Screening for lymphatic disorders involving the FLT4...

Details Screening for lymphatic disorders involving the FLT4... Screening for lymphatic disorders involving the FLT4...

1999-08-16

2004-07-20

Forman, BJ (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300, C536S024310, C536S024330

Reexamination Certificate

active

06764820

ABSTRACT:

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, Fla. (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, Fla. (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-like tyrosine kinase 4), cloned from human erythroleukaemia cell and placental cDNA libraries. [See U.S. Pat. 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); andPajusola et al.,
Cancer Res.,
52:5738-5743 (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 Flt4l (long, Genbank Accession Nos. X68203 and S66407, 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,
11: 921-931 (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 Flt4l [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: 3545-3555 (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: 548-553 (1998); Joukov et al, EAfBOJ, 16: 3898-3911; and Joukov et al.,
EMBO J,
15: 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 transgenic 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 of the 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 lymnphedema 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 5q34-q35; and th

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