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LYMPHEDEMA GENETICS

FOXC2 GENE - VEGFC2 / VEGFR3 - SOX18 - GJC2 - FLT4 - GATA2 - CCBE1 - FLT4

This page has been updated, for current information please see: 

Lymphedema Gene  CCBE1

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_ccbe1

Lymphedema Gene GATA2

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_gata2

Lymphedema Gene GJC2 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_gjc2

Lymphedema Gene  FLT4

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_flt4

Lymphedema Gene FOXC2

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2

Lymphedema Gene VEGFC

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc

Lymphedema Gene SOX18 Lymphedema Gene

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18

Home page: Lymphedema People

http://www.lymphedemapeople.com/

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There is finally much research going on regarding genetics and lymphedema. The specific gene (FOXC2) that is responsible for LE has been identified and experiments are being conducted in gene therapy with mice.

The FOXC2 is referred to as a forkhead gene, one of 17 thus far identified in humans.  Because it is a pleiotrophic developmental gene, a mutation can cause multiple effects. 

While this research is in its infancy, it does bring a very big light of hope that one day primary lymphedema can be stopped or prevented.

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Advances from Molecular to Clinical Lymphology

Marlys H. Witte, M.D.

University of Arizona HSC
Tucson, Arizona


The lymphatic system–composed of lymphatic vessels, lymph, lymph nodes, and lymphocytes (and other immunocytes)—is a distinctive vasculature (open junctions, anchoring filaments, valves, and intrinsic contractility), different from but similar to the blood vasculature; an integral component of the plasma-tissue fluid-lymph circulation (the “blood-lymph loop”); and the center of the immune system. Interference with the blood-lymph loop produces swelling, scarring, nutritional and immunodysregulatory disorders as well as disturbances in (lymph-hem)angiogenesis (“lymphedema-angiodysplasia [LE-AD] syndromes”).From a physiologic standpoint, edema represents an imbalance between the amount of “lymph” entering a tissue or organ (“lymph formation,” a process regulated by Starling’s law of transcapillary fluid exchange), and the amount of lymph exiting through the draining lymphatics (“lymph absorption”). Whereas “high output failure of the lymph circulation” can result from a wide variety of disturbances (e.g., venous hyper-tension, capillary hyperpermeability, hypoproteinemia) that promote increased lymph formation and thereby may overwhelm the limited capacity of the lymphatic circulation to handle an increased lymphatic load, “lymphedema” represents a “low output failure of the lymph circulation” due to a reduced capacity to handle a normal lymph load (e.g., either from a primary, at times hereditary, disturbance in lymphatic growth or secondary to extirpative operations, radiation damage, or filarial infection). Inadequate or reduced lymphatic capacity need not manifest as overt edema or lymphedema unless the lymph load is so exces-sive that it precipitates “system failure.” Successful treatment of either high or low output failure of the lymph circulation by past, current, or future methods depends on restoring “lymph balance” by reducing lymph formation, enhancing lymph absorption, or both, or preferably, by preventing the imbalance from occurring in the first place.Recent advances in molecular biology and the unlocking of the human genome have ushered in the era of “molecular lymphology.” These discoveries, new concepts, and techniques, viewed in the light of pioneering studies by the founders of the discipline of lymphology, are beginning to unravel the poorly understood embryonic development, physiology and pathophysiology of the lymphatic vascular system. Aside from the chromosomal aneuploidies commonly associated with lymphatic anomalies and even fetal demise, through a “reverse genetics” approach, specific genes have now been identified for three monogenic LE-AD conditions, and loci have been mapped for several others. Furthermore, there are close to 40 distinct familial syndromes, most OMIM-listed or cross-referenced, affecting the lymphatic segment of the vascu-lature. Mutations have been identified in endothelial receptor VEGFR3 for lymphatic growth factor VEGF-C in a subpopulation of Milroy syndrome of lymphatic hypoplasia; winged helix transcription factor FOXC2 uniformly in hundreds of patients with lymphedema-distichiasis syndrome with a hyperplastic lymphatic system; and transcription factor SOX18 in 2 families with autosomal recessive hypotrichosis-lymphedema-telangiectasia syndrome. Through a “forward genetics” approach, transgenic mouse models of LE-AD have implicated still other growth factor ligand-receptor families (e.g., the angiopoietin-tie system) and transcription factors in lymphatic development. The combination of these advances in “molecular lym-phology” with fresh insights and refined tools in “clinical lymphology,” particularly in non-invasive lymphatic 92system imaging, has opened up unparalleled opportunities in “translational lymphology”—bench to bed-side to community—for early detection, monitoring, and more rational classification of lymphatic disease. In addition, novel and improved therapeutic approaches including designer drugs, gene transfer, stem cell therapy, and tissue engineering, to control and modulate lymphatic growth and function should result.

Society for Vascular Medicine and Biology

2004

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LYMPHEDEMA, HEREDITARY, I

Alternative titles; symbols

NONNE-MILROY LYMPHEDEMA
LYMPHEDEMA, EARLY-ONSET
PRIMARY CONGENITAL LYMPHEDEMA;
PCLGene map locus 5q35.3

TEXT

A number sign (#) is used with this entry because hereditary lymphedema type I is caused by mutation in the FLT4 gene (136352), which encodes the vascular endothelial growth factor receptor-3.

See hereditary lymphedema type II, also known as Meige lymphedema (153200), and the lymphedema-distichiasis syndrome (153400) for disorders with related phenotypes.

CLINICAL FEATURES

Milroy (1928), a physician in Omaha, Nebraska, described the disorder in a family in which many of the affected persons were prominent in public and professional life. Rosen et al. (1962) observed congenital chylous ascites in an affected infant whose father had recurrent swelling of the scrotum beginning at the age of 20 years. Marked loss of albumin into the intestinal tract with consequent hypoproteinemia was demonstrated. In 2 patients, Hurwitz and Pinals (1964) observed persistent bilateral pleural effusion in which the protein content of the pleural fluid was high. Esterly (1965) described a family with 15 affected members of 3 generations. One child had striking congenital edema of the hands as a main feature and a second had similar swelling of the hands, as well as bilateral involvement of the legs and feet. A sib of the proposita had no apparent lymphedema, although 2 of his 4 children had bilateral swelling of the legs and feet. He was regarded at first as a 'skipped' generation similar to those noted in previous pedigrees of Milroy disease. Closer examination, however, demonstrated a definite 3 x 5 cm area of slight edema on the medial aspect of the left lower leg. This area was warm to the touch and could be pitted against the underlying tibia. High blood flow in the leg affected by congenital lymphedema has been thought to be due to accumulation of vasodilatory metabolites. Lymphedematous legs generally feel warm and the patients have warm feet. The proposita in the family reported by Esterly (1965) could recover the newspaper from her front walk in her bare feet in winter without discomfort. Esterly (1965) reviewed 22 previously documented pedigrees which, with his own family, gave a total of 152 affected persons. 30 MEDLINE Neighbors

Ferrell et al. (1998) studied 13 lymphedema families from the U.S. and Canada. All members of these families were of western European ancestry. 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 ranged from prenatal (diagnosed by ultrasound) to age 55 years. When affected x normal matings were analyzed, 76 of 191 children were affected, yielding a penetrance of 80%. 30 MEDLINE Neighbors

INHERITANCE

Holberg et al. (2001) performed a complex segregation analysis and a genomewide search for linkage in 6 previously described families with Milroy congenital lymphedema. Results confirmed that Milroy lymphedema is generally inherited as a dominant condition, but this mode of inheritance did not account for all observed familial correlations. The authors suggested that shared environmental or additional genetic factors may also be important in explaining the observed familial aggregation. 30 MEDLINE Neighbors

MAPPING

In linkage studies of 3 multigeneration families demonstrating hereditary lymphedema segregating as an autosomal dominant with incomplete penetrance, Ferrell et al. (1998) demonstrated a 2-point lod score of 6.1 at theta = 0.0 for marker D5S1354 and a maximum multipoint lod score of 8.8 at marker D5S1354 located at 5q34-q35. Linkage analysis in 2 additional families using markers from the linked region showed 1 family consistent with linkage to distal chromosome 5; in the second family, linkage to 5q was excluded for all markers in the region. 30 MEDLINE Neighbors

Evans et al. (1999) carried out a genomewide search in a 4-generation North American family with what they termed 'dominantly inherited primary congenital lymphedema.' They established linkage to markers from the 5q35.3 region in this family and in 4 additional British families. The locus appeared to be situated in the most telomeric region of 5q35.3. No recombination was observed with D5S408 (lod = 10.03) and D5S2006 (lod = 8.46), with a combined multipoint score of 16.55. Four unaffected subjects were identified as gene carriers and provided an estimated penetrance ratio of 0.84 for this disorder. 30 MEDLINE Neighbors

MOLECULAR GENETICS

In a family with hereditary lymphedema, Ferrell et al. (1998) identified a mutation in the FLT4 gene (136352.0001). In several families with autosomal dominant hereditary lymphedema, Karkkainen et al. (2000) identified different mutations in the FLT4 gene (see, e.g., 136352.0002).

ANIMAL MODEL

Congenital lymphedema is autosomal dominant in the pig (9,10:Van der Putte, 1978, 1978).

REFERENCES

1. Esterly, J. R. :
Congenital hereditary lymphoedema. J. Med. Genet. 2: 93-98, 1965.
2. Evans, A. L.; Brice, G.; Sotirova, V.; Mortimer, P.; Beninson, J.; Burnand, K.; Rosbotham, J.; Child, A.; Sarfarazi, M. :
Mapping of primary congenital lymphedema to the 5q35.3 region. Am. J. Hum. Genet. 64: 547-555, 1999.
PubMed ID : 9973292
3. Ferrell, R. E.; Levinson, K. L.; Esman, J. H.; Kimak, M. A.; Lawrence, E. C.; Barmada, M. M.; Finegold, D. N. :
Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum. Molec. Genet. 7: 2073-2078, 1998.
PubMed ID : 9817924
4. Holberg, C. J.; Erickson, R. P.; Bernas, M. J.; Witte, M. H.; Fultz, K. E.; Andrade, M.; Witte, C. L. :
Segregation analyses and a genome-wide linkage search confirm genetic heterogeneity and suggest oligogenic inheritance in some Milroy congenital primary lymphedema families. Am. J. Med. Genet. 98: 303-312, 2001.
PubMed ID : 11170072
5. Hurwitz, P. A.; Pinals, D. J. :
Pleural effusion in chronic hereditary lymphedema (Nonne, Milroy, Meige's disease): report of two cases. Radiology 82: 246-248, 1964.
PubMed ID : 14115303
6. Karkkainen, M. J.; Ferrell, R. E.; Lawrence, E. C.; Kimak, M. A.; Levinson, K. L.; McTigue, M. A.; Alitalo, K.; Finegold, D. N. :
Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nature Genet. 25: 153-159, 2000.
PubMed ID : 10835628
7. Milroy, W. F. :
Chronic hereditary edema: Milroy's disease. J.A.M.A. 91: 1172-1175, 1928.
8. Rosen, F. S.; Smith, D. H.; Earle, R., Jr.; Janeway, C. A.; Gitlin, D. :
The etiology of hypoproteinemia in a patient with congenital chylous ascites. Pediatrics 30: 696-706, 1962.
9. Van der Putte, S. C. J. :
Congenital hereditary lymphedema in the pig. Lymphology 11: 1-9, 1978.
PubMed ID : 642582
10. Van der Putte, S. C. J. :
The pathogenesis of congenital hereditary lymphedema in the pig. Lymphology 11: 10-21, 1978.
PubMed ID : 642583

CONTRIBUTORS

Cassandra L. Kniffin - reorganized : 11/19/2003
Sonja A. Rasmussen - updated : 3/12/2001
Victor A. McKusick - updated : 2/10/1999
Victor A. McKusick - updated : 1/6/1999

http://omim.org/entry/153100

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LYMPHEDEMA, HEREDITARY, II

Alternative titles; symbols

MEIGE LYMPHEDEMA
LYMPHEDEMA, LATE-ONSET
LYMPHEDEMA PRAECOX 
Gene map locus 16q24.3

TEXT

A number sign (#) is used with this entry because of evidence that hereditary lymphedema type II is caused by mutation in the forkhead family transcription factor gene MFH1 (FOXC2; 602402). Allelic disorders with overlapping features include the lymphedema-distichiasis syndrome (153400), lymphedema and ptosis (153000), and lymphedema and yellow nail syndrome (153300). Also see hereditary lymphedema type I, or Milroy disease (153100). 30 MEDLINE Neighbors

CLINICAL FEATURES

Edema, particularly severe below the waist, develops about the time of puberty. Meige (1898) described 8 cases in 4 generations without male-to-male transmission. Goodman (1962) reported the condition in 2 sisters and a brother with presumed normal parents who were not known to be related. Herbert and Bowen (1983) described a kindred with many cases of lymphedema of postpubertal onset. Involvement of the upper limbs (as well as the lower limbs), face, and larynx and, in one, a persistent pleural effusion were notable features. Scintilymphangiography indicated paucity or absence of lymph nodes in the axillae and above the inguinal ligaments. Chronic facial swelling resulted in a characteristic appearance of affected members including puffiness, shiny skin, deep creases, and, in some, excessive wrinkling. Emerson (1966) noted similar facial features and remarked on the possible erroneous diagnosis of myxedema. 30 MEDLINE Neighbors

Herbert and Bowen (1983) noted the difficulties of nosology. For example, because lymphedema and yellow nail syndrome has yellow or dystrophic nails as a variable feature, this could be the same disorder. They pointed also to the association of late-onset lymphedema with deafness (Emberger et al., 1979) and with primary pulmonary hypertension and cerebrovascular malformations (152900; Avasthey and Roy, 1968). 30 MEDLINE Neighbors

Figueroa et al. (1983) reported the association of cleft palate. In their family, the mother, with only lymphedema praecox of the legs, gave birth to 5 sons, 3 of whom had both lymphedema of the legs and cleft palate. A mild form of lymphedema affecting mainly the medial aspect of both ankles in a 21-year-old son was pictured. 30 MEDLINE Neighbors

Andersson et al. (1995) described a family in which 3 individuals, a grandmother, her son and her grandson, had onset of lymphedema in their mid-20s or 30s. The grandson was 23 years old when he had his first episode of lymphedema, which was thought to be due to thrombophlebitis. During the ensuing decade, he had episodic waxing and waning of lymphedema of both lower limbs and was treated with anticoagulant therapy. At the age of 35, he developed lymphangiosarcoma on the inner right thigh and died of metastases some months later. Lymphangiosarcoma, usually associated with postmastectomy lymphedema, had not been described previously in late-onset hereditary lymphedema. Andersson et al. (1995) raised the question of whether a genetic predisposition to malignancy combined with the lymphedema was etiologically significant. There seemed to be an unusually high frequency of cancer (uterine, colon, lung, prostate, breast, and bone) in the proband's family. 30 MEDLINE Neighbors

MOLECULAR GENETICS

Finegold et al. (2001) found a mutation in the FOXC2 gene (602402.0007) in a family with Meige lymphedema and also in a family with yellow nail syndrome.

HETEROGENEITY

Finegold et al. (2001) noted that the phenotypic classification of dominantly inherited lymphedema includes Milroy disease (hereditary lymphedema I), Meige lymphedema (hereditary lymphedema II), lymphedema-distichiasis syndrome, lymphedema and ptosis, and yellow nail syndrome. The phenotypes reported in their 11 families overlapped the findings reported in Meige lymphedema, lymphedema-distichiasis syndrome, lymphedema and ptosis, and yellow nail syndrome, but not in Milroy disease. Milroy disease is associated with mutation in the FLT4 gene (136352), whereas mutations in the FOXC2 gene were observed in the 4 lymphedema syndromes that had phenotypic overlap. 30 MEDLINE Neighbors

SEE ALSO

Juchems (1963); Osterland (1961); Wheeler et al. (1981)

REFERENCES

1. Andersson, H. C.; Parry, D. M.; Mulvihill, J. J. :
Lymphangiosarcoma in late-onset hereditary lymphedema: case report and nosological implications. Am. J. Med. Genet. 56: 72-75, 1995.
PubMed ID : 7747790
2. Avasthey, P.; Roy, S. B. :
Primary pulmonary hypertension, cerebrovascular malformation, and lymphoedema of the feet in a family. Brit. Heart J. 30: 769-775, 1968.
PubMed ID : 5718986
3. Emberger, J. M.; Navarro, M.; Dejean, M.; Izarn, P. :
Surdi-mutite, lymphoedeme des membres inferieurs et anomalies hematologiques (leucose aigue cytopenies) a transmission autosomique dominante. J. Genet. Hum. 27: 237-245, 1979.
PubMed ID : 295075
4. Emerson, P. A. :
Yellow nails, lymphoedema, and pleural effusions. Thorax 21: 247-253, 1966.
PubMed ID : 5914998
5. Figueroa, A. A.; Pruzansky, S.; Rollnick, B. R. :
Meige disease (familial lymphedema praecox) and cleft palate: report of a family and review of the literature. Cleft Palate J. 20: 151-157, 1983.
PubMed ID : 6342849
6. Finegold, D. N.; Kimak, M. A.; Lawrence, E. C.; Levinson, K. L.; Cherniske, E. M.; Pober, B. R.; Dunlap, J. W.; Ferrell, R. E. :
Truncating mutations in FOXC2 cause multiple lymphedema syndromes. Hum. Molec. Genet. 10: 1185-1189, 2001.
PubMed ID : 11371511
7. Goodman, R. M. :
Familial lymphedema of the Meige's type. Am. J. Med. 32: 651-656, 1962.
8. Herbert, F. A.; Bowen, P. A. :
Hereditary late-onset lymphedema with pleural effusion and laryngeal edema. Arch. Intern. Med. 143: 913-915, 1983.
PubMed ID : 6679236
9. Juchems, R. :
Das hereditaere Lymphoedem, Typ Meige. Klin. Wschr. 41: 328-332, 1963.
10. Meige, H. :
Dystrophie oedemateuse hereditaire. Presse Med. 6: 341-343, 1898.
11. Osterland, G. :
Beobachtungen zum Nonne-Milroy-Meige-Syndrom. Z. Menschl. Vererb. Konstitutionsl. 36: 108-117, 1961.
PubMed ID : 14482593
12. Wheeler, E. S.; Chan, V.; Wassman, R.; Rimoin, D. L.; Lesavoy, M. A. :
Familial lymphedema praecox: Meige's disease. Plast. Reconst. Surg. 67: 362-364, 1981.
PubMed ID : 7232571

CONTRIBUTORS

George E. Tiller - updated : 10/22/2001

http://omim.org/entry/153200

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FMS-LIKE TYROSINE KINASE 4; FLT4

Alternative titles; symbols

VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3; VEGFR3 Gene map locus 5q35.3

TEXT

CLONING

By screening a placenta cDNA library with a mouse Flt3 probe, Galland et al. (1992) isolated a human gene encoding a putative receptor-type tyrosine kinase. The deduced amino acid sequence of the intracellular portion of the molecule showed that it was strongly related to FLT1 (165070) and KDR (191306) and to a lesser degree to members of the class III receptor-type tyrosine kinases: FMS (164770), PDGFR (173490, 173410), KIT (164920), and FLT3 (136351). 30 MEDLINE Neighbors

MAPPING

Galland et al. (1992) mapped FLT4 to 5q34-q35, telomeric to the FMS and PDGFRB genes, by in situ hybridization. They assigned the mouse homolog to chromosome 11 by the same method. In the process of creating a radiation hybrid map of 18 genes, Warrington et al. (1992) demonstrated that the FLT4 gene is located on distal 5q between GABRA1 (137160) at 5q34-q35 and DRD1 (126449) at 5q35.1. Aprelikova et al. (1992) also mapped the FLT4 gene to 5q33-qter. 30 MEDLINE Neighbors

GENE FUNCTION

Among the factors stimulating angiogenesis, the acidic and basic fibroblast growth factors FGF1 (131220) and FGF2 (134920) and the vascular endothelial growth factor VEGF (192240) exert their effects via specific cell surface receptor tyrosine kinases: for FGF1 and FGF2, FGF receptor-1 (FGFR1; 136350), also known as FLT2, and the endothelial-specific FMS-like tyrosine kinase-1; and for VEGF, the KDR/FLK1 receptor. The protein product of the FLT4 receptor tyrosine kinase cDNA is structurally similar to the FLT1 and KDR/FLK1 receptors (Pajusola et al., 1992), but FLT4 does not bind VEGF (Pajusola et al., 1994). Lee et al. (1996) identified and characterized a vascular endothelial growth factor-related protein (VEGFC; 601528) that specifically binds to the extracellular domain of Flt4 and stimulates tyrosine phosphorylation and mitogenesis of endothelial cells. 30 MEDLINE Neighbors

Kaipainen et al. (1995) analyzed the expression of FLT4 by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. In adult human tissues, only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. The results suggested that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also supported the theory of the venous origin of lymphatic vessels. 30 MEDLINE Neighbors

Vascular endothelial growth factor is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGFC stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR3. Dumont et al. (1998) showed that targeted inactivation of the VEGFR3 gene in mice resulted in defective blood vessel development in early embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels. 30 MEDLINE Neighbors

MOLECULAR GENETICS

In affected members of a family with hereditary lymphedema type I (153100), Ferrell et al. (1998) identified a mutation in the FLT4 gene (136352.0001).

Karkkainen et al. (2000) identified mutations at the FLT4 locus in several families with hereditary lymphedema type I. They found that all disease-associated alleles analyzed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. These studies established that vascular endothelial growth factor receptor-3 is important for normal lymphatic vascular function. 30 MEDLINE Neighbors

In a family with hereditary lymphedema, Irrthum et al. (2000) identified a mutation in the FLT4 gene (136352.0006) that cosegregated with the disease. In vitro expression showed that this mutation inhibited the autophosphorylation of the receptor.

Kim and Dumont (2003) reviewed molecular mechanisms in lymphangiogenesis and their implications for human disease. In addition to VEGFR3 and FOXC2 (602402), 6 'lymphangiogenic markers' were reviewed. The role of some of these lymphangiogenetic mechanisms in cancer and metastasis was also reviewed. 30 MEDLINE Neighbors

ANIMAL MODEL

The Chy mouse mutant, characterized by accumulation of chylous ascites and swelling of the limbs, was obtained by ethylnitrosourea-induced mutagenesis (12,13:Lyon and Glenister, 1984, 1986). The phenotype is linked to mouse chromosome 11. Karkkainen et al. (2001) sequenced the Vegfr3 candidate gene on chromosome 11 in Chy mice and found a heterozygous 3157A-T mutation resulting in an ile1053-to-phe (I1053F) substitution in the tyrosine kinase domain. This mutation was located in a highly conserved catalytic domain of the receptor, in close proximity to the VEGFR3 mutations in human primary lymphedema. The I1053F mutant receptor was tyrosine kinase inactive. Although lymphedema patients with heterozygous missense mutations of VEGFR3 retain some receptor activity because of the presence of the wildtype allele (Karkkainen et al., 2000), the mutant VEGFR3 can be classified as a dominant-negative receptor similar to certain mutant KIT receptors in piebaldism (172800) and RET receptors (164761) in Hirschsprung disease (142623). Karkkainen et al. (2001) found that neuropilin-2 (NRP2; 602070) bound VEGFC and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia. This may explain why hypoplastic cutaneous, but not visceral, lymphatic vessels were found in the Chy mice. Using virus-mediated VEGFC gene therapy, Karkkainen et al. (2001) generated functional lymphatic vessels in the lymphedema mice. The results suggested that growth factor gene therapy is applicable to human lymphedema as well and provided a paradigm for other diseases associated with mutant receptors, i.e., ligand therapy. 30 MEDLINE Neighbors

ALLELIC VARIANTS (selected examples)

.0001 LYMPHEDEMA, HEREDITARY, I [FLT4, PRO1126LEU]

In a nuclear family with hereditary lymphedema type I (153100), Ferrell et al. (1998) identified a 3360G-A transition in the FLT4 gene, predicted to cause a nonconservative pro1126-to-leu (P1126L) substitution in the mature receptor.

.0002 LYMPHEDEMA, HEREDITARY, I [FLT4, GLY857ARG ]

In a family with hereditary lymphedema (153100) in members of 3 generations, Karkkainen et al. (2000) identified a heterozygous G-A transition in the FLT4 gene, resulting in a gly857-to-arg (G857R) substitution.

.0003 LYMPHEDEMA, HEREDITARY, I [FLT4, ARG1041PRO ]

In a family with hereditary lymphedema (153100) in at least 4 generations, Karkkainen et al. (2000) identified a mutation in the FLT4 gene, resulting in an arg1041-to-pro (R1041P) substitution.

.0004 LYMPHEDEMA, HEREDITARY, I [FLT4, LEU1044PRO ]

In a large family with autosomal dominant lymphedema (153100) in 5 generations and many different sibships, Karkkainen et al. (2000) identified a transition in the FLT4 gene, resulting in a leu1044-to-pro (L1044P) substitution.

.0005 LYMPHEDEMA, HEREDITARY, I [FLT4, PRO1114LEU ]

In a mother and 2 daughters with primary lymphedema (153100), Karkkainen et al. (2000) identified a pro1114-to-leu (P1114L) missense mutation of the FLT4 gene.

.0006 LYMPHEDEMA, HEREDITARY, I [FLT4, HIS1035ARG]

In a family in which the father and 4 of 7 children had congenital lymphedema (153100), Irrthum et al. (2000) demonstrated a his1035-to-arg (H1035R) missense mutation in the FLT4 gene.

.0007 HEMANGIOMA, CAPILLARY INFANTILE, SOMATIC [FLT4, PRO954SER ]

In 1 of 15 infantile hemangioma (602089) specimens, Walter et al. (2002) found a pro954-to-ser (P954S) missense mutation in the kinase insert of the FLT4 gene. This result, and the finding of a somatic missense mutation in the VEGFR2 gene (191306.0001) in another of the 15 specimens, suggested that alteration of the FLT4 signaling pathway in endothelial and/or pericytic cells may be a mechanism involved in hemangioma formation. 30 MEDLINE Neighbors

SEE ALSO

Evans et al. (1999); Milroy (1892); Offori et al. (1993)

REFERENCES

1. Aprelikova, O.; Pajusola, K.; Partanen, J.; Armstrong, E.; Alitalo, R.; Bailey, S. K.; McMahon, J.; Wasmuth, J.; Huebner, K.; Alitalo, K. :
FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res. 52: 746-748, 1992.
PubMed ID : 1310071
2. Dumont, D. J.; Jussila, L.; Taipale, J.; Lymboussaki, A.; Mustonen, T.; Pajusola, K.; Breitman, M.; Alitalo, K. :
Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282: 946-949, 1998.
PubMed ID : 9794766
3. Evans, A. L.; Brice, G.; Sotirova, V.; Mortimer, P.; Beninson, J.; Burnand, K.; Rosbotham, J.; Child, A.; Sarfarazi, M. :
Mapping of primary congenital lymphedema to the 5q35.3 region. Am. J. Hum. Genet. 64: 547-555, 1999.
PubMed ID : 9973292
4. Ferrell, R. E.; Levinson, K. L.; Esman, J. H.; Kimak, M. A.; Lawrence, E. C.; Barmada, M. M.; Finegold, D. N. :
Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum. Molec. Genet. 7: 2073-2078, 1998.
PubMed ID : 9817924
5. Galland, F.; Karamysheva, A.; Mattei, M.-G.; Rosnet, O.; Marchetto, S.; Birnbaum, D. :
Chromosomal localization of FLT4, a novel receptor-type tyrosine kinase gene. Genomics 13: 475-478, 1992.
PubMed ID : 1319394
6. Irrthum, A.; Karkkainen, M. J.; Devriendt, K.; Alitalo, K.; Vikkula, M. :
Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet. 67: 295-301, 2000.
PubMed ID : 10856194
7. Kaipainen, A.; Korhonen, J.; Mustonen, T.; van Hinsbergh, V. W. M.; Fang, G.-H.; Dumont, D.; Breitman, M.; Alitalo, K. :
Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Nat. Acad. Sci. 92: 3566-3570, 1995.
PubMed ID : 7724599
8. Karkkainen, M. J.; Ferrell, R. E.; Lawrence, E. C.; Kimak, M. A.; Levinson, K. L.; McTigue, M. A.; Alitalo, K.; Finegold, D. N. :
Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nature Genet. 25: 153-159, 2000.
PubMed ID : 10835628
9. Karkkainen, M. J.; Saaristo, A.; Jussila, L.; Karila, K. A.; Lawrence, E. C.; Pajusola, K.; Bueler, H.; Eichmann, A.; Kauppinen, R.; Kettunen, M. I.; Yla-Herttuala, S.; Finegold, D. N.; Ferrell, R. E.; Alitalo, K. :
A model for gene therapy of human hereditary lymphedema. Proc. Nat. Acad. Sci. 98: 12677-12682, 2001.
PubMed ID : 11592985
10. Kim, H.; Dumont, D. J. :
Molecular mechanisms in lymphangiogenesis: model systems and implications in human disease. Clin. Genet. 64: 282-292, 2003.
PubMed ID : 12974730
11. Lee, J.; Gray, A.; Yuan, J.; Luoh, S.-M.; Avraham, H.; Wood, W. I. :
Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc. Nat. Acad. Sci. 93: 1988-1992, 1996.
PubMed ID : 8700872
12. Lyon, M. F.; Glenister, P. H. :
New Mutants. Mouse Newsletter 71: 26 only, 1984.
13. Lyon, M. F.; Glenister, P. H. :
Gene order of Chy-vt-Re on chromosome 11. Mouse Newsletter 74: 96 only, 1986.
14. Milroy, W. F. :
An undescribed variety of hereditary oedema. New York Med. J. 56: 505-508, 1892.
15. Offori, T. W.; Platt, C. C.; Stephens, M.; Hopkinson, G. B. :
Angiosarcoma in congenital hereditary lymphoedema (Milroy's disease): diagnostic beacons and a review of the literature. Clin. Exp. Derm. 18: 174-177, 1993.
PubMed ID : 8482001
16. Pajusola, K.; Aprelikova, O.; Korhonen, J.; Kaipainen, A.; Pertovaara, L.; Alitalo, R.; Alitalo, K. :
FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res. 52: 5738-5743, 1992.
PubMed ID : 1327515
17. Pajusola, K.; Aprelikova, O.; Pelicci, G.; Weich, H.; Claesson-Welsh, L.; Alitalo, K. :
Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 9: 3545-3555, 1994.
PubMed ID : 7970715
18. Walter, J. W.; North, P. E.; Waner, M.; Mizeracki, A.; Blei, F.; Walker, J. W. T.; Reinisch, J. F.; Marchuk, D. A. :
Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma. Genes Chromosomes Cancer 33: 295-303, 2002.
PubMed ID : 11807987
19. Warrington, J. A.; Bailey, S. K.; Armstrong, E.; Aprelikova, O.; Alitalo, K.; Dolganov, G. M.; Wilcox, A. S.; Sikela, J. M.; Wolfe, S. F.; Lovett, M.; Wasmuth, J. J. :
A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5. Genomics 13: 803-808, 1992.
PubMed ID : 1322355

CONTRIBUTORS

Cassandra L. Kniffin - reorganized : 11/19/2003
Victor A. McKusick - updated : 11/4/2003
Victor A. McKusick - updated : 3/14/2002
Victor A. McKusick - updated : 1/14/2002
Victor A. McKusick - updated : 10/3/2000
Victor A. McKusick - updated : 5/25/2000
Victor A. McKusick - updated : 2/10/1999
Victor A. McKusick - updated : 1/6/1999
Victor A. McKusick - updated : 11/10/1998
Victor A. McKusick - updated : 10/27/1998

http://omim.org/entry/136352

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Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

Anne Saaristo1, Tanja Veikkola1, Tuomas Tammela1, Berndt Enholm1, Marika J. Karkkainen1, Katri Pajusola2, Hansruedi Bueler2, Seppo Ylä-Herttuala3 and Kari Alitalo1

1 Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, the Haartman Institute and Helsinki University Central Hospital, University of Helsinki, 00014 Helsinki, Finland
2 Institute of Molecular Biology, University of Zurich, 8057 Zurich, Switzerland
3 A.I. Virtanen Institute and Department of Medicine, University of Kuopio, 70211 Kuopio, Finland

Address correspondence to Dr. Kari Alitalo, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, P.O.B. 63 (Haartmaninkatu 8), University of Helsinki, 00014 Helsinki, Finland. Phone: 358-9-1912 5511; Fax: 358-9-1912 5510; E-mail: Kari.Alitalo@helsinki.fi

ABSTRACT

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Key Words: lymphedema • lymphatic endothelium • VEGF-C • VEGFR-2 • VEGFR-3

INTRODUCTION

Proangiogenic gene therapy, developed first in the pioneering work of Dr. Jeffrey Isner, has shown great promise in the treatment of cardiovascular ischemic diseases (13). In such studies, angiogenesis has been stimulated for example by overexpression of vascular endothelial growth factor (VEGF)* or various fibroblast growth factors (FGFs). More recent developments also include the use of modified forms of the hypoxia-induced transcription factor (HIF)-1{alpha}, which may orchestrate the induction of several angiogenic mechanisms (4, 5). However, although VEGF is a potent inducer of angiogenesis, the vessels it helps to create are immature, tortuous, and leaky, often lacking perivascular support structures (68). Only a fraction of the blood vessels induced in response to VEGF in the dermis and in subcutaneous fat tissue were stabilized and functional after adenoviral treatment of the skin of nude mice (9, 10), while intramuscular vessels developed into an angioma-like proliferation or regressed with a resulting scar tissue (9, 11). Furthermore, edema induced by VEGF overexpression complicates VEGF-mediated neovascularization, although recent evidence suggests that it can be avoided by providing angiopoietin-1 for vessel stabilization (12, 13).

Lymphatic vessels play an important physiological role in homeostasis, regulation of tissue fluid balance, and in the immune responses to pathogens, yet the molecular mechanisms that control their development and function are only beginning to be elucidated. So far, only two peptide growth factors have been found capable of inducing the growth of new lymphatic vessels in vivo. These factors, VEGF-C and VEGF-D (1416), belong to the larger VEGF family of growth factors which also includes VEGF, placenta growth factor (PlGF), and VEGF-B. VEGF-C and VEGF-D are ligands for the endothelial cell–specific tyrosine kinase receptors VEGFR-2 and VEGFR-3 (17, 18). In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in the lymphatic endothelial cells which line the inner surface of lymphatic vessels (19, 20). Whereas VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development and maintenance of the lymphatic vessels (21). Inhibition of VEGFR-3 signaling using soluble VEGFR-3 which competes for ligand binding with the endogenous receptors led to lymphatic vessel regression in a transgenic mouse model (22). Other molecules that have been reported to be necessary for normal lymphatic development include the transcription factor Prox-1 (23), the integrin {alpha}9 (24), and angiopoietin-2 (25).

Impairment of lymphatic function, which results in inadequate transport of fluid, macromolecules, or cells from the interstitium, is associated with a variety of diseases and leads to tissue edema, impaired immunity and fibrosis (26). Development of strategies for local and controlled induction of lymphangiogenesis would thus be of major importance for the treatment of such diseases. Adenoviral gene transfer of VEGF-C in the skin has been shown to result in a strong lymphangiogenic response (27, 28), but high levels of VEGF-C also lead to blood vascular effects such as increased vessel leakiness, presumably through the interaction of VEGF-C with the VEGFR-2 expressed on blood vascular endothelium (28). To develop a lymphatic-specific gene therapy approach without the unwanted blood vascular side effects, we have studied the potential of a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) as a therapeutic agent in lymphedema. We demonstrate that stimulation of VEGFR-3 alone by VEGF-C156S potently induces lymphangiogenesis both in transgenic embryos and after virus-mediated gene transfer. In a lymphedema mouse model functional cutaneous lymphatic vessels formed after intradermal infection with adeno-associated virus (AAV) encoding VEGF-C156S. Most importantly, VEGF-C156S essentially lacked the blood vascular effects of native VEGF-C.

MATERIAL AND METHODS

Generation and In Vitro Analysis of Recombinant Adenoviruses and AAVs.
For the adenovirus construct, the full-length human VEGF-C156S cDNA (29) was cloned as a BamHI/NotI fragment into the corresponding sites of the pAD BglII vector. Replication-deficient E1-E3 deleted adenoviruses were produced in 293 cells and concentrated by ultracentrifugation (30). Adenoviral preparations were analyzed to be free of helper viruses, lipopolysaccharide, and bacteriological contaminants (31). The adenoviruses encoding human VEGF-C and nuclear targeted LacZ were constructed as described (27, 30). For the AAV construct, the full-length human VEGF-C156S was cloned as a blunt-end fragment into the MluI site of psub-CMV-WPRE plasmid and the rAAV type 2 was produced as described previously (32). AAVs encoding human VEGF-C and EGFP were used as controls (32, 33).

For the analysis of protein expression, 293EBNA cells were infected with recombinant adenoviruses for 2 h in serum-free medium or by AAVs for 8 h in 2% FCS medium. After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig (18) fusion proteins. AdLacZ and AAV-EGFP infected cells were used as negative controls. The bound proteins were precipitated with protein G Sepharose, separated in 15% SDS-PAGE, and analyzed by autoradiography. To compare the protein production levels of AdVEGF-C156S and AdVEGF-C viruses, 20-µl aliquots of the media from AdVEGF-C156S, AdVEGF-C, and AdLacZ infected cell cultures were separated in 15% SDS-PAGE gel and subjected to Western blotting using polyclonal anti–VEGF-C antibodies (R&D Systems).

In Vivo Use and Analysis of the Viral Vectors.
All the studies were approved by the Committee for Animal Experiments of the University of Helsinki. 5 x 108 pfu of the recombinant adenoviruses or 5 x 109-1 x 1011 rAAV particles were injected intradermally into the ears of NMRI nu/nu mice (Harlan) or Chy lymphedema mice (32). The infected nude mice were killed 3, 5, 7, 10, 14, 21, 42, or 56 d after adenoviral infection and 3, 6, or 8 wk after AAV infection. The AAV-infected Chy mice were killed 1, 2, 4, 6, or 8 mo after infection. Total RNA was extracted from the ears (RNAeasy Kit; QIAGEN) 1 to 8 wk after adenoviral infection and 10 wk after AAV-infection. 10 µg of RNA was subjected to Northern blotting and hybridization with a mixture of [{alpha}32P]dCTP (Amersham Biotech) labeled cDNAs specific for VEGF-C. The glyceraldehyde-3-phosphate dehydrogenase cDNA probe was used as an internal control for equal loading. The adenoviral protein expression was confirmed by whole mount ß-galactosidase staining (34) of the AdLacZ-infected ears 1 to 7 wk after gene transfer. The AAV-EGFP-infected ears were studied under the fluorescence microscope at 3 wk to 8 mo after infection.

ß-Galactosidase and Lectin Staining of Vessels.
For visualization of the superficial lymphatic vessels in the K14-VEGF-C156S and K14-VEGF-C embryos (15, 16), staged VEGFR-3+/LacZ embryos were dissected, fixed in 0.2% glutaraldehyde, and stained with X-gal ( Sigma-Aldrich) for ß-galactosidase activity at +37°C. For the analysis of the adult cutaneous lymphatic phenotype, ß-galactosidase staining was performed for dissected adult mouse ear skin.

In some of the adenovirus-infected mice, Lycopersicon esculentum lectin staining was used to visualize the blood vessels in whole mount (12). Biotinylated lectin (1 mg/ml; Vector Laboratories) was injected into the femoral veins of the mice under anesthesia and after 2 min the mice were killed and perfusion fixed with 1% paraformaldehyde (PFA)/0.5% glutaraldehyde in PBS. The tissues were dissected and the biotinylated lectin was visualized by the ABC-DAB peroxidase method (Vectastain and Sigma-Aldrich). Finally the tissues were dehydrated and mounted on slides.

For the gene expression studies of different types of lymphatic vessels, a combination of biotinylated lectin and whole mount ß-galactosidase staining was performed for VEGFR-2+/LacZ (35) and VEGFR-3+/LacZ (36) adult mouse tissues.

Analyses of the Lymphatic and Blood Vessels.
For immunohistochemical analysis the mouse ears were dissected and fixed in 4% PFA. Those ears that were analyzed in whole mount were incubated in 5% H2O2 in methanol for 1 h to block endogenous peroxidase activity. The tissues were then blocked in 3% milk 0.3% Triton-X in PBS overnight, and antibodies against the vascular endothelial marker PECAM-1 ( BD Biosciences) or VEGFR-3 (R&D Systems) were applied overnight at +4°C. The visualization was achieved with either the ABC-DAB peroxidase method or with ABC-alkaline phosphatase using the alkaline phosphatase substrate kit II ( Vector Laboratories). Finally the tissues were flattened and mounted on slides.

5-µm deparaffinized tissue sections were subjected to heat induced epitope retrieval treatment or to an alternative enzyme treatment. The endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 20 min. Antibodies against VEGFR-3 (19), PECAM-1, podoplanin (a gift from Dr. Miguel Quintanilla, Alberto Sols Biomedical Research Institute, Madrid, Spain), or LYVE-1 (a gift from Dr. Erkki Ruoslahti, Burnham Institute, La Jolla, CA) were applied overnight at +4°C and staining was performed using the tyramide signal amplification kit ( NEN Life Science Products) and 3-amino-9-ethyl carbazole ( Sigma-Aldrich). Hematoxylin was used for counterstaining.

To study the function of the cutaneous lymphatic vessels in the Chy lymphedema mice, a small volume of FITC-labeled dextran (MW 464 000; Sigma-Aldrich) was injected intradermally to the periphery of mouse ear. Drainage of the dye via the lymphatic vessels was followed under a fluorescence microscope.

Quantification of the Lymphangiogenic Response.
To quantify the number of lymphatic vessels and branch points at 1 wk after adenoviral infection, six histological sections from ear midline with the highest vessel density were chosen from each study group (AdVEGF-C156S, AdVEGF-C, AdLacZ). The number of LYVE-1–positive vessels and the number of branches in these vessels were counted under a high power microscope. The area analyzed in each sample was 4 mm2.

Permeability Assay.
The right ear of each mouse was infected with AdVEGF-C156S, AdVEGF-C, or AdLacZ virus (5 x 108 pfu). The left ear received either AdLacZ (5 x 108 pfu) or PBS (in the AdLacZ group). 2 wk after the infection a modified Miles permeability assay was performed as described previously (12). 1 µl per gram of mouse weight of 3% Evans Blue was injected into the femoral vein and after 2 min the mice were perfusion fixed with 0.05 M citrate buffer (pH 3.5) in 1% PFA. The ears were dissected, washed, weighed and extracted in formamide at +55°C overnight. The Evans Blue absorbance of the formamide was then measured with a spectrophotometer set at 610 nm, and the leakage (ng/mg) was compared between the right and left ear of the same mouse.

RESULTS

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FORKHEAD BOX C2; FOXC2

Alternative titles; symbols

FORKHEAD, DROSOPHILA, HOMOLOG-LIKE 14; FKHL14
MESENCHYME FORKHEAD 1
; MFH1Gene map locus 16q24.3

TEXT

The 'forkhead' (or winged helix) gene family, originally identified in Drosophila, encodes transcription factors with a conserved 100-amino acid DNA binding motif.

CLONING

Miura et al. (1993) used RT-PCR of brain mRNA to isolate a mouse gene containing a forkhead domain that they designated MFH1 for 'mesenchyme forkhead-1.' They found that MFH1 is expressed temporally in mouse embryos, first in non-notochordal mesoderm and later in developing mesenchyme.

Miura et al. (1997) used the mouse gene to clone the human MFH1 gene, which encodes a predicted 501-amino acid protein with 94% sequence identity to mouse MFH1. Both human and mouse MFH1 are intronless and act as transactivators of transcription in transfected cells.

GENE FUNCTION

Cederberg et al. (2001) identified FOXC2 as a key regulator of adipocyte metabolism. In mice overexpressing Foxc2 in white adipose tissue (WAT) and brown adipose tissue (BAT), the intraabdominal WAT depot was reduced and had acquired a brown fat-like histology, whereas interscapular BAT was hypertrophic. Increased Foxc2 expression had a pleiotropic effect on gene expression in BAT and WAT. There was an induction of the BAT-specific gene Ucp1 (113730) in the intraabdominal WAT depot. The authors also demonstrated a change in steady-state levels of several WAT- and BAT-derived mRNAs that encode genes of importance for adipocyte insulin action, differentiation, metabolism, sensitivity to adrenergic stimuli, and intracellular signaling. The nature of these Foxc2-generated responses was consistent with protection against obesity and related symptoms, such as diet-induced insulin resistance. Furthermore, in wildtype mice, Foxc2 mRNA levels were upregulated by high fat diet, whereas mice with targeted disruption of 1 Foxc2 allele had a decreased interscapular BAT cell mass. Cederberg et al. (2001) concluded that FOXC2 affects adipocyte metabolism by increasing the sensitivity of the beta-adrenergic cAMP protein kinase A (PKA; see 176911) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Furthermore, they stated that increased FOXC2 levels induced by high fat diet seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance, and a likely consequence would be protection against type II diabetes. 30 MEDLINE Neighbors

MAPPING

Kaestner et al. (1996) mapped the respective MFH1 genes to mouse chromosome 8 by linkage analysis and to human chromosome 16q22-q24 by fluorescence in situ hybridization. In mouse, MFH1 is 8 kb from another forkhead family member, designated fkh6; the 2 genes are similarly arranged in humans.

GENE STRUCTURE

Fang et al. (2000) determined that the FOXC2 gene contains a single coding exon and spans approximately 1.5 kb.

MOLECULAR GENETICS

The lymphedema-distichiasis syndrome (153400) is an autosomal dominant disorder that presents with lymphedema of the limbs, with variable age at onset, and double rows of eyelashes. The complications may include cardiac defects, cleft palate, extradural cysts, and photophobia, suggesting a defect in a gene with pleiotropic effects acting during development. Mangion et al. (1999) mapped the disorder to 16q24.3. Fang et al. (2000) found 2 inactivating mutations (602402.0001 and 602402.0002) in the FOXC2 gene in 2 families with lymphedema-distichiasis syndrome. 30 MEDLINE Neighbors

Bell et al. (2001) reported the mutation analysis of 14 families with lymphedema-distichiasis syndrome. All but 1 of these pedigrees had small insertions or deletions in the FOXC2 gene, which seemed likely to produce haploinsufficiency. The mutation sites were scattered throughout the gene. The exceptional family had a missense mutation in the forkhead domain of the protein. 30 MEDLINE Neighbors

Finegold et al. (2001) identified mutations in FOXC2 in 11 of 86 families with lymphedema-distichiasis syndrome; mutations were predicted to disrupt the DNA binding domain and/or C-terminal alpha-helices essential for transcription activation by FOXC2. Broad phenotypic heterogeneity was observed within these families. The authors observed 4 overlapped phenotypically defined lymphedema syndromes: Meige lymphedema (153200), lymphedema-distichiasis syndrome, lymphedema and ptosis (153000), and yellow nail syndrome (153300), but not Milroy disease (153100). The authors stated that the phenotypic classification of autosomal dominant lymphedema does not appear to reflect the underlying genetic causation of these disorders. 30 MEDLINE Neighbors

ANIMAL MODEL

Smith et al. (2000) reported that Mfh1 +/- mice have anterior segment abnormalities similar to those reported in humans with Axenfeld-Rieger anomaly: small or absent canal of Schlemm, aberrantly developed trabecular meshwork, iris hypoplasia, severely eccentric pupils, and displaced Schwalbe line, but with normal intraocular pressure. The penetrance of clinically obvious abnormalities varied with genetic background. In some affected eyes, collagen bundles were half normal diameter, or collagen and elastic tissue were very sparse, suggesting that abnormalities in extracellular matrix synthesis or organization may contribute to development of the ocular phenotypes. No disease-associated mutations were identified in the human homolog FKHL14 in 32 Axenfeld-Rieger anomaly patients. Similar abnormalities were found in Foxc1 +/- (FKHL7; 601090) mice. 30 MEDLINE Neighbors

ALLELIC VARIANTS (selected examples)

.0001 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, TYR99TER]

In a family with lymphedema-distichiasis syndrome (153400), Fang et al. (2000) found that affected members had a C-G change at nucleotide 297, resulting in a tyr99-to-ter (Y99X) substitution in the FOXC2 gene. The first member of this family studied was a fetus that, because of hydrops fetalis, was electively aborted at 17 weeks' gestation. The fetal karyotype was 46,XX. The father was diagnosed with hereditary lymphedema-distichiasis, and 2 sons had distichiasis. An earlier pregnancy was electively aborted because of the presence of hydrops and presumed Turner syndrome, although subsequent pathologic examination did not show internal abnormalities compatible with Turner syndrome. The family history suggested that the hydrops fetalis seen in the 2 fetuses was a result of the lymphedema-distichiasis gene mutation. 30 MEDLINE Neighbors

.0002 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, 4-BP DUP, NT1093]

In affected members of a family with lymphedema-distichiasis syndrome (153400), Fang et al. (2000) found a 4-nucleotide (GGCC) duplication at position 1093 of the coding region of the FOXC2 gene. The mutation, which would create 98 novel amino acids before truncating the protein, lay in the carboxy-terminal region after the forkhead domain. In addition to lymphedema and distichiasis, affected members of the family had cystic hygroma, arachnoid cysts, and cleft palate. 30 MEDLINE Neighbors

.0003 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, 11-BP DEL, NT290]

In a family in which multiple members had LD (153400), Bell et al. (2001) found an 11-bp deletion involving nucleotides 290-300 and resulting in the creation of 361 novel amino acids beginning at codon 96.

.0004 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, 1-BP DEL, 1331A]

In a family in which members in 3 successive generations had LD (153400), Bell et al. (2001) found deletion of 1331A, disrupting codon 443, producing a frameshift, and adding 27 novel amino acids.

.0005 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, 1-BP INS, 209T]

In a family with cases of LD (153400) in 3 successive generations, Bell et al. (2001) found insertion of a T after nucleotide 209, causing disruption of codon 70 and a frameshift with addition of 391 novel amino acids.

.0006 LYMPHEDEMA-DISTICHIASIS SYNDROME [FOXC2, 2-BP INS, 201CT]

In a sporadic case of LD (153400), Bell et al. (2001) found a dinucleotide insertion of CT after nucleotide 201, disrupting codon 67 and causing a frameshift with production of 4 novel amino acids.

.0007 LYMPHEDEMA, HEREDITARY, II [FOXC2, 1-BP INS, 589C]

YELLOW NAIL SYNDROME, INCLUDED

In a family with hereditary lymphedema II (153200), Finegold et al. (2001) found a single base insertion of C after nucleotide 589, causing a frameshift with premature termination at amino acid 463. Age of onset was after puberty, and 1 affected family member had a cleft palate.

In a family with lymphedema and yellow nail syndrome (153300), Finegold et al. (2001) found the same mutation. Three of 7 affected family members also exhibited ptosis, thus demonstrating phenotypic overlap between yellow nail syndrome and lymphedema and ptosis (153000).

.0008 LYMPHEDEMA AND PTOSIS [FOXC2, 1-BP DEL, 505A]

In a family with lymphedema and ptosis (153000), Finegold et al. (2001) found a single base deletion of 505A, causing a frameshift with premature termination at amino acid 202. Age of onset of lymphedema ranged from 8 to 13 years among affected family members.

.0009 DISTICHIASIS, LYMPHEDEMA, AND CLEFT PALATE [FOXC2, 8-BP DEL, NT914]

Bahuau et al. (2002) reported a family showing autosomal dominant segregation of upper- and lower-eyelid distichiasis in 7 relatives over 3 generations, in addition to below-knee lymphedema of pubertal onset in 3. Two children had cleft palate in addition to distichiasis, but without the previously reported association of Pierre Robin sequence (Bell et al., 2001; Brice et al., 2002). Other ophthalmologic anomalies included divergent strabismus and early-onset myopia. Although no family member had pterygium colli, congenital heart disease, or facial dysmorphism, the disorder was linked to markers on chromosome 16q24.3 and was thus proposed to be allelic to lymphedema-distichiasis syndrome (153400). Bahuau et al. (2002) demonstrated an out-of-frame deletion of the FOXC2 gene, 914-921del, segregating with the syndrome. Whether the heterogeneity observed was related to genotype-phenotype correlation, a hypothesis not primarily supported by the apparent loss-of-function mechanism of the mutations, or governed by modifying genes, was undetermined. 30 MEDLINE Neighbors

REFERENCES

1. Bahuau, M.; Houdayer, C.; Tredano, M.; Soupre, V.; Couderc, R.; Vazquez, M.-P. :
FOXC2 truncating mutation in distichiasis, lymphedema, and cleft palate. Clin. Genet. 62: 470-473, 2002.
PubMed ID : 12485195
2. Bell, R.; Brice, G.; Child, A. H.; Murday, V. A.; Mansour, S.; Sandy, C. J.; Collin, J. R. O.; Brady, A. F.; Callen, D. F.; Burnand, K.; Mortimer, P.; Jeffery, S. :
Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene. Hum. Genet. 108: 546-551, 2001.
PubMed ID : 11499682
3. Brice, G.; Mansour, S.; Bell, R.; Collin, J. R. O.; Child, A. H.; Brady, A. F.; Sarfarazi, M.; Burnand, K. G.; Jeffery, S.; Mortimer, P.; Murday, V. A. :
Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J. Med. Genet. 39: 478-483, 2002.
PubMed ID : 12114478
4. Cederberg, A.; Gronning, L. M.; Ahren, B.; Tasken, K.; Carlsson, P.; Enerback, S. :
FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell 106: 563-573, 2001.
PubMed ID : 11551504
5. Fang, J.; Dagenais, S. L.; Erickson, R. P.; Arlt, M. F.; Glynn, M. W.; Gorski, J. L.; Seaver, L. H.; Glover, T. W. :
Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am. J. Hum. Genet. 67: 1382-1388, 2000. Note: Erratum: Am. J. Hum. Genet. 68: 818 only, 2001.
PubMed ID : 11078474
6. Finegold, D. N.; Kimak, M. A.; Lawrence, E. C.; Levinson, K. L.; Cherniske, E. M.; Pober, B. R.; Dunlap, J. W.; Ferrell, R. E. :
Truncating mutations in FOXC2 cause multiple lymphedema syndromes. Hum. Molec. Genet. 10: 1185-1189, 2001.
PubMed ID : 11371511
7. Kaestner, K. H.; Bleckmann, S. C.; Monaghan, A. P.; Schlondorff, J.; Mincheva, A.; Lichter, P.; Schutz, G. :
Clustered arrangement of winged helix genes fkh-6 and MFH-1: possible implications for mesoderm development. Development 122: 1751-1758, 1996.
PubMed ID : 8674414
8. Mangion, J.; Rahman, N.; Mansour, S.; Brice, G.; Rosbotham, J.; Child, A. H.; Murday, V. A.; Mortimer, P. S.; Barfoot, R.; Sigurdsson, A.; Edkins, S.; Sarfarazi, M.; Burnand, K.; Evans, A. L.; Nunan, T. O.; Stratton, M. R.; Jeffery, S. :
A gene for lymphedema-distichiasis maps to 16q24.3. Am. J. Hum. Genet. 65: 427-432, 1999.
PubMed ID : 10417285
9. Miura, N.; Iida, K.; Kakinuma, H.; Yang, X.-L.; Sugiyama, T. :
Isolation of the mouse (MFH-1) and human (FKHL14) mesenchyme fork head-1 genes reveals conservation of their gene and protein structures. Genomics 41: 489-492, 1997.
PubMed ID : 9169153
10. Miura, N.; Wanaka, A.; Tohyama, M.; Tanaka, K. :
MFH-1, a new member of the fork head domain family, is expressed in developing mesenchyme. FEBS Lett. 326: 171-176, 1993.
PubMed ID : 8325367
11. Smith, R. S.; Zabaleta, A.; Kume, T.; Savinova, O. V.; Kidson, S. H.; Martin, J. E.; Nishimura, D. Y.; Alward, W. L. M.; Hogan, B. L. M.; John, S. W. M. :
Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum. Molec. Genet. 9: 1021-1032, 2000.
PubMed ID : 10767326

CONTRIBUTORS

Victor A. McKusick - updated : 4/10/2003
Victor A. McKusick - updated : 12/26/2002
George E. Tiller - updated : 10/17/2001
Stylianos E. Antonarakis - updated : 10/8/2001
Victor A. McKusick - updated : 9/20/2001
Victor A. McKusick - updated : 12/12/2000
George E. Tiller - updated : 5/2/2000

CREATION DATE

Rebekah S. Rasooly : 2/26/1998

http://omim.org/entry/602402

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Truncating mutations in FOXC2 cause multiple lymphedema syndromes

David N. Finegold1,2,+, Mark A. Kimak1, Elizabeth C. Lawrence1, Kara L. Levinson1, Elizabeth M. Cherniske3, Barbara R. Pober3, Jean W. Dunlap1 and Robert E. Ferrell1
1Department of Human Genetics, Graduate School of Public Health and 2Department of Pediatrics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA and 3Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA

Received 14 February 2001; Revised and Accepted 16 March 2001.

DDBJ/EMBL/GenBank accession no. NM_005251.

Abstract

Hereditary lymphedemas are developmental disorders of the lymphatics resulting in edema of the extremities due to altered lymphatic flow. One such disorder, the lymphedema-distichiasis syndrome, has been reported to be caused by mutations in the forkhead transcription factor, FOXC2. We sequenced the FOXC2 gene in 86 lymphedema families to identify mutations. Eleven families were identified with mutations predicted to disrupt the DNA binding domain and/or C-terminal -helices essential for transcription activation by FOXC2. Broad phenotypic heterogeneity was observed within these families. The phenotypes observed overlapped four phenotypically defined lymphedema syndromes. FOXC2 appears to be the primary cause of lymphedema-distichiasis syndrome and is also a cause of lymphedema in families displaying phenotypes attributed to other lymphedema syndromes. Our data demonstrates that the phenotypic classification of autosomal dominant lymphedema does not reflect the underlying genetic causation of these disorders.

Introduction

Hereditary lymphedema is a chronic disabling condition which results in swelling of the extremities due to altered lymphatic flow. Patients with lymphedema suffer from recurrent local infections, physical impairment and social anxiety, and may be at increased risk for developing cancers such as lymphangiosarcoma. Hereditary lymphedema may occur as an isolated condition, examples of which include Milroy disease (OMIM 153100) and lymphedema praecox (OMIM 153200), or as a component of a complex syndrome. We have demonstrated that mutations in the kinase domain of the vascular endothelial growth factor receptor-3 (VEGFR3) gene causes Milroy disease (1), and this finding has been confirmed (2). Syndromic lymphedema-cholestasis (OMIM 214900) has been mapped to a 6 cM region on chromosome 15 (3). The syndrome of lymphedema-distichiasis (OMIM 153400) was mapped to a narrow region of chromosome 16 (4), containing the FOXC2(MFH-1) gene, and mutations in FOXC2 have been identified in families with lymphedema-distichiasis (5). We sequenced the FOXC2 gene in a series of 86 families ascertained through an individual identified with lymphedema to determine the extent of allelic heterogeneity in the FOXC2 gene, and subsequently examined the extent of phenotypic heterogeneity in families with FOXC2 mutations

Results

Mutations in the coding region of the FOXC2 gene were identified in 11 families of mixed European ancestry ascertained through a proband with lymphedema. Detailed results of the mutation screening are given in Table 1. Each mutation was found to segregate with lymphedema risk in the family in which it was observed, although DNA samples were not available for every family member on whom phenotypic information was available. One mutation, a single nucleotide insertion, was observed in two independently ascertained families (families D and E). The exact nucleotide position of this insertion cannot be specified as it occurs in a contiguous sequence of five cytosines. Genotyping of a series of flanking microsatellite markers (D16S2624, D16S511 and D16S402) over a 19 cM region and a FOXC2 single nucleotide polymorphism (SNP) located between D16S2624 and D16S511 suggest that the mutation in these two families arose independently, as they do not share flanking marker haplotypes. The coding region of FOXC2 was sequenced in 75 randomly ascertained, unrelated individuals of mixed European ancestry, and none of the variations described in Table 1 were observed. The absence of the mutations noted in Table 1 in unaffected individuals, cosegregation of these mutations with lymphedema risk in families, and the fact that the mutations seen in the patients with lymphedema are predicted to lead to protein truncation, support the causative nature of these mutations. Mutations included a 7 bp (family I) and a 14 bp (family A) duplication-insertion, four single base insertions (families D, E, F and J), two single base deletions (families C and H), deletions of 16 bp (family K) and 19 bp (family G), and a CT transition (family B). The net effect of these mutations was predicted to create a premature termination of the mature protein. The forkhead domain of FOXC2 is reported to extend from nucleotides 211 to 510 (GenBank accession no. NM_005251). Three of the mutations occurred within the forkhead domain and would be likely to disrupt DNA binding. The remaining eight mutations occurred following the forkhead domain and lead to truncations of the mature protein and elimination of key -helical domains required for the interaction of FOXC2 with the transcription complex (6).

Onset of lymphedema in affected members of these families was between birth and 30 years of age. The average age of onset was 13.7 years and the median onset was 13 years. Of the 44 individuals with lymphedema, three had an unknown age of onset and 29 had a peripubertal onset (lymphedema praecox). Four individuals (ages 6–12 years) with distichiasis but not lymphedema have not reached the median age at onset for lymphedema and may still develop lymphedema. As observed in families with congenital lymphedema due to VEGFR3 mutations, not all mutation carriers express lymphedema. One FOXC2 mutation carrier, a 41-year-old female (family A), failed to show any clinical phenotype. A 22-year-old female (family G), for whom DNA was not available, was reported to have ptosis as the only clinical finding. Other features observed in these families included distichiasis, cleft palate, ptosis, yellow nails, congenital heart defects and cystic hygroma.
Among the 86 families screened, distichiasis was reported in three families in which we did not detect a mutation in the FOXC2 coding sequence. Sequencing of 1168 bp of the 5'-flanking region of FOXC2 in these families did not reveal further mutations. The phenotypic features of distichiasis families without FOXC2 mutations were indistinguishable from those families with FOXC2 mutations (data not shown). The families without FOXC2 mutations were too small to exclude linkage to the chromosome 16q24 region and the possibility of undetected FOXC2 mutations cannot be excluded. Four families with lymphedema-yellow nails syndrome did not demonstrate a FOXC2 mutation.

Discussion

We report the occurrence of mutations in the FOXC2 gene in families with the lymphedema-distichiasis syndrome, as well as in a family with lymphedema and without distichiasis. The mutations reported and the truncated proteins predicted to result from these mutations appear causal for the phenotypes seen and confirm FOXC2 as a causal gene for developmental abnormalities in the lymphatic system. The finding of mutations in a lymphedema family without distichiasis highlights the phenotypic variability associated with FOXC2 mutations.

The phenotypic classification of dominantly inherited lymphedema includes Milroy disease (OMIM 153100), Meige lymphedema (lymphedema praecox) (OMIM 153200), lymphedema-distichiasis syndrome (OMIM 153400), lymphedema and ptosis (OMIM 15300) and yellow-nail syndrome (OMIM 153300). The age at onset data from Table 2 and data from the two families described by Fang et al. (5) suggest that FOXC2 mutations are not etiologic of Milroy disease, which is associated with early childhood onset (pre-pubertal) lymphedema. However, the phenotypes observed in our 11 families overlap the findings reported in Meige syndrome, lymphedema-distichiasis syndrome, lymphedema-ptosis syndrome and yellow nail syndrome. Hence, the phenotypic classification of autosomal dominant lymphedema does not reflect the underlying genetic causation of these disorders.

The mutations identified in families with and without distichiasis occur within or shortly after the critically conserved forkhead domain, where they are expected to interfere with DNA binding or to disrupt C-terminal -helices critical for transcription activation by the forkhead transcription factor. The forkhead/hepatic nuclear factor motif is found in a family of transcription factors with unique DNA binding characteristics, first described by Weigel et al. (7). The forkhead domain is characterized by a highly conserved 110 amino acid sequence, the structure of which consists of -helices and ß-strands separated by two wing-like domains. Since the three-dimensional structure can be visualized in the shape of a butterfly, the region has been referred to as a ‘winged helix’ (8). Fourteen contact points define the interaction with DNA resulting in high specificity of binding. Footprint and deletion studies confirm the necessity to maintain this motif as a structural unit (9–11). While the flanking regions of the forkhead domain have not been as extensively studied as the forkhead region itself, regions in both the C- and N-terminus are known to be essential for transcriptional activation (6). Mutations in members of this diverse gene family have been shown to cause a variety of disease phenotypes (5,12–19). No distinct phenotypic features distinguished our families with mutations directly within the forkhead domain from those where the mutation was observed 3' following the forkhead region. The majority of mutations identified would be predicted to generate a normal core forkhead domain followed by a variable length nonsense peptide. We agree with the conclusion reached by Fang et al. (5) that FOXC2 mutations may exert their actions through a mechanism of haploinsufficiency. However, the possibility exists that the C-terminal missense peptide which results from downstream truncations following insertion and deletion mutations may exert a dominant gain of function effect in some families. The identification of FOXC2 gene mutations in our pedigrees which are characterized by multiple features of varied lymphedema syndromes supports the hypothesis that classification of lymphedema syndromes by phenotypic features is inconsistent with the genetic variations determined through mutational analysis.

Subjects and Methods

Subjects

All families were ascertained based on the presence of primary lymphedema in at least two family members. Families were ascertained through the Lymphedema Family Study website (www.pitt.edu/~genetics/lymph) through local referral, and two families were referred through GeneTests, an online genetic testing resource (www.genetests.org), by Yale University School of Medicine and Stanford University. Of the 86 families screened, 71 were of mixed European ancestry. These 71 families included the families identified with mutations (11) and polymorphisms (3). The remaining 15 families were of mixed ethnicity. This study was reviewed and approved by the Institutional Review Board of the University of Pittsburgh and written informed consent was obtained for each individual who participated. Medical records were requested to confirm medical diagnoses of lymphedema and associated phenotypes.

Mutation detection

Sequencing of FOXC2 was performed on 86 probands ascertained with lymphedema, who were found to be negative for mutations in VEGFR3 by direct sequencing. A subset of this group also reported evidence of distichiasis and/or other features of the lymphedema-distichiasis syndrome. Amplification and sequencing primers were designed from the FOXC2 cDNA (GenBank accession no. NM_005251) and from Fang et al. (5). Exonic sequences were amplified in two overlapping segments using the following primer combinations: 1F, 5'-TCTCTCGCGCTCTCTCGCTC-3' and 1R2, 5'-CGTTCGCAGGGTCATGATGTT-3' (62°ta, 1.5 mM Mg++, 6% final concentration DMSO); and 1F2, 5'-GTCATCACCAAGGTGGAGACG-3' with 1R, 5'-CTTTTTTGCGTCTCTGCAGCCC-3' (60°ta, 1.0 mM Mg++, 6% final concentration DMSO). These primers amplify a sequence beginning 90 bp 5' to the reported FOXC2 ATG start site and ending 95 bp 3' from the end of the coding sequence. This provided an overlap of 170 bp in the middle of the single exon. The same primers and conditions were used to sequence 75 unrelated, healthy control subjects of mixed European ancestry.

Primers to amplify additional 5' sequence containing potential control elements were designed from a bacterial artificial chromosome clone (GenBank accession no. AC009108.8) containing FOXC2. This clone, which also contained homologs FOXL1 and FOXF1, was acquired using the DoubleTwist biologic search service. Promoter region PCR was performed using the following combinations: PF1, 5'-CAGTCAGCACGTTGCTAC-3' with PR1, 5'-CTTCTTGCTGAAAGCGAG-3' and PF2, 5'-GATTGGCTCAAAGTTCCG-3' (55°ta, 2.0 mM Mg++, 8% final concentration DMSO) with PR2, 5'-GCATGCTGCTTCCGAGAC-3' (55°ta, 1.25 mM Mg++, 8% final concentration DMSO). These primer sets amplify 1168 bp of 5'-flanking sequence with an overlap of 66 bp in the middle of this region.

Acknowledgments

We thank the family members who participated in this study. We thank Peter Chase, M.D. for examining key family members. We acknowledge the sequencing performed by the University of Pittsburgh Center for Genomic Sciences Sequencing Core Facility. This work was supported by NIH Grant R01 HD37243.

Footnotes

To whom correspondence should be addressed. Tel: +1 412 624 3018; Fax: +1 412 624 3020; Email: dnf@mars.upmc.edu

References

1 Karkkainen, M.J., Ferrell, R.E., Lawrence, E.C., Kimak, M.A., Levinson, K.L., McTigue, M.A., Alitalo, K. and Finegold, D.N. (2000) Missense mutations interfere with VEGFR-3 signalling in primary lymphedema. Nat. Genet., 25, 153–159. [Medline]

2 Irrthum, A., Karkkainen, M.J., Devriendt, K., Alitalo, K. and Vikkula, M. (2000) Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet., 67, 295–301. [Medline]

3 Bull, L.N., Roche, E., Song, E.J., Pedersen, J., Knisely, A.S., van der Hagen, C.B., Eiklid, K., Aagenaes, O. and Freimer, N.B. (2000) Mapping of the locus for Cholestasis-Lymphedema Syndrome (Aagenaes Syndrome) to a 6.6-cM interval on chromosome 15q. Am. J. Hum. Genet., 67, 994–999. [Medline]

4 Mangion, J., Rahman, N., Mansour, S., Brice, G., Rosbotham, J., Child, A.H., Murday, V.A., Mortimer, P.S., Barfoot, R., Sigurdsson, A. et al. (1999) A gene for lymphedema-distichiasis maps to 16q24.3. Am. J. Hum. Genet., 65, 427–432. [Medline]

5 Fang, J., Dagenais, S.L., Erickson, R.P., Arlt, M.F., Glynn, M.W., Gorski, J.L., Seaver, L.H. and Glover, T.W. (2000) Mutations in FOXC2(MFH-1), a forkhead family transcription factor, are responsible for the hereditary Lymphedema-Distichiasis Syndrome. Am. J. Hum. Genet., 67, 1382–1388. [Medline]

6 Kaufmann, E. and Knochel, W. (1996) Five years on the wings of forkhead. Mech. Dev., 57, 3–20. [Medline]

7 Weigel, D., Jürgens, G., Küttner, F., Seifert, E. and Jäckle, H. (1989) The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell, 57, 645–658. [Medline]

8 Clark, K.L., Halay, E.D., Lai, E. and Burley, S.K. (1993) Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature, 364, 412–420. [Medline]

9 Lai, E., Prezioso, V.R., Smith, E., Litvin, O., Costa, R.H. and Darnell, J.E.,Jr (1990) HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. Genes Dev., 4, 1427–1436. [Abstract]

10 Lai, E., Prezioso, V.R., Tao, W.F., Chen, W.S. and Darnell, J.E.,Jr (1991) Hepatocyte nuclear factor 3 {alpha} belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene forkhead. Genes Dev., 5, 416–427. [Abstract]

11 Kaufmann, E., Hoch, M. and Jäckle, H. (1994) The interaction of DNA with the DNA-binding domain encoded by the Drosophila gene fork head. Eur. J. Biochem., 223, 329–337. [Abstract]

12 Galili, N., Davis, R.J., Fredericks, W.J., Mukhopadhyay, S., Rauscher, F.J.,III, Emanuel, B.S., Rovera, G. and Barr, F.G. (1993) Fusion of a fork head domain gene to PAX 3 in the solid tumour alveolar rhabdomyosarcoma. Nat. Genet., 5, 230–235. [Medline]

13 Hillion, J., De Coniat, M., Jonveaux, P., Berger, R. and Bernard, O.A. (1997) AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. Blood, 90, 3714–3719. [Abstract/Free Full Text]

14 Nishimura, D., Swiderski, R., Alward, W., Searby, C.C., Patil, S.R., Bennet, S.R., Kanis, A.B., Gastier, J.M., Stone, E.M. and Sheffeld, V.C. (1998) The forkhead transcription factor gene FKHL-7 is responsible for glaucoma phenotypes which map to 6p25. Nat. Genet., 19, 140–147. [Medline]

15 Lehmann, O.J., Ebenezer, N.D., Jordan, T., Fox, M., Ocaka, L., Payne, A., Leroy, B.P., Clark, B.J., Hitchings, R.A., Povey, S. et al. (2000) Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma. Am. J. Hum. Genet., 67, 1129–1135. [Medline]

16 Mirzayans, F., Gould, D.B., Heon, E., Billingsley, G.D., Cheung, J.C., Mears, A.J. and Walter, M.A. (2000) Axenfeld-Rieger syndrome resulting from mutation of the FKHL-7 gene on chromosome 6p25. Eur. J. Hum. Genet., 8, 71–74. [Medline]

17 Nishimura, D.Y., Searby, C.C., Alward, W.L., Walton, D., Craig, J.E., Mackey, D.A., Kawase, K., Kanis, A.B., Patil, S.R., Stone, E.M. and Sheffield, V.C. (2001) A spectrum of FOXC1 mutations suggest gene dosage as a mechanism for developmental defects of the anterior chamber of the eye. Am. J. Hum. Genet., 68, 364–372. [Medline]

18 Wildin, R.S., Ramsdell, F., Peake, J., Faravelli, F., Casanova, J.L., Buist, N., Levy-Lahad, E., Mazzella, M., Goulet, O., Perroni, L. et al. (2001) X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet., 27, 18–20. [Medline]

19 Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L., Kelly, T.E., Saulsbury, F.T., Chance, P.F. and Ochs, H.D. (2001) The immune disregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet., 27, 20–21. [Medline]

http://hmg.oupjournals.org/cgi/content/full/10/11/1185

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Evidence for genetic heterogeneity in lymphedema-cholestasis syndrome.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12712065&dopt=Abstract

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FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance.
Cederberg A, Gronning LM, Ahren B, Tasken K, Carlsson P, Enerback S.

Medical Genetics, Department of Medical Biochemistry, Goteborg University, Box 440, SE-405 30, Goteborg, Sweden.

Obesity, hyperlipidemia, and insulin resistance are common forerunners of type 2 diabetes mellitus. We have identified the human winged helix/forkhead transcription factor gene FOXC2 as a key regulator of adipocyte metabolism. Increased FOXC2 expression, in adipocytes, has a pleiotropic effect on gene expression, which leads to a lean and insulin sensitive phenotype. FOXC2 affects adipocyte metabolism by increasing the sensitivity of the beta-adrenergic-cAMP-protein kinase A (PKA) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Increased FOXC2 levels, induced by high fat diet, seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance--a likely consequence hereof would be protection against type 2 diabetes.

PMID: 11551504 [PubMed - indexed for MEDLINE

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11551504&dopt=Abstract

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FOXC2 mRNA expression and a 5' Untranslated region polymorphism of the gene are associated with insulin resistance

Diabetes,  Dec, 2002  by Martin Ridderstrale,  Emma Carlsson,  Mia Klannemark,  Anna Cederberg,  Christina Kosters,  Hans Tornqvist,  Heidi Storgaard,  Allan Vaag,  Sven Enerback,  Leif Groop

http://diabetes.diabetesjournals.org/content/51/12/3554.long

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Expression of FOXC2 in Adipose and Muscle and its Association with Whole-Body Insulin Sensitivity

Gina B. Di Gregorio1*, Rickard Westergren2, Sven Enerback2, Tong Lu1, and Philip A. Kern1

1 The Central Arkansas Veterans Healthcare System, Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2 Medical Genetics, Department of Medical Biochemistry, Goteborg University, Goteborg, Sweden

* To whom correspondence should be addressed. E-mail: Digregorioginab@uams.edu.

FOXC2 is a winged helix/forkhead transcription factor involved in PKA signaling. Overexpression of FOXC2 in the adipose tissue of transgenic mice protected against diet-induced obesity and insulin resistance. We examined the expression of FOXC2 in fat and muscle of non-diabetic humans with varying obesity and insulin sensitivity. There was no relation between BMI and FOXC2 mRNA in either adipose or muscle. There was a strong inverse relation between adipose FOXC2 mRNA and insulin sensitivity, using the frequently sampled IV glucose tolerance test (r=-0.78, P<0.001). However, there was no relationship between muscle FOXC2 and any measure of insulin sensitivity. To separate insulin resistance from obesity, we examined FOXC2 expression in pairs of subjects who were matched for BMI, but who were discordant for SI. When compared to the insulin sensitive subjects, the insulin resistant subjects had 3-fold higher levels of adipose FOXC2 mRNA (p=0.03). In contrast, muscle FOXC2 mRNA expression was no different between insulin resistant and insulin sensitive subjects. There was no association of adipose or muscle FOXC2 mRNA with either circulating or adipose-secreted TNF{alpha}, IL6, leptin, adiponectin, or non-esterified fatty acids. Thus, adipose FOXC2 is more highly expressed in insulin resistant subjects, and this effect is independent of obesity. This association between FOXC2 and insulin resistance may be related to the role of FOXC2 in PKA signaling.

http://ajpendo.physiology.org/cgi/content/short/00155.2004v1

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FOXC2 - Familial congenital non-immune hydrops, chylothorax, and pulmonary lymphangiectasia.

Stevenson DA, Pysher TJ, Ward RM, Carey JC.

Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, Utah.

Pulmonary lymphangiectasia is an uncommon congenital anomaly, and familial occurrence has rarely been reported. We report on two sibs with bilateral pleural effusion/chylothorax and hydrops who died neonatally. One sib required prenatal intrauterine hemithoracic drainage. Autopsy confirmed congenital pulmonary lymphangiectasia (CPL) histologically in the first case. Hydrops, characterized as subcutaneous edema and effusions in two or more body cavities, may be due to a variety of factors, but the co-occurrence of CPL in one of these sibs, although rare, supports the notion that chylothorax and hydrops may be caused by structural lesions of lymph channels. Although most cases of CPL are sporadic, the reported sibs support autosomal recessive inheritance, with intrafamilial variability of a lymphatic disorder on a genetic basis. Mutations in vascular endothelial growth factor receptor-3 (VEGFR3) in families with Milroy disease, mutations of FOXC2 in the lymphedema-distichiasis syndrome, and fatal chylothorax in alpha9-deficient mice are potential candidate genes. (c) 2006 Wiley-Liss, Inc.

PMID: 16419129 [PubMed - in process]

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VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain.

Abstract - Nature Neuroscience

Feb. 5, 2006

Le Bras B, Barallobre MJ, Homman-Ludiye J, Ny A, Wyns S, Tammela T, Haiko P, Karkkainen MJ, Yuan L, Muriel MP, Chatzopoulou E, Breant C, Zalc B, Carmeliet P, Alitalo K, Eichmann A, Thomas JL.

[1] Institut National de la Sante et de la Recherche Medicale (INSERM), U711, Paris F-75013, France. [2] Universite Pierre & Marie Curie, Faculte de Medecine Pitie Salpetriere, IFR 70, Paris F-75005, France. [3] These authors contributed equally to this work.

Vascular endothelial growth factor C (VEGF-C) was first identified as a regulator of the vascular system, where it is required for the development of lymphatic vessels. Here we report actions of VEGF-C in the central nervous system. We detected the expression of the VEGF-C receptor VEGFR-3 in neural progenitor cells in Xenopus laevis and mouse embryos. In Xenopus tadpole VEGF-C knockdowns and in mice lacking Vegfc, the proliferation of neural progenitors expressing VEGFR-3 was severely reduced, in the absence of intracerebral blood vessel defects. In addition, Vegfc-deficient mouse embryos showed a selective loss of oligodendrocyte precursor cells (OPCs) in the embryonic optic nerve. In vitro, VEGF-C stimulated the proliferation of OPCs expressing VEGFR-3 and nestin-positive ventricular neural cells. VEGF-C thus has a new, evolutionary conserved function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3.

PMID: 16462734 [PubMed - as supplied by publisher]

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Microarray analysis of VEGF-C responsive genes in human lymphatic endothelial cells.

2005

Yong C, Bridenbaugh EA, Zawieja DC, Swartz MA.

Integrative Biosciences Institute, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

Vascular endothelial growth factor-C (VEGF-C) is considered one of the most important factors influencing lymphatic endothelial cell biology. The goal of this work was to characterize the gene expression response by lymphatic endothelial cells (LECs) to VEGF-C. Primary cultures of human microvascular LECs were exposed to 100 ng/mL VEGF-C for 30 minutes and 6 hours, and their lysates were evaluated by microarray analysis to determine changes in mRNA expression induced by VEGF-C. Characteristic of a response to a growth factor stimulus, the largest number of differentially expressed genes were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and the transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis and neurodegeneration were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C.

PMID: 16379588 [PubMed - indexed for MEDLINE]

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The association between vascular endothelial growth factor-C, its corresponding receptor, VEGFR-3, and prognosis in primary breast cancer: A study with 193 cases.

2006

Bando H, Weich HA, Horiguchi S, Funata N, Ogawa T, Toi M.

Department of Surgery, Tokyo Metropolitan Komagome Hospital, Tokyo Metropolitan Cancer and Infectious Disease Center,Tokyo 113-8677, Japan.

Lymphangiogenesis plays an important role in several normal and pathological conditions, such as wound healing, pathogen infection, inflammation or the metastasis formation of endothelial malignancies. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4). In this study, we measured intratumoral levels of VEGF-C and VEGFR-3 through enzyme-linked immunosorbent assay (ELISA) in 193 primary breast cancer tissues and examined their prognostic values. A significant correlation was found between the VEGF-C and VEGFR-3 protein levels. High VEGF-C levels were associated with low-grade tumors and a smaller size. Univariate analysis showed that high VEGF-C was significantly associated with a favourable prognosis for disease-free survival (DFS) and overall survival (OS). No significant prognostic value of VEGFR-3 was detected. Multivariate analysis confirmed the independent prognostic value of VEGF-C. The intratumoral VEGF-C level is a significant prognostic indicator of primary breast cancer. An investigation of the mechanisms of VEGF-C protein processing in human cancer tissue should be carried out in the future.

PMID: 16465426 [PubMed - in process]

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LYMPHEDEMA FAMILY STUDY

http://www.pitt.edu/AFShome/g/e/genetics/public/html/lymph/

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GENOMICS: Gene Found for Lymphedema.

Applied Genetics News, July, 2000

http://www.findarticles.com/cf_dls/m0DED/12_20/63802638/p1/article.jhtml

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To locate a genetic counselor or geneticist in your area

http://www.kumc.edu/gec/support/lymphede.html

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Lymphovenous Canada: A Canadian in Washington - The Genetics of Rare Diseases Conference

http://www.lymphovenous-canada.ca/geneticsrarediseases.htm

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Scientists find genetic mutations that cause a form of hereditary lymphedema

http://www.umich.edu/~urecord/0001/Nov13_00/7.htm


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November 24, 2003

Novel Discoveries Leading To Targeted Treatment Of Lymphatic
Diseases

A gene responsible for lymphatic vessel formation

Novel discoveries at the University of Helsinki, Finland, about the
development of the lymphatic network may help researchers to better
understand the mechanisms of cancer and its metastasis, and also
diseases such as lymphedema, wound healing and inflammatory and
autoimmune disorders.

Lymphatic vascular network is essential in transporting the tissue
fluids and immune cells from tissues to the nearby lymph nodes and
back to the blood circulation. Thus, this network of vessels is
crucial in provoking body's immune defense mechanisms. A study
published as advance online publication of `Nature Immunology' (web
edition 23.11.2003) describes VEGF-C as an essential regulator of
lymphatic vessel development. Dr. Karkkainen and collaborators from
the University of Helsinki show that in the absence of this growth
factor the lymphatic development is interrupted, which results in
fluid accumulation in tissues and embryonic lethality. VEGF-C
haploinsufficiency gene results in delayed and abnormal lymphatic
development and swelling of the limbs, in a disease called
lymphedema.

Within the past couple of years, there has been an unprecedented
explosion of lymphatic biology research. The current study was done
in one of the leading laboratories in the field of
lymphangiogenesis, in the group lead by Dr. Kari Alitalo from the
University of Helsinki, which has reported major advances in the
fields of angiogenesis, lymphangiogenesis and cancer biology in
recent years. The authors believe that the current study helps in
developing more targeted treatments of various lymphatic diseases.
Now, for the first time there are exciting new developments making
treatment possible for the over one hundred million people worldwide
who suffer from diseases related to the lymphatic system.

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Sprouting lymphatics VEGF-C is required for the
initiation of lymphatic vessel development | By Tudor
Toma



The lymphatic vessels drain extracellular fluid and
play an essential role in immune surveillance. Defects
in lymphatic vessel formation or function can cause
lymphedema, a clinically well characterized condition,
the underlying molecular mechanisms of which have been
unclear. In the November 23 Nature Immunology, Marika
Karkkainen and colleagues at the University of
Helsinki show that vascular endothelial growth factor
C (VEGF-C) is required for sprouting of the first
lymphatic vessels from embryonic veins (Nature
Immunology, DOI:10.1038/ni1013, November 23, 2003).

Karkkainen et al. targeted the Vegfc locus in mouse
chromosome 8 and analyzed the lymphatic vascular
development in embryos deficient in VEGF-C. They
observed that in Vegfc-/- mice endothelial cells
commit to the lymphatic lineage, but do not sprout to
form lymph vessels. The lack of lymphatic vessels
resulted in prenatal death due to fluid accumulation
in tissues. Vegfc+/– mice developed cutaneous
lymphatic hypoplasia and lymphedema. In addition, the
authors showed that sprouting was rescued by VEGF-C
and VEGF-D, but not by VEGF, indicating VEGF receptor
3 specificity.

“Our results indicate that VEGF-C is not needed for
cell commitment to the lymphatic endothelial lineage,
but that paracrine VEGF-C signaling is required for
the migration and eventual survival of
Prox-1-expressing endothelial cells from the cardinal
vein and for the subsequent formation of lymph sacs,”
conclude the authors.

Links for this article
G. Oliver, N. Harvey, “A stepwise model of the
development of lymphatic vasculature,” Annals of the
New York Academy of Sciences, 979:159-165, December
2002.
[PubMed Abstract]

M. Karkkainen et al., “Vascular endothelial growth
factor C is required for sprouting of the first
lymphatic vessels from embryonic veins,” Nature
Immunology, DOI:10.1038/ni1013, November 23, 2003.

LINKS FOR THIS ARTICLE

http://www.nature.com/natureimmunology

University of Helsinki
http://www.helsinki.fi/university/

http://www.innovations-report.com/html/reports/medicine_health/report-23550.html

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Hereditary lymphedema genetic mutations found

ANN ARBOR---University of Michigan scientists have identified genetic mutations that cause a serious medical condition called hereditary lymphedema-distichiasis or LD. Discovering the gene is the first step toward a future diagnostic test for LD and increased scientific understanding of the gene's impact on early development of the heart and lymphatic system.

People with the mutated gene often develop severe lymphedema, or fluid retention in their arms and legs. They also have double rows of eyelashes, a condition called distichiasis. Some members of LD families have other complications---including heart defects, spinal abnormalities and cleft palate.

U-M scientists and collaborators from the University of Arizona discovered that the mutations responsible for LD are located in the FOXC2 gene on chromosome 16. FOXC2 is one of a large group of related transcription factors in what scientists call the forkhead/winged helix family. Like all transcription factors, forkhead genes serve as master control switches that regulate the activity of other genes.

An article describing the FOXC2 mutations will be published in the December 2000 issue of the
American Journal of Human Genetics, which is available now on the journal's Web site at http://www.ajhg.org. (Access the "AJHG Electronic Edition" and click on "Latest articles.")

"Forkhead genes are important, because they play a major role in regulating embryonic development in all animal species," says Thomas W. Glover, Ph.D., a professor of human genetics, pediatrics and communicable diseases in the U-M Medical School, who directed the study. "At least 17 forkhead genes have been identified in humans and 80 in other species, but we know little about how they affect human development and genetic disease."

Because the physical effects of LD are variable and often don't appear until adolescence, Glover says scientists don't know how common the syndrome is. "While LD is a relatively rare genetic disorder, it is probably more common than we think," he says. "Since lymphedema is usually a side-effect of surgery, injury or infection, many physicians don't realize it also can be a genetic disorder. We need greater awareness of LD and other forms of congenital lymphedema, so patients can be diagnosed accurately and treated effectively. In addition, what we learn from this genetic disorder may apply to other more common forms of lymphedema, as well."

In two families in the study, children received an inactive form of the FOXC2 gene from one parent. A child born with lymphedema in a third family had a different type of mutation called a chromosomal translocation, in which a chromosome break shut down the FOXC2 gene. The discovery of the patient with a translocation mutation---which was made by Robert Erickson, M.D., a University of Arizona scientist---was a key to finding the location of the gene, according to Glover.

To scientists, the study is significant, because it is the first discovery of a FOXC2 mutation in humans and only the second known example of a forkhead gene mutation in humans. It also provides important clues to the molecular events involved in development of the heart and lymphatic system---about which little is known.

"FOXC2 is a pleiotrophic developmental gene," Glover says. "This means that a mutation in a single gene produces multiple effects. Studying the effects of FOXC2 mutations in LD families will help us understand why LD shows variable expression from individual to individual. Some children who inherit the mutated gene develop lymphedema only, while others also show severe heart defects and cleft palate."

In future research, Glover and his research associates plan to study laboratory mice in which the FOXC2 gene has been removed---both as a model for lymphedema and to identify other genes regulated by FOXC2 during embryonic development. Glover also hopes to find more families with hereditary lymphedema who are willing to participate in future studies.

"Our immediate goal is to apply a molecular diagnostic test for LD to get a more accurate definition of the syndrome and its frequency," Glover says. "We also hope to learn more about the mechanisms of primary lymphedema and the role of FOXC2 and other forkhead genes in human development and genetic disease."


###
Funding for the study was provided by the U-M Medical School and the National Institutes of Health. Lymphedema-distichiasis was first identified as a hereditary syndrome in 1954 by James V. Neel, M.D., former U-M professor emeritus of human genetics; Harold Falls, U-M professor emeritus of ophthalmology; and William J. Schull, professor emeritus of human genetics at the University of Texas-Houston.

Jianming Fang, Ph.D., a U-M postdoctoral fellow, is first author on the study. Other U-M collaborators include postdoctoral fellows Susan L. Dagenais, Ph.D., and Martin F. Arlt, Ph.D.; graduate student Michael W. Glynn; Jerome L. Gorski, M.D., U-M professor of pediatrics and communicable diseases and associate professor of human genetics; Robert P. Erickson, M.D., professor of pediatrics at the University of Arizona; and Laurie H. Seaver, M.D., of the J.C. Self Research Institute of Human Genetics in Greenwood, S.C.

NOTE: If you believe LD runs in your family and are interested in participating in future research studies, call the University of Michigan Health System TeleCare line at 1-800-742-2300, category #6245.

http://www.eurekalert.org/pub_releases/2000-11/UoM-Hlgm-0711100.php

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A model for gene therapy of human hereditary lymphedema

Article

www.pnas.org/cgi/content/full/221449198v1

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Hereditary Lymphedema

www.ajhg.org

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Genes and Development

The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature

http://www.genesdev.org/cgi/content/full/16/7/773

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Truncating mutations in FOXC2 cause multiple lymphedema syndromes

http://hmg.oupjournals.org/cgi/content/full/10/11/1185

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Sprouting lymphatics

http://cmbi.bjmu.edu.cn/news/0311/124.htm

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Genetic Professional Societies
Human, Medical, and Clinical Genetics

http://www.kumc.edu/gec/prof/soclist.html

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Genetic clinics, centers, departments

http://www.kumc.edu/gec/prof/genecntr.html

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Gene symbol : FOXC2 - Mutations

Mutations in this gene were first reported in 2000

http://www.journals.elsevierhealth.com/periodicals/ympa/article/S1091-8531(03)00144-7/abstract

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Mutations in FOXC2

Abstract

http://note.cellbio.duke.edu/Faculty/Hogan/hogan4.pdf

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VEGFR-3 in primary lymphedema

Marika J Karkkainen

Academic Dissertation, December 2001.
University of Helsinki, Haartman Institute, Molecular and Cancer Biology Laboratory, Biomedicum Helsinki, Faculty of Medicine and Helsinki University Central Hospital.

http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/karkkainen/

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VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer

Abstract

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=10329591&dopt=Abstract

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VEGFR-3 Ligands and Lymphangiogenesis

Michael Jeltsch

University of Helsinki 2002

http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/jeltsch/

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Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24

G Brice1, S Mansour2, R Bell2, J R O Collin3, A H Child1, A F Brady4, M Sarfarazi5, K G Burnand6, S Jeffery2, P Mortimer7 and V A Murday2

1 Department of Cardiological Sciences, St George's Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK
2 Medical Genetics Unit, St George's Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK
3 Moorfields Eye Hospital, City Road, London EC1V 2PD, UK
4 Kennedy Galton Centre, Northwick Park Hospital, Watford Road, Harrow, UK
5 University of Connecticut Health Center, Farmington, Connecticut, USA
6 Department of Academic Surgery, St Thomas' Hospital, UMDS, London, UK
7 Department of Dermatology, St George's Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK

Correspondence to:
Dr S Mansour, South West Thames Regional Genetic Service, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK;
smansour@sghms.ac.uk

Revised version received 28 March 2002
Accepted for publication 2 April 2002

ABSTRACT

Introduction: Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts.

Subjects: We report clinical findings in 74 affected subjects from 18 families and six isolated cases. All of them were shown to have mutations in FOXC2 with the exception of one family who had two affected subjects with lymphoedema and distichiasis and linkage consistent with the 16q24 locus.

Results: The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with a normal or increased number of lymphatic vessels rather than the hypoplasia or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%), and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins.

Conclusion: Unlike previous publications, the thorough clinical characterisation of our patients permits more accurate prediction of various phenotypic abnormalities likely to manifest in subjects with FOXC2 mutations.

Keywords: primary lymphoedema; distichiasis; FOXC2; varicose veins

Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Distichiasis is a congenital anomaly in which accessory eyelashes occur along the posterior border of the lid margins in the position of the Meibomian gland orifices. The accessory eyelashes may be represented by a few cilia or (less commonly) an additional regular, well formed row. Normally, the Meibomian glands act as modified sebaceous glands in which the ducts open directly onto the ocular surface to release sebum. The association of distichiasis of the lids with lymphoedema of the lower limbs was probably first described in 1899.1 Since that time, numerous clinical associations have been reported in this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts.

The familial nature of primary lymphoedema in general and lymphoedema-distichiasis in particular has long been recognised.2–4 The gene for Milroy disease (congenital familial lymphoedema) has recently been mapped to chromosome 5q35.3 and probable causative mutations found in the VEGFR3 gene.5,6 Most families sharing this phenotype appear to be consistent with linkage to this region (A Evans, personal communication). The most common type of primary, familial lymphoedema is that first described by Meige (isolated pubertal onset lymphoedema),3 but to date no locus has been reported.

Recently, lymphoedema-distichiasis was mapped to chromosome 16q247 using three families with clear dominant inheritance of the condition. With the addition of further members from the largest family, the map distance was reduced to less than 2 Mb.8 A previously reported neonate with congenital lymphoedema was found to have a de novo balanced translocation involving chromosome 16 and the Y chromosome (t(Y;16)(q12;q24.3). Further studies showed that the chromosomal breakpoint was in the critical region for lymphoedema-distichiasis. The breakpoint did not appear to disrupt a gene, so candidate genes in the immediate region were considered. Two unrelated families with LD were found to have mutations in one of these genes, FOXC2. Subsequently, seven mutations were found in this gene in seven additional families with lymphoedema-distichiasis.9,10 A further 34 novel mutations in FOXC2, primarily small insertions and deletions, have now been characterised in our series of patients11 ( R Bell, personal communication). Another recent study looked at 86 families with primary lymphoedema and eleven of these families were shown to have a mutation in FOXC2. All but one of these families had distichiasis in one or more affected member.

In this paper we describe the clinical and molecular features of this condition as found in a group of 18 families and six isolated cases of lymphoedema-distichiasis syndrome.

METHODS

Eighteen familial and six isolated cases of distichiasis were ascertained from lymphoedema clinics (PSM and KGB), the operating lists of an ophthalmologist specialising in adnexal surgery (JROC), and from clinical geneticists in the UK. Probands were included if they had distichiasis and primary lymphoedema or if they had one of these features and a family history of the other feature. Informed consent was obtained from all participants and Ethics Committee approval obtained. Most probands and family members were examined looking specifically for signs of distichiasis, lymphoedema, and other known associations with LD. The authors examined all but six subjects. These six are included as reliable clinical information was available from hospital notes, photographs, and family members. In addition, all six had FOXC2 mutations. Blood or buccal samples were collected from all willing subjects and screened for mutations in the transcription factor gene FOXC2. The methods used for identifying the mutations have been previously described in detail.11 Families and sporadic cases were included in the study if a mutation in FOXC2 was identified or linkage to the gene confirmed (one family). There were a total of 74 affected subjects (43M:31F). Isotope lymphoscintigraphy was obtained on 16 subjects and 10 patients had previously had direct radiocontrast lymphangiography.

Penetrance for lymphoedema and difference in age of onset between males and females was calculated by excluding probands, to avoid ascertainment bias. Chi-squared and Cox regression analysis was used to assess statistical significance.

RESULTS

The mutations identified in our series of patients are listed in table 1. Most have previously been reported,11 but there are nine novel mutations. These are three point mutations, three single base insertions, two deletions (one 1 bp and one 8 bp deletion), and one 4 bp duplication. Two of the point mutations caused premature stop codons. The third was a missense mutation, and is only the second in FOXC2 so far identified as likely to give rise to LD, all the remainder being nonsense or frameshifts.9–12 As the G362A (R121H) change is found in an isolated case of LD, it is impossible to prove that this is the causative mutation in this person. However, this is a highly conserved amino acid, being one of only 11 that are identical in 24 members of the forkhead family in the 100 amino acid DNA binding domain.13 It is also adjacent to the less well conserved isoleucine that is mutated to methionine in Axenfeld-Rieger syndrome with glaucoma in FOXC1.14 Together with the fact that this mutation was not seen in 100 control chromosomes, this is compelling evidence that this missense mutation does produce LD in this person.

One family showed no mutations but was consistent with linkage to the FOXC2 locus. There were two affected and three unaffected subjects. The two affected brothers both had bilateral lower limb lymphoedema, distichiasis, and ptosis. This pedigree generated a two point maximum lod score of 1.0 at {theta}=0 for D16S520. As there has been no evidence for genetic heterogeneity in this condition, and both affected subjects had distichiasis and lymphoedema, this was felt to be highly suggestive of linkage to FOXC2.

Lymphoedema, Lymphedema

The severity of the lymphoedema varied within and between families. Mild lymphoedema was defined as swelling confined to the lower leg, moderate lymphoedema to the knee, while marked lymphoedema was defined as swelling extending into the thigh. This information was not available for one affected female. Eleven percent (4/36) of males and 25% of females (5/20) had mild lymphoedema. There were 58.3% of males (21/36) and 60% of females (12/20) with moderate lymphoedema. There was a greater proportion of males with marked lymphoedema, 30.5% (11/36) compared with 15% of females (3/20), but this did not reach statistical significance ({chi}2, p=0.25).

The lymphoedema was usually bilateral and predominantly asymmetrical. In a minority (three patients) the lymphoedema was unilateral.

In most cases no precipitating factor was identified (37); however, in 27 (20M:7F) of these cases the age of onset was between 10 and 19 years (that is, may have been related to puberty). Precipitating factors included injury or infection (5) (such as insect bites, ingrowing toe nails, surgery) and in females the oral contraceptive pill and pregnancy (6).

Males were much more likely than females to develop cellulitis or infection in the oedematous leg. Where the information was available, 64.7% (22 of 34) of males and 25% (4 of 16) of females reported one or more episodes of infection. This was a statistically significant finding ({chi}2, p= 0.02).

Management of the lymphoedema included compression stockings, bandaging, and massage. The stockings provided some reduction in swelling and discomfort but were often uncomfortable in the summer months and cosmetically unacceptable to many patients. Two patients underwent bulk reducing surgery which was unsuccessful.

Clinical aspects
In our series of 74 patients, 57 patients had clinical evidence of lymphoedema (36M:21F). Fourteen patients had no evidence of lymphoedema (9F:5M), two were thought probably to have lymphoedema, and one patient refused to be examined. Of the 14 with no evidence of lymphoedema (age range 1 to 34 years), six were under the age of 11 and therefore could yet develop lymphoedema.

The age of onset of the lymphoedema is shown by the cumulative risk graph (fig 1). There were 20 informative males and 22 informative females. Only three of the males were unaffected with lymphoedema at the time of the study, whereas eight women were unaffected. However, the age of onset in males is earlier and this may account for the difference. The earlier age of onset in males was statistically significant (p=0.015, using Cox regression analysis with one degree of freedom on SPSS version 10, Exp (B) = 2.628 with 95% confidence limits of 1.209 to 5.711). Half of the males are already affected by the age of 11 years, whereas only half the females are affected by their early 20s. The penetrance in this series appeared to be complete by the 40s, but there are very few informative older subjects.

The severity of the lymphoedema varied within and between families. Mild lymphoedema was defined as swelling confined to the lower leg, moderate lymphoedema to the knee, while marked lymphoedema was defined as swelling extending into the thigh. This information was not available for one affected female. Eleven percent (4/36) of males and 25% of females (5/20) had mild lymphoedema. There were 58.3% of males (21/36) and 60% of females (12/20) with moderate lymphoedema. There was a greater proportion of males with marked lymphoedema, 30.5% (11/36) compared with 15% of females (3/20), but this did not reach statistical significance ({chi}2, p=0.25).

The lymphoedema was usually bilateral and predominantly asymmetrical. In a minority (three patients) the lymphoedema was unilateral.

In most cases no precipitating factor was identified (37); however, in 27 (20M:7F) of these cases the age of onset was between 10 and 19 years (that is, may have been related to puberty). Precipitating factors included injury or infection (5) (such as insect bites, ingrowing toe nails, surgery) and in females the oral contraceptive pill and pregnancy (6).

Males were much more likely than females to develop cellulitis or infection in the oedematous leg. Where the information was available, 64.7% (22 of 34) of males and 25% (4 of 16) of females reported one or more episodes of infection. This was a statistically significant finding ({chi}2, p= 0.02).

Management of the lymphoedema included compression stockings, bandaging, and massage. The stockings provided some reduction in swelling and discomfort but were often uncomfortable in the summer months and cosmetically unacceptable to many patients. Two patients underwent bulk reducing surgery which was unsuccessful.

Isotope lymphoscintigraphy and lymphangiography
Lymphoscintigram results were consistent with lymphoedema in nine of 11 subjects tested, with abnormally low uptake of radioactive colloid in ilioinguinal nodes at both 30 minutes and one hour. Two females, aged 23 and 24, showed normal uptake at both 30 and 60 minutes and no clinical evidence of lymphoedema; however both were found to have asymptomatic distichiasis. In some cases lymphatic function was within normal limits in terms of nodal uptake, but images showed increased lymph conducting pathways and dermal backflow indicating lymph reflux (figs 2 and 3).

Lymphangiograms, now rarely performed, had previously been performed on 10 subjects. Five of these were reviewed and showed increased nodal tissue with small multiple nodes extending into the mesentery. The number of lymph channels was at the upper limit of normal (figs 4 and 5). One of these lymphangiograms has been previously reported.15

Distichiasis
Distichiasis (fig 6) was present in 38 males (92.7%) and 28 females (96.6%) (overall prevalence of distichiasis was 94.3%). It was not present in four patients (3M:1F) and two patients were not examined for distichiasis. In two further patients, the findings were uncertain (there were one or two accessory eyelashes in the inner canthus only). The distichiasis was not always diagnosed at birth but usually during childhood or puberty. However, it is felt that the distichiasis is probably present at or shortly after birth but frequently not diagnosed until later. Six affected patients had no symptoms of distichiasis and were only discovered on clinical examination during this study. The distichiasis was usually confined to a few sparse, fine eyelashes. They occurred on the inferior and superior eyelid and were more often central and lateral than medial. In one male carrier of a FOXC2 mutation, there was no distichiasis but the Meibomian glands appeared prominent.

Complications occurred in 73.7% (45 of 61) of the patients in whom information was available. These included mild corneal irritation, photophobia, recurrent conjunctivitis, and styes. There was no significant difference in the incidence of complications between males and females. The distichiasis was managed in a number of different ways including epilation (plucking), cryotherapy, electrolysis, lid splitting operations, and laser treatment. Most of these measures gave only temporary relief.

Varicose veins
Varicose veins are common in the general population, increasing in prevalence with age. A study in 1978 showed a prevalence of 10% in the population between the ages of 25 and 34 years rising to 50% over the age of 64.16 Previously, varicose veins have been shown to be more common in patients with lymphoedema, with a prevalence of 25%,17 although the type of lymphoedema was not specified. In one large lymphoedema-distichiasis family, linked to chromosome 16q24, all subjects affected with lymphoedema also had abnormal findings when venous function was tested using light reflective rheography.18 The findings on doppler ultrasound of the veins in a 19 year old, of bilateral incompetence at the saphenofemoral junction and incompetent mid-calf perforator veins plus moderate reflux in the deep veins, suggested congenital abnormality of the deep and superficial veins.

In our group of subjects, varicose veins were notable owing to both early onset and increased prevalence compared with the general population. Forty-nine percent (33 of 67) of subjects in this study were found to have varicose veins with age of onset between 7 and 28 years. Males and females were found to be affected equally often and the varicose veins were frequently present before the onset of lymphoedema.

Ptosis
Congenital ptosis is not an uncommon finding in the general population. However, it was noted in 31% (14M:9F) of patients with LD. Severity ranged from mild asymptomatic cases to extreme bilateral cases requiring early surgery to allow correct development of the visual pathway. Sixty percent (14 of 23) of cases of ptosis were bilateral but most were mild. Six of the 23 (26%) affected patients had marked ptosis, all of whom required surgery (three bilateral, three unilateral).

Congenital heart disease
Congenital heart disease occurs in the white population at the rate of approximately 1%.18 In this series, the rate was considerably higher at 6.8% (five cases). There were two patients with Fallot's tetralogy, one ventricular septal defect, one pulmonary defect (not specified), and one patent ductus arteriosus. Cardiac arrhythmias have been reported in association with this condition.20 Four of these patients have been investigated in the past for palpitations, and two from the same family have had episodes of sinus bradycardia. None of these patients required treatment.

Cleft palate
Cleft lip and/or palate occurs in the European population at a rate of 7.9 per 10 000 live births.21 In this study group, three males (no females) had a cleft palate (4%). Each of these occurred in isolation (that is, not within the same family). One of these had Pierre-Robin sequence, one had a cleft of the hard palate, and another soft palate involvement only.

In one family, the father required a tracheostomy in infancy for a "throat blockage". His medical records were not available but it is possible that he had Pierre-Robin sequence. His son was reported as having a subglottic stenosis.

Other associations
Scoliosis was reported in two patients. One previously reported patient had scoliosis in association with rib fusion, neck webbing, double uterus, and bilateral, severe ptosis.22 Only one other subject had neck webbing.

Five patients reported renal abnormalities. Two had nephritis in early childhood, one had a duplex kidney, one had multiple urinary tract infections, and one patient required a renal transplant for recurrent pyelonephritis.

There were no reports of spinal extradural cysts, but there was one spinal tumour (fibrillary astrocytoma).

There was only one affected subject who had a hydrocele at birth. An affected female had an ovarian varicocele.

Three patients had strabismus, one requiring an operation at the age of 2 years. Other eye abnormalities included early cataracts (n=1) and corneal dystrophy (n=1).

An affected 4 year old boy with very marked distichiasis also had moderate learning difficulties and autistic features. One of the affected males had a daughter with learning difficulties but examination and testing were refused. There were no dysmorphic facies associated with this condition, but synophrys was noted in a number of cases.

DISCUSSION

Clinical practice
Lymphoedema-distichiasis is said to be a very rare cause of primary lymphoedema. However, as is clear in this report, 18 families and six isolated cases have been identified in the United Kingdom. There are very few publications on this condition and the phenotype is not well characterised. Many cases of LD have possibly been labelled as Meige disease as the patients have not been asked about or examined for distichiasis. The lymphoscintigrams may give an indication that this is the underlying condition. In LD they characteristically show initial uptake of tracer from the feet to the groin nodes with subsequent reflux of tracer back down into the lower leg (fig 3), whereas patients with Milroy and Meige syndromes simply show lack of uptake of the isotope and little or no node uptake. Lymphangiography, which is rarely performed now, shows that the number of lymphatic vessels is at the upper limit of normal (compared with the hypoplasia found in Meige syndrome) and multiple small lymph nodes in the mesentery. Therefore, although both may present with pubertal lymphoedema, the underlying mechanism appears to be very different. The cause of the lymphatic malfunction in the presence of normal or increased numbers of lymph vessels is not clear. It is likely that these lymphatic vessels function poorly or they may be the result of a proximal abnormality or obstruction.

The lymphoedema in LD usually appears during puberty or later (occasionally it presents in childhood). Results here prove, for the first time, that it is possible to develop lymphoedema of genetic origin well into adult life. This has been suspected but there has been no evidence to support the hypothesis. Unlike Meige syndrome, which has an increased incidence and severity in females, LD affects males at an earlier age and possibly more severely. Infections, such as cellulitis, seem to cause an irreversible exacerbation of the swelling and are more frequent in males. There may be hormonal factors involved which are not yet understood. This is suggested by the fact that most subjects develop lymphoedema around the time of puberty and females report onset associated with the oral contraceptive pill and pregnancy.

Distichiasis is highly penetrant. It is present from an early age and probably at birth. It is often the first indicator that a person is affected. Frequently, distichiasis is associated with corneal irritation, photophobia, and conjunctivitis. Occasionally it is asymptomatic, usually because the lashes curl away from the cornea, but in some cases this may be the result of corneal hypoaesthesia.23 The problems associated with the distichiasis are, therefore, unrelated to the number of aberrant eyelashes present. The extra eyelashes can often be seen on close inspection by the naked eye, but in some cases slit lamp examination is required. It has been proposed by Fox24 that distichiasis occurs separately from lymphoedema. His report looked at 78 patients with a diagnosis of distichiasis; however, there is no mention of lymphoedema. In our series, many ascertained from an adnexal surgeon, there were no families in which lymphoedema was absent, except in those cases where the only affected members were very young and in whom the lymphoedema may not yet have developed.

There was a very high frequency of varicose veins of early onset and often with no predisposing factors. Lymphatic vessels and veins have the same embryological origin, so FOXC2 probably has a role in the development of both.25 This is the first gene that has been implicated in the aetiology of chronic venous disease. It is likely that the abnormality in the veins aggravates the lymphoedema by increased capillary filtration owing to the venous hypertension.

Interestingly, there were no cases of spinal extradural cysts (despite a number of published reports); however, these could be asymptomatic, as noted by Schwartz et al.26 Recently, a FOXC2 mutation has been described in a family with dominant inheritance of cleft palate and distichiasis.27 Cleft palate occurred in three of our patients from different families. All three had novel FOXC2 mutations and were in different domains to the mutation described by Bahau et al.27

Ptosis occurred frequently in this group of patients. This suggests another developmental role for FOXC2. Interestingly, blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) has been shown to be caused by mutations in another forkhead transcription factor gene, FOXL2.13

There were a number of unusual clinical features in this series of patients. Many of these are likely to be coincidental findings. Renal abnormalities and learning difficulties have not been previously reported and may not be related to the lymphoedema-distichiasis syndrome.

Genetics
The gene for this condition, FOXC2, formerly known as MFH-1, is a forkhead transcription factor gene encoding a 2.2 kb transcript with a single exon coding region which is highly GC rich. It has not previously been linked to any human disease. However, another forkhead transcription factor gene, FOXC1 (FKHL-7), which is highly homologous to FOXC2, has been reported as causing anomalies of the anterior segment of the eye, which are dominantly inherited.14

In families with FOXC2 mutations, there appears to be no clear genotype-phenotype correlation. There was much variation in expression of the disease even within families in this series. This is consistent with a model of haploinsufficiency, with the intra- and interfamilial variation most likely the result of stochastic effects or interaction with other genes in the FOXC2 pathway.

Genetic heterogeneity has been postulated in a single family with no identified mutation of FOXC2.28 However, no linkage data were available and three members of the family had keratoconus (two required corneal transplantation), a feature not seen in this series of 74 patients. From our series we have been unable to show heterogeneity. In one family, with two subjects clearly affected with both distichiasis and lymphoedema, a FOXC2 mutation was not found. In this family, linkage data supported linkage to the FOXC2 locus. Further analysis will be required as our analyses so far would not exclude large deletions or rearrangements or mutations in the promoter or 3` untranslated region.

FOXC2 has recently been proposed to be important in the prevention of obesity and diet induced insulin resistance.29 It is postulated that an increase in the expression of FOXC2 leads to a lean and insulin sensitive phenotype and, conversely, a decrease, as would be expected in the haploinsufficiency model, should lead to obesity. Although in our series body mass index was not recorded, there was no obvious excess of obese subjects or diabetics.

Understanding development
LD is an uncommon cause of primary lymphoedema but probably not as rare as previously considered. The distichiasis is a highly penetrant feature and an important indicator of this disease. Much of the genetics of primary lymphoedema of pubertal onset remains unknown, presumably because this group has genetic heterogeneity. The distichiasis in this condition has been a distinguishing feature identifying a particular group of primary lymphoedema and therefore leading to identification of the causative gene.

The aspects of the phenotype shown here are of interest when considering the possible developmental roles of FOXC2 in human development. This is especially true of the venous insufficiency associated with LD, as it suggests that the gene plays an important role in the genesis of both venous cardiovascular and lymphatic systems. A recent publication supports this.30

The lymphoedema, in itself, is not life threatening but is the cause of much morbidity. It is difficult to manage and many of the more severely affected patients suffer from its cosmetic appearance. Understanding the function of this gene, and its developmental pathway, may help in identifying other important genes involved in the development of the lymphatic system.

ACKNOWLEDGEMENTS

RB and GB were supported by the British Heart Foundation. AHC wishes to acknowledge the support of the Bluff Field Charitable Trust. We wish to thank The Birth Defects Foundation for funding, Dr David Goudie, Consultant Clinical Geneticist, Ninewells Hospital and Medical School, Dundee for referral of patients, and John Simpson for statistical advice.

REFERENCES

http://jmg.bmjjournals.com/cgi/content/full/39/7/478

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SRY-BOX 18; SOX18
Gene map locus 20q13.33

TEXT

DESCRIPTION

The testis-determining gene SRY (480000) encodes a transcription factor characterized by a DNA-binding motif known as the HMG (high mobility group) domain. The SOX gene family consists of genes related to SRY, with a sequence identity of more than 60% to the SRY HMG box. See 600898.

CLONING

Greenfield et al. (1996) described a novel member of the mouse Sox gene family, Sox18, which is transcribed in adult lung and in cardiac and skeletal muscle. They reported that the Sox18 protein binds the sequence AACAAAG in vitro and that it is capable of transactivating gene expression. Pennisi et al. (2000) found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. 30 MEDLINE Neighbors

By EST database searching for sequences with an HMG-box, followed by 3-prime and 5-prime RACE, Azuma et al. (2000) identified a partial cDNA sequence which they then used to screen a human heart cDNA library. They isolated a cDNA clone encoding a deduced 384-amino acid protein that shares 83% identity with the mouse Sox18 protein. Northern blot analysis showed that a 1.9-kb SOX18 transcript is predominantly expressed in the heart, although weak signals are seen in brain, liver, testis, and leukocytes. 30 MEDLINE Neighbors

MAPPING

On the basis of linkage analysis, Greenfield et al. (1996) mapped the Sox18 gene to distal mouse chromosome 2. They noted that distal mouse chromosome 2 includes 1 of 10 known imprinted regions in the mouse. By homology, the human SOX18 gene would map to chromosome 20q. By PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, Azuma et al. (2000) mapped the SOX18 gene to 20q13.33. Pennisi et al. (2000) also confirmed the assignment of the SOX18 gene to 20q13.3 by radiation hybrid analysis. 30 MEDLINE Neighbors

MOLECULAR GENETICS

Using microsatellite analysis in 3 families with hypotrichosis-lymphedema-telangiectasia syndrome (HLTS; 607823), Irrthum et al. (2003) excluded the VEGFR3 (136352) and FOXC2 (602402) genes, which are related to other disorders involving lymphedema, as candidate genes. They identified the murine 'ragged' phenotype, which is caused by mutation in the Sox18 gene, as a likely counterpart of HLTS because it presents a combination of hair and cardiovascular anomalies, including symptoms of lymphatic dysfunction. By sequencing the SOX18 gene in the 3 HLTS families, they identified homozygous missense mutations (601618.0001 and 601618.0002) in affected members of 2 consanguineous families and a heterozygous nonsense mutation (601618.0003) in an affected child and his brother, who died in utero with hydrops fetalis, of a nonconsanguineous family. The nonsense mutation, which truncated the SOX18 protein in its transactivation domain, was not found in genomic DNA from either parent and was thought to constitute a de novo germline mutation. 30 MEDLINE Neighbors

ANIMAL MODEL

Greenfield et al. (1996) noted that Sox18 is a candidate for 2 mouse mutants, 'ragged' and 'wasting.' 'Ragged' heterozygous mice are viable and healthy with thin ragged coats comprised of guard hairs and awls. Homozygotes almost completely lack vibrissae and coat hairs, display generalized edema and cyanosis, rarely survive past weaning, and, depending on the genetic background, may have an accumulation of chyle in the peritoneum. Ragged(J) mice have a phenotype indistinguishable from that of ragged mice. The ragged mutation is semidominant. Pennisi et al. (2000) identified mutations in the Sox18 gene that underlie the cardiovascular and hair follicle defects in ragged mice. The ragged phenotype is caused by deletion of a cytosine at nucleotide 960 of the murine Sox18 gene; ragged(J) is caused by deletion of a guanine at nucleotide 959. Fusion proteins containing these mutations lacked the ability to activate transcription relative to wildtype controls in an in vitro assay. 30 MEDLINE Neighbors

ALLELIC VARIANTS
(selected examples)

.0001 HYPOTRICHOSIS-LYMPHEDEMA-TELANGIECTASIA SYNDROME [SOX18, ALA104PRO]

In a family reported by Devriendt et al. (2002) in which a male and female child of first-cousin Belgian parents were affected with hypotrichosis-lymphedema-telangiectasia syndrome (607823), Irrthum et al. (2003) identified homozygosity for a 455G-C transversion in the SOX18 gene, resulting in an ala104-to-pro (A104P) substitution. The mutation was present in heterozygous state in the unaffected parents. 30 MEDLINE Neighbors

.0002 HYPOTRICHOSIS-LYMPHEDEMA-TELANGIECTASIA SYNDROME [SOX18, TRP95ARG]

In a 12-year-old Turkish girl with hypotrichosis-lymphedema-telangiectasia syndrome (607823) described by Glade et al. (2001), Irrthum et al. (2003) identified a homozygous 428T-A transversion in the SOX18 gene, resulting in a trp95-to-arg (W95R) substitution. Her parents were first cousins. 30 MEDLINE Neighbors

.0003 HYPOTRICHOSIS-LYMPHEDEMA-TELANGIECTASIA SYNDROME [SOX18, CYS240TER]

In a boy with hypotrichosis-lymphedema-telangiectasia syndrome (607823) and in tissue from his deceased brother, who died in utero at 30 weeks' gestation, Irrthum et al. (2003) identified a heterozygous 865C-A transversion in the SOX18 gene, resulting in a cys240-to-ter (C240X) substitution. The mutation was not present in the genomic DNA of the unaffected nonconsanguineous parents. 30 MEDLINE Neighbors

REFERENCES

1. Azuma, T.; Seki, N.; Yoshikawa, T.; Saito, T.; Masuho, Y.; Muramatsu, M. :
cDNA cloning, tissue expression, and chromosome mapping of human homolog of SOX18. J. Hum. Genet. 45: 192-195, 2000.
PubMed ID : 10807548
2. Devriendt, K.; Vikkula, M.; Irrthum, A.; Mattjijs, G.; Mertens, A.; Fryns, J.-P. :
Autosomal recessive alopecia and lymphedema. (Abstract) Genet. Counsel. 13: 74-75, 2002.
3. Glade, C.; van Steensel, M. A.; Steijlen, P. M. :
Hypotrichosis, lymphedema of the legs and acral telangiectasias--new syndrome? Europ. J. Derm. 11: 515-517, 2001.
4. Greenfield, A.; Dunn, T.; Muscat, G.; Koopman, P. :
The Sry-related gene Sox18 maps to distal mouse chromosome 2. Genomics 36: 558-559, 1996.
PubMed ID : 8884288
5. Irrthum, A.; Devriendt, K.; Chitayat, D.; Matthijs, G.; Glade, C.; Steijlen, P. M.; Fryns, J.-P.; Van Steensel, A. M.; Vikkula, M. :
Mutations in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosis-lymphedema-telangiectasia. Am. J. Hum. Genet. 72: 1470-1478, 2003.
PubMed ID : 12740761
6. Pennisi, D.; Gardner, J.; Chambers, D.; Hosking, B.; Peters, J.; Muscat, G.; Abbott, C.; Koopman, P. :
Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nature Genet. 24: 434-437, 2000.
PubMed ID : 10742113
7. Pennisi, D. J.; James, K. M.; Hosking, B.; Muscat, G. E. O.; Koopman, P. :
Structure, mapping, and expression of human SOX18. Mammalian Genome 11: 1147-1149, 2000.
PubMed ID : 11130989

CONTRIBUTORS

Victor A. McKusick - updated : 5/21/2003
Victor A. McKusick - updated : 12/15/2000
Victor A. McKusick - updated : 6/12/2000
Ada Hamosh - updated : 3/30/2000

CREATION DATE

Moyra Smith : 1/8/1997

EDIT HISTORY

carol : 5/28/2003
tkritzer : 5/23/2003
terry : 5/21/2003
carol : 2/18/2002
carol : 12/19/2000
terry : 12/15/2000
alopez : 6/30/2000
carol : 6/13/2000
terry : 6/12/2000
alopez : 3/31/2000
terry : 3/30/2000
alopez : 6/11/1998
mark : 1/11/1997
jamie : 1/8/1997
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&d=OMIM&dopt=Detailed&tmpl=dispomimTemplate&list_uids=601618

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Assessment of genetic predisposition to secondary lymphedema as a potential tool in personalizing cancer treatment and survivorship.


Author(s):
J. M. Armer, B. R. Stewart; University of Missouri, Columbia, MO

Abstract Disclosures

Abstracts that were granted an exception in accordance with ASCO's Conflict of Interest Policy are designated with a caret symbol (^) here and in the printed Proceedings.

Abstract:

Background: We know little about how genetic factors associated with other (primary) lymphatic disorders may impact the development of (secondary) lymphedema (LE) following breast cancer treatment. This study is a pilot for a larger-scale genetics study with primary aims to: (1) examine associations among specific candidate genes and human growth factors known to be associated with primary LE in a cohort of breast cancer patients with secondary LE, and (2) seek to identify novel genetic mutations associated with LE risk through Genome Wide Association Study (GWAS) analysis. Methods: Institutional funding was obtained for a GWAS-design feasibility study with 96 breast cancer survivors with and without LE (48/48). Genetic material (from buccal swabs), limb volume (by perometry and circumferences), and self-reported LE-related symptoms are collected in one laboratory appointment. Results: Ninety-fine percent of survivors participating in an ongoing longitudinal study have consented to participate in the genetic pilot. Buccal swabs have provided adequate yield for DNA extraction (concentration average 174.94 ng/ul). The Illumina HumanOmni1-Quad BeadChip is the microarray used for the GWAS analysis. Conclusions: These pilot findings form the basis for a larger multisite study aimed at examining genetic predisposition to secondary LE, leading to the design and timing of subsequent interventions aimed at reducing LE risk and improving overall survivorship quality of life. Additionally, findings concerning interactions among breast cancer treatments and LE genetic predisposition will have the potential to guide the selection of cancer treatment to minimize these complications when survival outcomes are equivalent across competing treatment approaches.

http://www.asco.org/ascov2/Meetings/Abstracts?&vmview=abst_detail_view&confID=100&abstractID=60361

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Cloning and functional analysis of the Sry-related HMG box gene, Sox18.

Hosking BM, Wyeth JR, Pennisi DJ, Wang SC, Koopman P, Muscat GE.

University of Queensland, Institute for Molecular Bioscience, Centre for Molecular and Cellular Biology, Ritchie Research Laboratories, B402A, St Lucia, 4072, Queensland, Australia.

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.

PMID: 11179689 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11179689&itool=iconabstr

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The human SOX18 gene: cDNA cloning and high resolution mapping.

Stanojcic S, Stevanovic M.

Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, PO Box 794, 11001 Belgrade, Yugoslavia.

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY and these genes play key roles during animal development. We report here cloning and characterisation of the human SOX18 gene. SOX18 gene is expressed in foetal brain as well as in a wide range of foetal and adult tissues indicating its function is not restricted to early development. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3, 27.29 cR distal from the marker D20S173.

PMID: 10858556 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10858556&dopt=Abstract

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SOX genes: architects of development.

Prior HM, Walter MA.

Ocular Genetics Research Group, University of Alberta, Edmonton, Canada.

Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease.

Publication Types:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8827711&itool=iconabstr

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Mutations in KIF11 Cause Autosomal-Dominant Microcephaly Variably Associated with CongenitalLymphedema and Chorioretinopathy.

Jan 2012

Ostergaard P, Simpson MA, Mendola A, Vasudevan P, Connell FC, van Impel A, Moore AT, Loeys BL, Ghalamkarpour A,Onoufriadis A, Martinez-Corral I, Devery S, Leroy JG, van Laer L, Singer A, Bialer MG, McEntagart M, Quarrell O, Brice G,Trembath RC, Schulte-Merker S, Makinen T, Vikkula M, Mortimer PS, Mansour S, Jeffery S. Source Medical Genetics Unit, Biomedical Sciences, St. George's University of London, London SW17 0RE, UK.

Abstract

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated withlymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of  retinal and lymphatic structures.

Science Direct

http://www.sciencedirect.com/science/article/pii/S0002929711005532

=======

Lymphedema People Pages on Lymphedema Genes:

Lymphedema Gene VEGFC

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc

Lymphedema Gene SOX18

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18

Lymphedema Gene CCBE1

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_ccbe1

Lymphedema Gene KIF11

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_kif11

Lymphedema Gene FLT4

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_flt4

Lymphedema Gene GATA2

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_gata2

Lymphedema Gene GJC2

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_gjc2

Lymphedema Gene FOXC2

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2

Lymphedema Genetics

http://www.lymphedemapeople.com/thesite/lymphedema_genetics.htm

=========================================

Join us as we work for lymphedema patients everywehere:

Advocates for Lymphedema

Dedicated to be an advocacy group for lymphedema patients. Working towards education, legal reform, changing insurance practices, promoting research, reaching for a cure.

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Pat O'Connor

Lymphedema People / Advocates for Lymphedema

=======================================================

For information about Lymphedema

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For Information about Lymphedema Complications

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For information about Lymphedema Treatment Options

http://www.lymphedemapeople.com/thesite/lymphedema_treatment_options_revised.htm

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Lymphedema Glossary

http://www.lymphedemapeople.com/forum/topic.asp?TOPIC_ID=247

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Lymphedema People

Support Groups

-----------------------------------------------

Children with Lymphedema

The time has come for families, parents, caregivers to have a support group of their own. Support group for parents, families and caregivers of chilren with lymphedema. Sharing information on coping, diagnosis, treatment and prognosis. Sponsored by Lymphedema People.

http://health.groups.yahoo.com/group/childrenwithlymphedema/

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......................

Lipedema Lipodema Lipoedema

No matter how you spell it, this is another very little understood and totally frustrating conditions out there. This will be a support group for those suffering with lipedema/lipodema. A place for information, sharing experiences, exploring treatment options and coping.

Come join, be a part of the family!

http://health.groups.yahoo.com/group/lipedema_lipodema_lipoedema/?yguid=209645515

Subscribe: lipedema_lipodema_lipoedema-subscribe@yahoogroups.com

......................

MEN WITH LYMPHEDEMA

If you are a man with lymphedema; a man with a loved one with lymphedema who you are trying to help and understand come join us and discover what it is to be the master instead of the sufferer of lymphedema.

http://health.groups.yahoo.com/group/menwithlymphedema/

Subscribe: menwithlymphedema-subscribe@yahoogroups.com

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All About Lymphangiectasia

Support group for parents, patients, children who suffer from all forms of lymphangiectasia. This condition is caused by dilation of the lymphatics. It can affect the intestinal tract, lungs and other critical body areas.

http://health.groups.yahoo.com/group/allaboutlymphangiectasia/

Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com

......................

Lymphatic Disorders Support Group @ Yahoo Groups

While we have a number of support groups for lymphedema... there is nothing out there for other lymphatic disorders. Because we have one of the most comprehensive information sites on all lymphatic disorders, I thought perhaps, it is time that one be offered.

DISCRIPTION

Information and support for rare and unusual disorders affecting the lymph system. Includes lymphangiomas, lymphatic malformations, telangiectasia, hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database of information available through sister site Lymphedema People.

http://health.groups.yahoo.com/group/lymphaticdisorders/

Subscribe: lymphaticdisorders-subscribe@yahoogroups.com
.

===========================

At our home page we have 18 categories with 218 articles

on lymphedema, edema, and related conditions:

           The Forums

          Lymphedema Information

          Lymphedema and Edema Related Conditions

          Hereditary Conditions of the Lymphatics

          Related Medical Conditions

          Complications of Lymphedema

          Lymphedema Treatment Options

         Complete Listings of Therapists and Links

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Updated Feb. 1, 2012