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
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 LYMPHEDEMAA 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.
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. 
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%.
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.
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.
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.
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).
Congenital lymphedema is autosomal dominant in the pig (9,10:Van der Putte, 1978, 1978).
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
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| LYMPHEDEMA, HEREDITARY, II |
Alternative titles; symbols
MEIGE LYMPHEDEMAA 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).
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.
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).
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.
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.
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.
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.
Juchems (1963); Osterland (1961); Wheeler et al. (1981)
George E. Tiller - updated : 10/22/2001
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| FMS-LIKE TYROSINE KINASE 4; FLT4 |
Alternative titles; symbols
VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3; VEGFR3 Gene map locus 5q35.3TEXT
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Evans et al. (1999); Milroy (1892); Offori et al. (1993)
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
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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 (1–3).
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
,
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
(6–8).
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 (14–16),
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 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 [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
FOR COMPLETE ARTICLE WITH ILLUSTRATIONS, GRAPHS - PLEASE CLICK ON THE LINK BELOW
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| FORKHEAD BOX C2; FOXC2 |
Alternative titles; symbols
FORKHEAD, DROSOPHILA, HOMOLOG-LIKE 14; FKHL14The 'forkhead' (or winged helix) gene family, originally identified in Drosophila, encodes transcription factors with a conserved 100-amino acid DNA binding motif.
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.
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.
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.
Fang et al. (2000) determined that the FOXC2 gene contains a single coding exon and spans approximately 1.5 kb.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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
Rebekah S. Rasooly : 2/26/1998
----------------------------------------------------------------
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
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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]
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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]
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Lai, E., Prezioso, V.R., Tao, W.F., Chen, W.S. and Darnell, J.E.,Jr
(1991) Hepatocyte nuclear factor 3 
belongs to a gene family in mammals that is homologous to the Drosophila
homeotic gene forkhead. Genes Dev., 5,
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[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]
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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),
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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
-----------------------------------------------------------------
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 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
----------------------------------------------------------------
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,
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
----------------------------------------------------------------
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]
----------------------------------------------------------------
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]
----------------------------------------------------------------
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]
----------------------------------------------------------------
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]
----------------------------------------------------------------
LYMPHEDEMA
FAMILY STUDY
http://www.pitt.edu/AFShome/g/e/genetics/public/html/lymph/
----------------------------------------------------------------
GENOMICS:
Gene Found for Lymphedema.
Applied Genetics News, July, 2000
http://www.findarticles.com/cf_dls/m0DED/12_20/63802638/p1/article.jhtml
----------------------------------------------------------------
To locate a
genetic counselor or geneticist in your area
http://www.kumc.edu/gec/support/lymphede.html
----------------------------------------------------------------
Lymphovenous
Canada: A Canadian in Washington - The Genetics of Rare Diseases
Conference
http://www.lymphovenous-canada.ca/geneticsrarediseases.htm
---------------------------------------------------------------------
Scientists
find genetic mutations that cause a form of hereditary lymphedema
http://www.umich.edu/~urecord/0001/Nov13_00/7.htm
******************************************************
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.
----------------------------------------------------------------
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
----------------------------------------------------------------
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
----------------------------------------------------------------
A
model for gene therapy of human hereditary lymphedema
Article
www.pnas.org/cgi/content/full/221449198v1
----------------------------------------------------------------
Hereditary
Lymphedema
www.ajhg.org
----------------------------------------------------------------
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
----------------------------------------------------------------
Truncating
mutations in FOXC2 cause multiple lymphedema syndromes
http://hmg.oupjournals.org/cgi/content/full/10/11/1185
----------------------------------------------------------------
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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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.
=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 (
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 (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.
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 (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.
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
----------------------------------------------------------------
| SRY-BOX 18; SOX18 |
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
Moyra Smith : 1/8/1997
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
----------------------------------------------------------------
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
----------------------------------------------------------------
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]
----------------------------------------------------------------
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]
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:
----------------------------------------------------------------
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.
http://health.groups.yahoo.com/group/AdvocatesforLymphedema/
| Subscribe: | AdvocatesforLymphedema-subscribe@yahoogroups.com |
Pat O'Connor
Lymphedema People / Advocates for Lymphedema
=======================================================
For information about Lymphedema
http://www.lymphedemapeople.com/thesite/all_about_lymphedema.htm
For Information about Lymphedema Complications
http://www.lymphedemapeople.com/thesite/lymphedema_complications.htm
For Lymphedema Personal Stories
http://www.lymphedemapeople.com/forum/forum.asp?FORUM_ID=7
For information about Lymphedema Wounds
http://www.lymphedemapeople.com/thesite/lymphedema_wound_care_revised.htm
For information about Lymphedema Treatment Options
http://www.lymphedemapeople.com/thesite/lymphedema_treatment_options_revised.htm
For information about Children's Lymphedema
http://www.lymphedemapeople.com/thesite/lymphedema_childrens_pediatric.htm
=======================================================
Lymphedema Glossary
http://www.lymphedemapeople.com/forum/topic.asp?TOPIC_ID=247
===================================================
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/
Subscribe: childrenwithlymphedema-subscribe@yahoogroups.com
......................
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
......................
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
Cellulitis and Related Infections
Wound Information, Care, Treatment
Skin Care, Conditions and Complications
Exercise, Diets, Nutrition
Miscellaneous Interesting Articles section
Resources, Organizations, Support Groups
Government Resources
Advocacy and Lobbying Resources
Resources for the Medical Community
===================================================
Updated Feb. 1, 2012