icated that this gene consists of thirty exons has ind separated by twenty-nine introns of varying size. The exon intron organization is summarized as follows:

EXON Bp of SEQ ID NO: 1 NUMBER size (bpl INTRON
SIZE

58 by unknown 97 by > 1 kb 245 by 218 by 113 by 120 by 163 by 107 by 140 by 269 by 169 by 261 by 118 by >1 kb 155 by unknown 163 by > 1 kb 127 by unknown 109 by unknown 363 by 293 by 147 by 99 by 132 by approx. 160 by 107 by 301 by 139 by >464 by 105 by unknown 114 by 143 by 89 by >1 kb 151 by unknown 95 by unknown 123 by unknown 112 by 974 by 100 by 400 by 107 by unknown 148 by > 1 kb 121 by unknown 86 by unknown 30a (Flt4 short) 3913-4111 199 by 3.7 kb 30b (Flt4long) 3913-4416 >504 by (CDS 504 bp) The foregoing information permits rapid design of oligonucleotides for amplifying select portions of the VEGFR-3 gene from genomic DNA, or RNA, or cDNA, to facilitate rapid analysis of an individual's VEGFR-3 coding sequence, to determine whether the individual possesses a mutation that correlates with a lymphedema phenotype.

Identification of additional non-conservative missense mutants Using procedures essentially as described in Example 1, the VEGFR-3 coding sequences from additional affected and unaffected individuals from families having members suffering from heritable lymphedema were studied. The analysis focused on families with statistical linkage to chromosome 5q as described in Example 1. The additional analysis included the PCR amplification and sequencing of Exon 17, Exon 22, and Exon 23 sequences with the following PCR primers:
Exon 17-1 S'-CATCAAGACGGGCTACCT-3' (SEQ B7 NO: 23) Exon 17-2 5'-CCGCTGACCCCACACCTT-3' (SEQ ID NO: 24) Exon 22-1 5'-GAGTTGACCTCCCAAGGT-3' (SEQ 1D NO: 25) Exon 22-2 5'-TCTCCTGGACAGGCAGTC-3' (SEQ >D NO: 26) Exon 23-1 5'-GAGTTGACCTCCCAAGGT-3' (SEQ m NO. 27) Exon 23-2 5'-TCTCCTGGACAGGCAGTC-3' (SEQ 1D NO. 28) These additional studies identified four additional non-conservative missense mutations in evolutionarily conserved amino acids in kinase domains I
and II
of human VEGFR-3. Each mutation, shown in Table 3 below, was observed in a single independently ascertained family, and in each family, the mutation co-segregates with individuals suffering from, or considered at risk for developing, lymphedema.
None of these mutations were observed in the VEGFR-3 genes in a random sample of more than 300 chromosomes from individuals from families unai~licted with heritable lymphedema.

Mutations in VEGFR-3 causing Hereditary Lymphedema*
Nucleotide Amino Acid Functional Exon Substitution**Substitution Domain 24 C3360T P1114L Kinase 2 17 G2588A G857R Kinase 1 23 G3141C R1041P Kinase 2 23 T3150C L1044P Kinase 2 23 G3164A D 1049N Kinase 2 * Numbers indicate nucleotide or amino acid positions in SEQ ID NOs: 1 and 2.
**It will be appeciated that, since DNA is double-stranded, each mutation could be characterized in two equivalent ways, depending on whether reference is being made to the coding or the non-coding strand.
Referring to SEQ >D NO: 2, the kinase domains of VEGFR-3 comprise approximately residues 843-943 and residues 1009-1165. Within these domains, molecular modeling suggests that residues 6852, 6854, 6857, K879, E896, H1035, D1037, N1042, D1055, F1056, 61057, E1084, D1096 and 81159 are of particular importance in comprising or shaping the catalytic pocket within the kinase domains.
See van Der Geer and Hunter, Ann. Rev. Cell. Biol., 10: 251-337 (1994); and Mohammadi et al., Cell 86: 577-587 (1996). Thus, this data identifying additional mutations implicate missense mutations within a kinase domain of the VEGFR-3 protein as correlating strongly with a risk for developing a heritable lymphedema phenotype. Mutations which affect residues in and around the catalytic pocket appear particularly likely to correlate with lymphedema. The P 1114L mutation, though not situated within the catalytic pocket, is postulated to cause a conformational alteration that affects the catalytic pocket. The G857R mutation is postulated to block the catalytic pocket and/or the ATP binding site of the kinase domain.

Functional Analysis of Additional VEGFR-3 missense mutations Using procedures essentially as described above in Example 2, the functional state ofthe G857R, L1044P, and D1049N mutations were analyzed.
(PLCLB buffer, comprising 150 mm NaCI, 5% glycerol, 1%Triton X-100, 1.SM
MgCl2, 50 mm HEPES, pH 7.5, was substituted for RIPA buffer described in Example 2 for immunoprecipitation and Western blotting protocols.) A VEGFR-3-encoding construct comprising the G857R mutation was generated from the long form of human VEGFR-3 cDNA using the oligonucleotide:
5'-CGG CGC CTT CAG GAA GGT GGT-3' (SEQ ID NO: 20) A construct comprising the L 1044P mutation was generated from the long form of human VEGFR-3 cDNA using the oligonucleotide:
5'-CGG AAC ATT CCG CTG TCG GAA-3' (SEQ ID NO: 21) A construct comprising the D 1049N mutation was generated from the long form of human VEGFR-3 cDNA using the oligonucleotide:
5'-GTC GGA AAG CAA CGT GGT GAA-3' (SEQ ID NO: 22).
The constructs were transiently transfected into 293T cells and harvested for Western blotting essentially as described in Example 2, except for the buffer substitution described above. In contrast to wild type VEGFR-3 and containing the P641S mutation, no phosphorylated G857R or L1044P mutant protein was detected using the phosphotyrosine antibodies, consistent with the results that had been observed for P1114L. The expression of similar amounts of the VEGFR-3 protein was confirmed using the monoclonal 9D9f~7 antibody, which is directed towards the extracellular domain of the VEGFR-3 in the Western blotting. This data suggested that these observed mutations did indeed affect VEGFR-3 kinase function.
The D1049N mutant appeared to retain at least some tyrosine kinase activity.
It is also noteworthy that VEGFR-1 and VEGFR-2 contain an asparagine residue at the position in their tyrosine kinase domains which corresponds to position 1049 of VEGFR-3.

a -46- i:
ata suggest that the D 1049N variation may only be an allelic variant ' that correlates with hereditary lymphedema, rather than a causative mutation.
To determine whether the VEGFR-3 mutants function in a dominant negative manner, each construct was co-transfected at varying ratios with wild type receptor into 293T cells essentially as described in Example 2. Unlike the results observed for P 1114L and described in Example 2, neither the G857R mutant nor the L1044P mutant seemed to interfere with phosphorylation of the co-transfected wild type receptor.
The absence of a dominant negative effect in these experiments does not foreclose a conclusion that the mutations described above are causative.
It has been found that a significant fraction of ligand-activated receptor tyrosine kinases traffic to the lysosomal compartment after internalization, where they are degraded.
However, receptors which are not ligand-activated preferentially recycle back to the cell surface after internalization. Thus, it is possible that the turnover time of the weakly phosphorylated mutant receptor is significantly longer than that of the wild type receptor protein. If this were true, the amount of the mutant receptor on the endothelial cell surface could be considerably higher than the amount of the phosphorylated and rapidly internalized wild type receptor, and any available ligand would thus bind a disproportionally high number of mutant receptors. Both a possible dominant negative effect of the mutant receptor and an abnormally long half life of the tyrosine kinase negative mutant receptor could eventually lead to lymphedema.
Alternatively, a mutation that merely decreases (but does not eliminate) VEGFR-tyrosine kinase activity may display a constitutive low level of internalization and degravdation that is insufficient to trigger sufficient downstream signalling, but decreases the effective concentration of VEGFR-3 on cell surfaces for ligand binding and effective activation, leading eventually to lymphedema.
While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those in the art, all of which are intended as aspects of the present invention.
Accordingly, only such limitations as appear in the claims should be placed on the invention.

' 47 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LUDWIG INSTITUTE FOR CANCER RESEARCH -AND- HELSINKI
UNIVERSITY LICENSING LTD. OY -AND- UNIVERSITY OF
PITTSBURG OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION
(ii) TITLE OF INVENTION: SCREENING AND THERAPY FOR LYMPHATIC DISORDERS

(VEGFR-3 ) ZO (111) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk 2 ~ (B) COMPUTER: IHM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,283,470 (B) FILING DATE: 29-SEPT-1999 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 09/375,248 (B) FILING DATE: 16-AUG-1999 30 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART 6 BIGGAR

(B) REGISTRATION NUMBER:

(C) REFERENCE/DOCI~T NUMBER: 64267-1017 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) INFORMATION FOR SEQ ID NO.: 1:.

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 4111 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (ix) FEATURE

(A) NAME/ID;Y: CDS

(B) LOCATION: (20)..(4111) 2 (C) OTHER INFORMATION: Human Flt4 (VEGFR-3) long form 0 cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:

CTG CGA CTG

Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu TAC TCC ATG

Trp Leu Cys Leu Gly Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met ATC GAC ACC

Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr CTC GAG TGG

Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp AAG GAC AGC

Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser ' 49 Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe 2 0 Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro 4 0 Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly 560 565 57p Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln lO TGC GAA TAC CTG TCC TAC GAT GCC AGC CAG TGG GAA TTC CCC CGA GAG 2548 Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg Pro Gly Ser Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln 1165 1170 ~ 1175 Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser Gln Val Ser Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser GAG CAT

Gly Met Val Leu Ala Ser Glu PheGlu Gln Ile Glu Ser Arg Glu His TGT GCT

Arg Gln Glu Ser Gly Phe Ser LysGly Pro Gly Gln Asn Val Cys Ala TCC GAG

Val Thr Arg Ala His Pro Asp GlnGly Arg Arg Arg Arg Pro Ser Glu GTG GAG

Arg Gly Ala Arg Gly Gly Gln PheTyr Asn Ser Glu Tyr Gly Val Glu GAC GTG

Leu Ser Glu Pro Ser Glu Glu HisCys Ser Pro Ser Ala Arg Asp Val TAC

Thr Phe Phe Thr Asp Asn Ser Tyr (2) INFORMATION FOR SEQ ID NO.:2:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1363 30 (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID
NO.:
2:

Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu Trp Leu Cys Leu Gly 4 0 Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val ' 55 Val Arg Aap Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu 2 0 Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp 2 0 Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu ' 57 Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile 2 0 Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala S~r Ala Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg pro Gly Ser SO Ser Asp A~g Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly Thr Arg Met Arg Ala 2 0 Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser Gln Val Ser Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln Glu Ser Gly Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala Val Thr Arg Ala His Pro Asp Ser Gln Gly Arg Arg Arg Arg pro Glu Arg Gly Ala Arg Gly Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly Glu Leu Ser Glu Pro Ser Glu Glu Asp His Cys Ser Pro Ser Ala Arg Val Thr Phe Phe Thr Asp Asn Ser Tyr ' 59 (2) INFORMATION FOR SEQ ID NO.: 3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1997 (H) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (ix) FEATURE

(A) NAME/KEY: CDS

(B) LOCATION: (352)..(1611) (C) OTHER INFORMATION: Human eDNA
for prepro-VEGF-C

(xi) SEQUENCE DESCRIPTION: SEQ ID
NO.: 3:

CCCGCCCCGC CTCTCCAAAA AGCTACACCG ACGCGGACCGCGGCGGCGTCCTCCCTCGCC

CTCGCTTCAC CTCGCGGGCT CCGAATGCGG GGAGCTCGGATGTCCGGTTTCCTGTGAGGC

TTTTACCTGA CACCCGCCGC CTTTCCCCGG CACTGGCTGGGAGGGCGCCCTGCAAAGTTG

GGAACGCGGA GCCCCGGACC CGCTCCCGCC GCCTCCGGCTCGCCCAGGGGGGGTCGCCGG

CCACCCCTGC CCCCGCCAGC GGACCGGTCC CCCACCCCCGGTCCTTCCACC ATG CAC

Met His TCT CTG GCT

Leu Leu Gly Phe Phe Ser Val Ala Cys Leu Ala Ala Leu Ser Leu Ala GCC GCC TTC

30 Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Glu Ser Ala Ala Phe GAC GCG GCC

Gly Leu Asp Leu Ser Asp Ala Glu Pro Gly Glu Thr Ala Asp Ala Ala TTA CGG TCC

Tyr Ala Ser Lys Asp Leu Glu Glu Gln Ser Val Ser Val Leu Arg Ser Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys TGT CAG CTA AGG AAA GGA TGG CAA
GGC CAT AAC
AGA GAA
CAG GCC

Cys Gln Leu Arg Lys Gly Trp Gln Asn Arg Glu Gln Ala Asn Gly His CTC AAC TCA AGG ACA GAA ACT ATA TTT GCT GCA GCA CAT TAT

Leu Asn Ser Arg Thr Glu Thr Ile Phe Ala Ala Ala His Tyr Glu Lys Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln 2 0 Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg 4 0 Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser (2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 419 (H) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY:

' 62 (ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 4:
Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Tyr Ser Asp Glu Glu Gln Ser Ala Ala Lys Leu Leu Arg Ser Val Ser Asp GluLeu Thr Leu Tyr Pro Tyr Trp Met Val Met Val Glu Lys Tyr Cys GlnLeu Lys Gly Trp Gln Asn Arg Gln Lys Arg Gly His Glu Ala Leu AsnSer Thr Glu Thr Ile Phe Ala Ala Asn Arg Glu Lys Ala His Asn ThrGlu Leu Ser Ile Asp Glu Trp Lys Tyr Ile Lys Asn Arg Thr Cys MetPro Glu Cys Ile Asp Gly Lys Phe Gln Arg Val Val Glu Gly Ala ThrAsn Phe Lys Pro Pro Val Ser Tyr Val Thr Phe Cys Val Arg Gly GlyCys Asn Glu Gly Leu Cys Met Thr Cys Cys Ser Gln Asn Ser Ser TyrLeu Lys Leu Phe Glu Thr Val Leu Thr Ser Thr Ile Pro Ser Gly ProLys Val Ile Ser Phe Asn His Ser Gln Pro Thr Ala Thr Cys Cys MetSer Leu Val Tyr Arg Val His Ile Arg Lys Asp Gln Ser Ile Arg SerLeu Ala Leu Pro Gln Gln Ala Asn Arg Pro Thr Cys Ala Lys Cys ProThr Tyr Trp Ile Cys Cys Thr Asn Met Asn Arg Asn His Leu Gln Glu Ser Ser Asp Gly Asp Ser Ala Asp Ala Asp Phe Met Phe ' 63 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 2 0 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met Ser (2) INFORMATION
FOR SEQ
ID NO.:
5:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 2029 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

4 0 (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (ix) FEATURE

(A) NAME/KEY: CDS

(8) LOCATION: (411)..(1475) (C) OTHER INFORMATION: Human prepro-VEGF-D
eDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.:
5:

' 64 CTTACAAAGC

TGAAGTAAAT

TTTTGGAGAA

AAAGCGTACC

GTTTTCTGTG

ATTTTCTCTA

Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg Ser Ala 70 75 eo Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro (2) INFORMATION FOR SEQ 6:
ID NO.:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 354 (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: ID 6:
SEQ NO.:

Met Tyr Arg Glu Trp Val AsnVal PheMet LeuTyrVal Val Val Met 2 Gln Leu Val Gln Gly Ser GluHis GlyPro LysArgSer 0 Ser Asn Val Ser Gln Ser Thr Leu Glu GluGln GlnIle AlaAlaSer Arg Ser Arg Ser Leu Glu Glu Leu Leu ThrHis SerGlu TrpLysLeu Arg Ile Asp Trp Arg Cys Arg Leu Arg SerPhe ThrSer AspSerArg Leu Lys Met Ser Ala Ser His Arg Ser PheAla AlaThr TyrAspIle Thr Arg Phe Glu Thr Leu Lys Val Ile GluTrp GlnArg GlnCysSer Asp Glu Thr Pro Arg Glu Thr Cys Val AlaSer GluLeu LysSerThr Glu Val Gly Asn Thr Phe Phe Lys Pro ValAsn ValPhe CysGlyGly Pro Cys Arg Cys Cys Asn Glu Glu Ser CysMet AsnThr ThrSerTyr Leu Ile Ser ' 67 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 2 0 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro (2) INFORMATION
FOR SEQ
ID NO.:
7:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 17 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE

(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 7:
TCACCATCGA TCCAAGC 1~
(2) INFORMATION FOR SEQ ID NO.: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 8:

(2) INFORMATION
FOR SEQ
ID NO.:
9:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE

' 69 (C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9:

(2) INFORMATION FOR SEQ ID NO.: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 10:

(2) INFORMATION
FOR SEQ
ID NO.:
11:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE

(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 11:

(2) INFORMATION FOR SEQ ID NO.: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 10 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 12:

(2) INFORMATION
FOR SEQ
ID NO.:
13:

(ij SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vij ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE

(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 13:

(2) INFORMATION FOR SEQ ID NO.: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 14:

(2) INFORMATION
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ID NO.:
15:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE

' 72 (C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 15:

(2) INFORMATION FOR SEQ ID NO.: 16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 16:

(2) INFORMATION
FOR SEQ
ID NO.:
17:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ixj FEATURE

' 73 (C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 17:

(2) INFORMATION FOR SEQ ID NO.: 18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide 2 O (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 18:

(2) INFORMATION
FOR SEQ
ID NO.:
19:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1363 (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY:

30 (ii) MOLECULE TYPE; polypeptide (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 19:
Met Gln Pro Gly Ala Ala Leu Asn Leu Arg Leu Trp Leu Cys Leu Gly Leu Leu Gln Gly Leu Ala Asn Gly Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu Asp Ser Tyr Val Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Thr Trp Pro Gly Ala Gln Glu Val Leu Thr Thr Gly Gly Lys Asp Ser Glu Asp Thr Arg Val Val His Asp Cys Glu Gly Thr Glu Ala Arg Pro Tyr Cys Lys Val Leu Leu Leu Ala Gln Thr His Ala Asn Asn Thr Gly Ser Tyr His Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Thr Tyr Val Phe Val Arg Asp Phe Lys His Pro Phe Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp Ser Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Ile Thr Leu Arg Ser Gln Ser Ser Ala Leu His Pro Asp Gly Gln Glu Val Leu Trp Asp Asp Arg Arg Gly Met Arg Val Pro Thr Gln Leu Leu Arg Asp Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asn Phe Leu Ser Asn Leu Phe Val Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Tyr Pro Lys Lys Ser Met Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala Glu Phe Asp Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Ala Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln Asn Asp ' 75 Leu Gly Pro Tyr Val Cys Glu Ala Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His Glu Lys Pro Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Val Leu Glu Ala Thr Ala Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp Tyr Lys Asp Arg Lys Ala Val Thr Gly Arg His Asn Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala Ser Ala Gly Val Tyr Thr Leu Ala 2 0 Leu Trp Asn Ser Ala Ala Gly Leu Arg Gln Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro His Ile His Glu Lys Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Thr Leu Thr Cys Thr Ala Tyr Gly Val Pro Gln Pro Leu Ser Val Gln Trp His Trp Arg Pro Trp Thr Pro Cys Lys Thr Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Arg Asp Gly Met Pro Gln Cys Arg Asp Trp Lys Glu Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu Asp Ser Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asp Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Val Asn Lys Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Ser Ile Glu Ser Glu Pro Ser Glu Asp Pro Leu Glu Gly Gln Ser Val Arg Leu Ser Cys Arg Ala Asp Asn Tyr Thr Tyr Glu His Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala Gln Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Glu Ala Asn Leu ' 76 Glu Glu Ala Glu Pro Gly Ala Arg His Ala Thr Leu Ser Leu Asn Ile Pro Arg Val Ala Pro Glu Asp Glu Gly Asp Tyr Val Cys Glu Val Gln Asp Arg Arg Ser Gln Asp Lys His Cys His Lys Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Arg Cys Pro Val Ala Gly Ala His Val Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Lys 2 0 Glu Ser Gly Ile Asp Leu Ala Asp Ser Asn Gln Arg Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Ile Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met Lys Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly Arg Val Leu Gly His Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala Phe Gly Ile Asn Lys Gly Ser Ser Gys Asp Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Asn Gly Pro Leu Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Val Lys Arg Asp ' 77 Thr Phe Asn Pro Tyr Ala Glu Lys Ser Pro Glu Gln Arg Arg Arg Phe Arg Ala Met Val Glu Gly Ala Lys Ala Asp Arg Arg Arg pro Gly Ser Ser Asp Arg Ala Leu Phe Thr Arg Phe Leu Met Gly Lys Gly Ser Ala Arg Arg Ala Pro Leu Val Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala 2 0 Arg Asn Ile Leu Leu Ser Glu Ser Asp Ile Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Lys Asp Gly Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg His Ile Met Gln Ser Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Asp Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Gly Gly Trp Gln Glu Glu Glu Glu Glu Arg Met Ala Leu His Ser Ser Gln Ser Ser Glu Glu Asp Gly Phe Met Gln Ala Ser Thr Thr Ala Leu His Ile Thr Glu Ala Asp Ala Asp Asp Ser Pro Pro Ser Met His Cys His Ser Leu Ala Ala Arg Tyr Tyr Asn Cys Val Ser Phe Pro Gly Arg Leu Ala Arg Gly Thr Lys Thr Pro Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Leu Pro Met Thr Pro Thr Thr Tyr Lys AlaSer Gly Leu Met Met Ala Asp Val Asn Gln Thr Asp Ser Ser Glu GluPheGlu Glu Leu Glu Ser Arg ArgPro Gly His Glu Ser Phe Ser CysLysGly Pro Gly Gln His Met IlePro Gly Asp Arg His Pro Asp ProGlnGly Arg Arg Arg Arg pro GlnGly Gln Thr Ala Gly Gly Lys ValPheTyr Asn Asn Glu Tyr Gly ValSer Pro Glu Gln Cys Thr Glu GlyAspCys Cys pro Ser Ala Gly ThrPhe Ala Ser Phe Asp 2 0 Ser Tyr Ser (2) INFORMATION FOR SEQ ID NO.: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
30 (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 20:

40 (2) INFORMATION FOR SEQ ID NO.: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 r Y
' 79 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 21:

(2) INFORMATION FOR SEQ ID NO.: 22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS:
2 O (D ) TOPOLOGY
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 22:

(2) INFORMATION FOR SEQ ID NO.: 23:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
10 (A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 23:

(2) INFORMATION FOR SEQ ID NO.: 24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 2 0 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 24:

(2) INFORMATION FOR SEQ ID NO.: 25:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 25:

(2) INFORMATION FOR SEQ ID NO.: 26:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 2 0 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 26:

(2) INFORMATION FOR SEQ ID NO.: 27:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 (8) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 27:

(2) INFORMATION FOR SEQ ID NO.: 28:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 2 0 (8) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 28:

Claims (36)

1. A method of screening a human subject for an increased risk of developing a lymphatic disorder, comprising the steps of:
(a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the sequence or expression of at least one allele; and (b) screening for an increased risk of developing a lymphatic disorder from the presence or absence of said mutation, wherein the presence of a mutation altering the encoded amino acid sequence or expression of at least one VEGFR-3 allele in the nucleic acid correlates with an increased risk of developing a lymphatic disorder.

2. A method according to claim 1 wherein the assaying step comprises determining the presence or absence of a mutation altering a tyrosine kinase domain amino acid sequence of the protein encoded by the VEGFR-3 allele.

3. A method according to claim 1 wherein the assaying step comprises determining the presence or absence of a missense mutation in a VEGFR-3 allele at a position corresponding to one of codons 857, 1041, 1044 and 1049 of the VEGFR-3-encoding sequence set forth in SEQ ID NO: 1.

4. A method according to claim 1 wherein the assaying step comprises determining the presence or absence of a missense mutation in the VEGFR-3 allele at a position corresponding to codon 1114 of the VEGFR-3-encoding sequence set forth in SEQ ID NO: 1.

5. A method according to claim 1 wherein the assaying step comprises at least one procedure selected from the group consisting of:
(a) determining a nucleotide sequence of at least one codon of at least one VEGFR-3 allele of the human subject;
(b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences;
(c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.

6. A method according to claim 1 wherein the assaying step comprises:
performing a polymerise chain reaction (PCR) to amplify nucleic acid comprising VEGFR-3 coding sequence, and determining nucleotide sequence of the amplified nucleic acid.

7. A method of screening for a VEGFR-3 hereditary lymphedema genotype in a human patient, comprising the steps of:
(a) providing a biological sample comprising nucleic acid from said patient, said nucleic acid including sequences corresponding to said patient's alleles;
(b) analyzing said nucleic acid for the presence of a mutation or mutations;
(c) determining a VEGFR-3 genotype from said analyzing step; and (d) correlating the presence of a mutation in a VEGFR-3 allele with a hereditary lymphedema genotype.

8. The method according to claim 7 wherein said biological sample is a cell sample.

9. The method according to claim 7 wherein said analyzing comprises sequencing a portion of said nucleic acid, said portion comprising at least one codon of said VEGFR-3 alleles.

10. The method according to claim 7 wherein said nucleic acid is DNA

11. The method according to claim 7 wherein said nucleic acid is RNA.

12. A method of treatment for hereditary lymphedema, comprising the step of administering to a patient with hereditary lymphedema a therapeutically effective amount of a growth factor product selected from the group consisting of vascular endothelial growth factor C (VEGF-C) protein products, vascular endothelial growth factor D (VEGF-D) protein products, VEGF-C gene therapy products, and VEGF-D
gene therapy protein products.

13. A therapeutic or prophylactic method of treating lymphedema, comprising the steps of:
providing isolated lymphatic endothelial cells or lymphatic endothelial progenitor cells;
transforming or transfecting the cells ex vivo with a polynucleotide comprising a nucleotide sequence that encodes a wild type VEGFR-3;
and administering the transformed or transfected cells to the mammalian subject.

14. An oligonucleotide useful as a probe for identifying polymorphisms in a human Flt4 receptor tyrosine kinase gene, the oligonucleotide comprising 6-50 nucleotides that have a sequence that is identical or exactly complementary to a portion of a wild type human VEGFR-3 gene sequence or VEGFR-3 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.

15. An oligonucleotide according to claim 14 wherein the nucleotide sequence is exactly identical or exactly complementary to a portion of the human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, except for a nucleotide substitution at a position corresponding to a nucleotide selected from the group consisting of bases 2546 through 2848 and bases 3044 through 3514.

16. An oligonucleotide according to claim 14 wherein the nucleotide sequence is exactly identical or exactly complementary to a portion of the human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, except for a nucleotide substitution at a position corresponding to nucleotide 3360 of SEQ ID NO: 1.

17. An oligonucleotide according to claim 14 wherein the nucleotide sequence is exactly identical or exactly complementary to a portion of the human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, except for a nucleotide substitution at a position corresponding to a nucleotide selected from the group consisting of position 2588, position 3141, position 3150 and position 3164 of SEQ ID
NO: 1.

18. A kit comprising at least two oligonucleotides of the formula X n YZ m or its complement;
where n and m are integers from 0 to 49;
where 5 ~ (n + m) ~ 49;
where X n is a stretch of n nucleotides identical to a first portion of SEQ
ID NO: 1, said first portion ending immediately upstream (5') of position 3360 of SEQ
ID NO: 1; and where Z m is a stretch of m nucleotides identical to a second portion of SEQ ID NO: 1, said second portion beginning immediately downstream (3') of position 3360 of SEQ ID NO: 1; and wherein Y represents a nucleotide selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil nucleotides.

19. A kit comprising at least two oligonucleotides of the formula X n YZ m or its complement;
where n and m are integers from 0 to 49;
where 5 ~ (n + m) ~ 49;
where X n is a stretch of n nucleotides identical to a first portion of SEQ
ID NO: 1, said first portion ending immediately upstream (5') of position W of SEQ
ID NO: 1; and where Z m, is a stretch of m nucleotides identical to a second portion of SEQ ID NO: 1,,said second portion beginning immediately downstream (3') of position W of SEQ ID NO: 1;
wherein position W of SEQ ID NO: 1 is selected from the group consisting of nucleotides 2588, 3141, 3150, and 3164 of SEQ ID NO: 1; and wherein Y represents a nucleotide selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil nucleotides.

20. An array of oligonucleotide probes immobilized on a solid support, wherein each probe occupies a separate known site in the array; and wherein the array includes at least one probe set comprising two to four probes, wherein one probe is exactly identical or exactly complementary to a wild type human VEGFR-3 coding sequence, and the other one to three members of the set are exactly identical to the first member, but for at least one different nucleotide, which different nucleotide is located in the same position in each of the one to three additional set members.

21. An array of oligonucleotide probes immobilized on a solid support according to claim 20, wherein each probe occupies a separate known site in the array;
and wherein the array includes at least one probe set comprising two to four probes, wherein one probe is exactly identical or exactly complementary to a portion of a human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, and the other one to three members of the set are exactly identical to the first member, but for at least one different nucleotide, which different nucleotide is located in the same position in each of the one to three additional set members, said position corresponding to a position selected from the group consisting of bases 2546 through 2848 and bases 3044 through 3514 of SEQ ID NO: 1.

22. A purified polynucleotide comprising a nucleotide sequence encoding a human VEGFR-3 protein variant, wherein said polynucleotide is capable of hybridizing to the complement of SEQ ID NO: 1 under the following hybridization conditions:
hybridization at 42°C in 50% formamide, 5X SSC, 20 mM Na~PO 4, pH 6.8;
and washing in 0.2X SSC at 55°C;
and wherein the encoded VEGFR-3 protein variant has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 2 at one or more positions selected from the group consisting of amino acids 843 to 943 of SEQ
ID
NO: 2 and amino acids 1009 to 1165 of SEQ ID NO: 2.

23. A purified polynucleotide according to claim 22, wherein the encoded VEGFR-3 protein variant has an amino acid sequence that differs at position from the amino acid sequence set forth in SEQ ID NO: 2.

24. A purified polynucleotide according to claim 22 wherein the encoded VEGFR-3 protein variant has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 2 at position selected from the group consisting of residues 857, 1041, 1044 and 1049 of SEQ ID NO: 2.

25. A purified polynucleotide comprising a nucleotide sequence encoding a VEGFR-3 protein of a human that is affected with heritable lymphedema;
wherein said polynucleotide is capable of hybridizing to the complement of SEQ ID NO: 1 under the following hybridization conditions: hybridization at 42 ° C
in 50% formamide, 5X SSC, 20 mM Na~PO 4, pH 6.8; and washing in 0.2X SSC at 55°C;
and wherein the polynucleotide encodes a VEGFR-3 amino acid sequence that differs from SEQ ID NO: 2 at at least one residue.

26. A purified polynucleotide according to claim 25 wherein the polynucleotide encodes an amino acid sequence that differs from SEQ ID NO: 2 at at least one residue selected from the group consisting of residues 843 to 943 and 1009 to 1165 of SEQ ID NO: 2.

27. A vector comprising a polynucleotide according to claim 25.

28. A host cell that has been transformed or transfected with a polynucleotide according to claim 25 and that expresses the VEGFR-3 protein encoded by the polynucleotide.

29. A host cell according to claim 28 that has been co-transfected with a polynucleotide encoding the VEGFR-3 amino acid sequence set forth in SEQ ID
NO:
2 and that expresses the VEGFR-3 protein having the amino acid sequence set forth in SEQ ID NO: 2.

30. A method for identifying a modulator of intracellular VEGFR-3 signaling, comprising the steps of:
a) contacting a cell expressing at least one mutant mammalian VEGFR-3 polypeptide in the presence and in the absence of a putative modulator compound;
b) detecting VEGFR-3 signaling in the cell; and c) identifying a putative modulator compound in view of decreased or increased signaling in the presence of the putative modulator, as compared to signaling in the absence of the putative modulator.

31. A method according to claim 30 wherein the cell expresses the mutant mammalian VEGFR-3 polypeptide and a wildtype mammalian VEGFR-3 polypeptide.

32. A method according to claim 31 wherein the mutant and wildtype VEGFR-3 polypeptides are human.

33. A method according to claim 32 wherein said mutant VEGFR-3 polypeptide is characterized by a substitution or deletion mutation in a kinase domain of the VEGFR-3 polypeptide.

34. A method according to claim 32 wherein said mutant VEGFR-3 polypeptide is characterized by at least one substitution or deletion of the wild type VEGFR-3 amino acid sequence set forth in SEQ ID NO: 2, said at least one substitution or deletion occurring at a position corresponding to a residue selected from positions 843 to 943 and positions 1009 to 1165 of SEQ ID NO: 2.

35. A method according to claim 32 wherein the mutant VEGFR-3 polypeptide comprises a leucine amino acid at the position corresponding to position 1114 of SEQ ID NO: 2.

36. A method according to claim 32 wherein said mutant VEGFR-3 polypeptide is characterized by at least one substitution or deletion of the wild type VEGFR-3 amino acid sequence set forth in SEQ ID NO: 2, said at least one substitution or deletion occurring at a position corresponding to a residue selected from positions 857, 1041, 1044 and 1049, and 1114 of SEQ ID NO: 2.