LYMPHEDEMA GENETICS
FOXC2 GENE - VEGFC2 / VEGFR3 - SOX18
This page has been updated, for current information please see:
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
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
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=153100
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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
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=153200
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| FMS-LIKE TYROSINE KINASE 4; FLT4 |
Alternative titles; symbols
VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3; VEGFR3 Gene map locus 5q35.3TEXT
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
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=136352
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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
http://www.jem.org/cgi/content/full/196/6/719
----------------------------------------------------------------
| 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
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602402
----------------------------------------------------------------
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
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