SOX18 and Hypotrichosis-Lymphedema-Telan­giectasia

Lymphangiogenic Gene Therapy, yellow nail syndrome, lymphatic vascular development, Intratumoral lymphatics, peritumoral lymphatics, stem cell research, Angiopoietins, VEGF, PIGF, FOXC1, FOXC2, Lymphatic Insufficiency. SOX18, lymphatic hyperplasia, Molecular lymphangiogenesis, PROX1, FLT3, Telan­giectasia, Lymphatic endothelial cells, adult vasculogenesis, LYVE1

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SOX18 and Hypotrichosis-Lymphedema-Telan­giectasia

Postby patoco » Wed Sep 20, 2006 8:56 pm

Mutations in the Transcription Factor Gene SOX18 Underlie Recessive and Dominant Forms of Hypotrichosis-Lymphedema-Telangiectasia

The American Society of Human Genetics.

1Laboratory of Human Molecular Genetics, Christian de Duve Institute of
Cellular Pathology and Université catholique de Louvain, Brussels;
2Center for Human Genetics, University of Leuven, Leuven, Belgium;
3Prenatal Diagnosis and Medical Genetics Program, Mount Sinai Hospital,
University of Toronto, Toronto; 4Medisch Spectrum Twente, Hengelo, The
Netherlands; and 5Department of Dermatology, Academisch Ziekenhuis
Maastricht, Maastricht, The Netherlands

Address for correspondence and reprints: Prof. Miikka Vikkula,
Laboratory of Human Molecular Genetics, Christian de Duve Institute of
Cellular Pathology and Université catholique de Louvain, Avenue
Hippocrate 74+5, bp 75.39, B-1200 Brussels, Belgium. E-mail:


Hereditary lymphedema is a developmental disorder characterized by
chronic swelling of the extremities due to dysfunction of the lymphatic
vessels. Two responsible genes have been identified: the vascular
endothelial growth factor receptor 3 (VEGFR3) gene, implicated in
congenital lymphedema, or Milroy disease, and the forkhead-related
transcription factor gene FOXC2, causing lymphedema-distichiasis. We
describe three families with an unusual association of hypotrichosis,
lymphedema, and telangiectasia. Using microsatellite analysis, we first
excluded both VEGFR3 and FOXC2 as causative genes; we then considered the murine ragged phenotype, caused by mutations in the Sox18 transcription factor, as a likely counterpart to the human disease,
because it presents a combination of hair and cardiovascular anomalies,
including symptoms of lymphatic dysfunction. Two of the families were
consanguineous; in affected members of these families, we identified
homozygous missense mutations in the SOX18 gene, located in 20q13.

The two amino acid substitutions, W95R and A104P, affect conserved residues in the first a helix of the DNA-binding domain of the transcription
factor. In the third family, the parents were nonconsanguineous, and
both the affected child and his brother, who died in utero with hydrops
fetalis, showed a heterozygous nonsense mutation that truncates the
SOX18 protein in its transactivation domain; this substitution was not
found in genomic DNA from either parent and hence constitutes a de novo
germline mutation. Thus, we show that SOX18 mutations in humans cause
both recessive and dominant hypotrichosis-lymphedema-telangiectasia,
suggesting that, in addition to its established role in hair and blood
vessel development, the SOX18 transcription factor plays a role in the
development and/or maintenance of lymphatic vessels.


Lymphedema, a chronic swelling of the extremities that is due to
impaired lymphatic drainage, causes cosmetic harm, disability, and
predisposition to infection and chronic ulceration. Primary lymphedema
can be noninherited or hereditary, and, recently, progress has been
made in understanding the molecular bases of hereditary forms.
Congenital hereditary lymphedema, or Milroy disease (MIM 153100), has
been found to be caused by missense inactivating mutations in the
kinase domain of vascular endothelial growth factor receptor 3 (VEGFR3)
(Irrthum et al. 2000; Karkkainen et al. 2000), and lymphedema
associated with distichiasis (MIM 153400), an additional row of
eyelashes originating from the meibomian glands, has been shown to be
caused by truncating mutations in the forkhead-related transcription
factor FOXC2 (Fang et al. 2000; Finegold et al. 2001). Lymphedema is
also observed in a few genetic syndromes-such as
lymphedema-cholestasis (MIM 214900), or Aagenaes syndrome, mapped to chromosome 15q (Bull et al. 2000), and lymphedema-microcephaly-chorioretinopathy (MIM 152950) (Feingold and Bartoshesky 1992). Finally, nuchal lymphedema and hydrops fetalis are frequently observed in patients with Noonan syndrome (MIM 163950) (Witt et al. 1987) and in patients with a variety of chromosomal abnormalities, including Turner syndrome (Chitayat et al. 1989; Boucher et al. 2001).

The interest in the identification of genes involved in hereditary
lymphedema extends beyond the mere understanding of this disorder.
Conditions associated with lymphedema, such as Noonan syndrome, may
have higher incidences of lymphoproliferative disorders (Choong et al.
1999). In addition, lymphangiogenesis, the development of lymphatic
vessels, has recently been linked to tumor growth and metastasis, and
lymphedema genes are prime candidates as regulators of this process. In
particular, the VEGFR3 gene, implicated in hereditary congenital
lymphedema, has attracted much attention as a promising anticancer-drug target (Plate 2001). Overexpression of the VEGFR3 natural ligands VEGFC and VEGFD has been shown to lead to an increase in tumor growth and metastasis via the lymphatic system (Skobe et al. 2001; Stacker et al. 2001), whereas a block of VEGFR3 signaling has been shown to counteract these effects in the mouse (He et al. 2002). Furthermore, the identification of genes causing hereditary lymphedema could lead to therapeutic advances in the management of secondary lymphedema, such as lymphedema resulting from anticancer surgery or parasitic disease.

We describe three families with an unusual hereditary condition in
which hypotrichosis, lymphedema, and telangiectasia are associated.
After excluding VEGFR3 and FOXC2 as potential disease-causing genes, we identified the murine ragged phenotype as a putative counterpart of the human disorder. We found that SOX18, the human orthologue of the ragged gene, is mutated in these three families and accounts for both a
recessive form and a dominant form of the disease.

Subjects and Methods


Clinical findings in all patients are summarized in table 1. Written
informed consent was obtained from all participants, and the present
study was approved by the ethical committee of the medical faculty at
the Université catholique de Louvain, Brussels, Belgium.
Family I.-The index patient is a boy, the first child born to
unaffected parents who are first cousins of Belgian descent (Devriendt
et al. 2002). He was born at 32 wk gestation, with birth weight of
1.650 kg (25th-50th percentile), and presented with respiratory
distress. A normal amount of scalp hair was present at birth but
decreased progressively to the extent of total alopecia at age 6 mo.
Some hair growth was noted later (at age 3.5 years), but it remained
very sparse (figs. 1A and 1B), with absent eyebrows and eyelashes.
Sweating, nails, and teeth were normal. The skin over the hands and
feet was thin and transparent, with visible blood vessels. A skin
biopsy revealed several abortive hair follicles, some atrophic
sebaceous glands, and normal sweat glands. A bilateral hydrocele was
surgically corrected at age 12 years. At age ~15 years, the patient
progressively developed lymphedema of the lower limbs. A Doppler
ultrasonography of the venous system of the lower limbs done at age 25
years was normal. A lymphatic scintigraphy was performed by
interdigital subcutaneous injection of a radio-labeled (Technetium 99m)
colloidal tracer into the dorsal part of the foot. No tracer was
detected in the groin during a monitoring period of 32.5 min,
indicating that there was no detectable lymphatic flow.

His younger sister presented a similar phenotype. She was born at 36 wk
gestation with birth weight 2.7 kg (50th percentile). Initially, she
had normal black hair. A vascular nevus was present on the palm of the
right hand and faded during childhood. During infancy, her hair
diminished progressively, and, around age 2 years, her hair was very
sparse and has remained so ever since. She had neither eyebrows nor
eyelashes, and she did not develop axillary or pubic hair at puberty.
Around puberty, she developed progressive lymphedema of the lower limbs (fig. 1C). Doppler study of the venous system of the legs was normal. Clinical examination at age 26 years showed, besides the hair
abnormalities, normal teeth and nails. Sweating was normal, but her
skin was thin. The skin over her hands and feet was transparent, and
dilated veins and varicosities were apparent on the palm of her right
hand (fig. 1D). The pedigree of family I is shown in figures 2A and 3B.

Family II.-The index patient has been described in detail elsewhere
(Glade et al. 2001). In summary, she is a 12-year-old child of
unaffected, first-cousin, Turkish parents. Her younger brother is
unaffected. Lymphedema appeared in the lower limbs at age 4 years.
Scalp hair has always been sparse, but with a normal appearance.
Eyebrows and eyelashes were missing. Palms and soles showed multiple
telangiectasias, with ectatic capillaries and cutis marmorata-like
lividity of the skin. Small dark-red papular vascular lesions were seen
on several toes. Her nails and teeth were normal. The pedigree of
family II is shown in figure 3D. DNA from the parents was not

Family III.-The index patient is a boy, born in 1997, with sparse
hair. He presented swelling of the upper eyelids, scrotal edema, and
very large bilateral hydroceles. Hair loss began at age ~6 mo,
accompanied by a lightening of its color. Now, at age ~6 years,
alopecia is almost complete (fig. 1E), including eyebrows and
eyelashes. The patient presented mild eczema on the cheeks and
telangiectasias on the scalp, scrotum (fig. 1F), and legs. His nails
and teeth were normal.

The brother of the index patient died in utero at 30 wk gestation. The
fetus had nonimmune hydrops fetalis, with chylous effusions in the
pleural and peritoneal cavities. The lungs presented generalized
vascular congestion and a mild dilatation of lymphatic vessels.
Parental physical examination showed no abnormalities. The pedigree of
family III is shown in figure 3F.

Microsatellite Analysis

Peripheral blood was obtained from participating family members. For
the deceased fetus of family III, a tissue sample was used. DNA was
extracted using standard laboratory procedures. Microsatellite markers
around the VEGFR3, FOXC2, and SOX18 genes were identified on the basis of Entrez Map View (National Center for Biotechnology Information),
build 28, and the Unified Database (UDB) (Weizmann Institute of Science
[WIS] Bioinformatics and Biological Computing), both as of January
2002. In addition, we used an intronic CA-repeat microsatellite
(VEGFR3-CA in fig. 2B) in the VEGFR3 gene (Iljin et al. 2001).
Genotyping, using 32P-labeled oligonucleotides, was performed as
described elsewhere (Boon et al. 1999). To rule out nonpaternity in
family III, in which a de novo mutation was observed, we genotyped the
four members of the family for 10 Weber set 8 microsatellites from
various chromosomes.

Database Search for Candidate Genes for

We searched for potential murine models for the disease in the Jackson
Laboratory Mouse Genome Informatics databases, using various available
query forms and the keywords "lymphedema," "lymphatic,"
"chylous," "chylothorax," "edema," "edematous," and
"swelling." Mouse phenotypes retrieved were evaluated for their
resemblance to the human condition.

SOX18 Gene Sequencing and Restriction-Enzyme Analyses of Mutations

SOX18 gene structure was obtained from the University of
California-Santa Cruz (UCSC) Genome Bioinformatics Web site (Human
Genome Project Working Draft, December 2001 freeze). A 1,550-bp PCR
fragment containing the full SOX18 coding sequence and the 196-bp
intervening intron was amplified using genomic DNA from patients and
control individuals, was purified using the Qiagen PCR purification
columns (Qiagen), and was sequenced on a CEQ2000 fluorescent capillary sequencer (Beckman Coulter). Sequences were further aligned and analyzed with Sequencher 3.1 (Gene Codes). The identified nucleotide substitutions G455C, T428A, and C865A created additional recognition sites for restriction enzymes MnlI, HphI, and DdeI, respectively. The presence of these RFLPs was tested in the members of the relevant family, as well as in 96 control individuals from the genetically heterogeneous Belgian population. Oligonucleotide sequences and reaction conditions for PCR amplification, sequencing, and restriction
analysis are available from the authors on request.


Exclusion of VEGFR3 and FOXC2-and Database Identification of SOX18

In families I and II, the parents were first cousins and presented no
signs of either lymphatic or hair anomalies, despite the marked
phenotype of their children. This strongly suggested an autosomal
recessive mode of inheritance. To assess the possibility that the
lymphedema-causing genes VEGFR3 and FOXC2 are involved in the syndrome, we determined the genotypes of the parents of family I and their children for microsatellite markers closely surrounding these genes
(fig. 2B). The affected siblings showed heterozygosity for markers at
both loci, excluding these genes as causative for the disease under a
recessive inheritance model.

A search in mouse databases identified the ragged phenotype, caused by
mutations in the Sox18 gene, as a putative murine counterpart of human
hypotrichosis-lymphedema-telangiectasia. The order of markers around
human SOX18 was inconsistent between integrated (UDB [WIS
Bioinformatics and Biological Computing]) and sequence-based (UCSC
Genome Bioinformatics) maps of the human genome, both as of January
2002, and genotyping revealed an intermingling of uninformative,
recombinant, and potentially linked markers (data not shown). This
prompted us to analyze SOX18 by direct sequencing in the patients with

Homozygous SOX18 Mutations in Patients from Families I and II

Sequencing of the SOX18 gene in family I, presenting consanguinity,
revealed a G455C transversion in the coding sequence (GenBank accession number NM_018419) (fig. 3A). This nucleotide substitution caused the replacement of alanine at position 104 in the SOX18 protein (GenBank accession number NP_060889) by a proline (A104P). The mutation was present at homozygous state in the two affected children and at heterozygous state in the unaffected parents (fig. 3B), in accordance with a recessive inheritance pattern in a consanguineous family.

In consanguineous family II, we identified a T428A transversion in the
coding sequence, resulting in the replacement of tryptophan at position
95 in the protein by an arginine (W95R). This mutation was present at
homozygous state in the affected patient (fig. 3C).

Neither mutation was observed in the genomic DNA from 96 control
individuals (data not shown).

Heterozygous SOX18 Mutation in Patients from Family III

In family III, we identified a C865A transversion in the coding
sequence (fig. 3E), transforming the codon for cysteine at position 240
of the protein (TGC) into a premature stop codon (TGA). The mutation
was detected at heterozygous state in DNA extracted from the blood of
the patient and the tissue of the deceased fetus and was not present in
genomic DNA from the parents (fig. 3F). This mutation, which truncates
the transcription factor in its transactivating domain, was not
observed in genomic DNA from 96 control individuals. The genotypes of
the four members of the family, for 10 microsatellite markers, were
compatible with paternity (data not shown).


involvement of VEGFR3 and FOXC2, the two genes associated with
lymphedema in humans (Fang et al. 2000; Irrthum et al. 2000; Karkkainen et al. 2000; Finegold et al. 2001). FOXC2, in particular, could be considered to be an interesting candidate, because it causes
lymphedema, with variable age at onset, associated with distichiasis
(the presence of an additional row of eyelashes). A hair abnormality
was also seen in the three families described here, and lymphedema was
not always present at birth. However, we were able to exclude the two
genes in one of the consanguineous families by using microsatellite

We then searched for an animal model of the disease in public
databases. The chy mouse has recently been identified as an animal
model for congenital hereditary lymphedema. As do human patients, the
chy mouse has an inactivating mutation in the VEGFR3 tyrosine kinase
domain and exhibits leg edema due to hypoplastic lymphatic vessels
(Karkkainen et al. 2001). An anomaly frequently observed in the chy
mouse is the occurrence of chylous ascites, which may be caused by
dysfunction of intestinal lymphatic vessels. Similarly, the murine
ragged phenotype, caused by a mutation in the gene encoding the
transcription factor Sox18, presents chylous ascites, in addition to
sparse fur (Pennisi et al. 2000b). Thus, we considered SOX18 to be a
remarkable candidate for the human hypotrichosis-lymphedema-telangiectasia syndrome.

In adults, the SOX18 gene is expressed in a broad range of tissues,
including brain, heart, skeletal muscle, spleen, kidney, liver, and
lung (Pennisi et al. 2000c; Hosking et al. 2001). It belongs to the SOX
(Sry-type high-mobility group [HMG] box) gene family, encoding
transcription factors required in diverse developmental processes, such
as lens formation, not allowed and neural determination, spermatogenesis,
chondrogenesis, and cardiac development (Wegner 1999). These
transcription factors are highly similar to each other in the HMG
domain, a 79-amino-acid DNA-binding motif that specifically binds to
the heptameric sequence (A/T)(A/T)CAA(A/T)G (Wegner 1999). Three
members of the SOX family are already associated with a human disease.
Mutations in SRY cause not allowed reversal and gonadal dysgenesis (Berta et
al. 1990; Jager et al. 1990), mutations in SOX9 cause campomelic
dysplasia with autosomal not allowed reversal (Foster et al. 1994; Wagner et
al. 1994), and mutations in SOX10 cause Waardenburg-Hirschsprung
syndrome (Pingault et al. 1998).

We identified recessive SOX18 missense mutations in affected
individuals from two consanguineous families (I and II) with
hypotrichosis-lymphedema-telangiectasia. The first recessive mutation,
A104P, affects an alanine that is conserved in most members of the SOX
family (fig. 4A). Because SOX18 and SRY are homologous and present high sequence similarity in their respective HMG domains, we used the
nuclear-magnetic-resonance model of SRY (Werner et al. 1995) to
visualize the positions of the two recessive mutations (fig. 4B). The
alanine at position 104 (shown in red in the model) is located in the
first a helix of the DNA-binding domain, and its replacement by a
proline, an amino acid that produces significant bends in a helices,
is very likely to affect the structure of this domain. The second
recessive mutation, W95R, substitutes a tryptophan residue (shown in
magenta in fig. 4) that is conserved in all SOX proteins. The
three-dimensional structure of SRY reveals that this tryptophan belongs
to a cluster of conserved aromatic and aliphatic residues that maintain
orientation of the three a helices of the DNA-binding domain through
their packing (Werner et al. 1995), and the mutation thus probably
destabilizes the domain.

The C240X SOX18 mutation in family III, located in the vicinity of the
murine ragged frameshift mutations (fig. 4C), causes a
most-likely-dominant form of the syndrome, since the patient and the
fetus are heterozygous for the substitution and since sequencing of the
complete coding region and splice sites did not reveal additional
genetic alterations. Moreover, the nucleotide substitution was not
found in DNA extracted from the blood of the parents, and nonpaternity
was ruled out, implying that this mutation arose de novo. This is
similar to the situation for the other SOX genes associated with human
disorders (i.e., SRY, SOX9, and SOX10), in which usually-de-novo
mutations are observed at the heterozygous state (Berta et al. 1990;
Jager et al. 1990; Foster et al. 1994; Wagner et al. 1994; Pingault et
al. 1998). Like the autosomal SOX9 and SOX10 genes, which presumably
exert their effect through haploinsufficiency (Foster et al. 1994;
Wagner et al. 1994; Pingault et al. 1998), the C240X mutation may
result in a null allele. In this case, the proteins with the recessive
mutations should retain some activity. However, a dominant-negative
effect cannot be ruled out, because the resulting protein is truncated
in its transactivation domain and, if still present, may bind the
promoters of target genes via its intact DNA-binding domain.

The first component of the syndrome, early-onset hypotrichosis with
absence of eyebrows and eyelashes, is present in all patients with
SOX18 mutations. During murine embryonic development, Sox18 is
transiently expressed in the mesenchyme underlying the vibrissae and
pelage hair follicles, and its role in hair development has been
demonstrated by its implication in the murine ragged phenotype (Pennisi
et al. 2000b). Its two closest paralogues, Sox7 and Sox17, have been
postulated to carry overlapping functions (Pennisi et al. 2000a). Human
SOX7 and Xenopus Sox17 physically interact with ß-catenin and
interfere with Wnt/ß-catenin signaling (Zorn et al. 1999; Takash et
al. 2001), a pathway essential for hair morphogenesis (Gat et al.
1998). ß-Catenin controls fate decision of the skin stem cells toward
epidermal or follicular keratinocyte differentiation (Huelsken et al.
2001), and the observed hypotrichosis could be due to abnormal
interference with this process by Sox18. Intriguingly, mice with
conditional inactivation of ß-catenin after hair-follicle formation
show complete loss of pelage after the first hair cycle, a phenotype
reminiscent of the human patients with SOX18 mutations.

The second component of the syndrome, lymphedema, presented differently in the three families. The age at onset of the lower-limb lymphedema was highly variable, ranging from 4 to 15 years. The affected boy from family III currently has no lower-limb lymphedema, but he presented with edema of the upper eyelids. A more severe failure of the lymphatic system presented in his late brother, who had nonimmune hydrops fetalis.

This severe presentation was also observed in two patients in
a family with hereditary lymphedema-distichiasis caused by a mutation
in the FOXC2 gene (Fang et al. 2000). The frequent involvement of the
lymphatic system among patients implies SOX18 in development and/or
maintenance of lymphatic vessels. Because SOX proteins constitute a
family of 20 members in humans (Schepers et al. 2002), have overlapping expression patterns, and bind to the same DNA heptamer motif, they are supposed to achieve specificity through combinatorial associations with other factors (Wilson and Koopman 2002). Interaction with, for example, FOXC2 or the lymphatic vessel regulator PROX1 (Wigle and Oliver 1999) might enable SOX18 to specifically activate lymphatic-related target genes. Such candidate targets are the genes encoding vascular endothelial growth factors VEGFC and VEGFD, as well as their receptor, VEGFR3. Indeed, direct regulation of the VEGFC and VEGFD genes might be possible, because they contain the heptameric binding site for SOX proteins in their promoters, as revealed by examination of current sequence databases (data not shown).

The third component of the syndrome is an anomaly of peripheral blood
vessels, manifesting as telangiectasia in three of the four patients.
Involvement of blood vessels is not surprising, since Sox18 mRNA is
transiently detected in endothelial cells during mouse embryonic
development and since the Sox18-mutated ragged mouse exhibits
conspicuous vascular anomalies, such as hematomas and dilation or
rupture of peripheral blood vessels (Pennisi et al. 2000b); however, no
signs of comparable vascular abnormalities were observed in the
patients. The telangiectasias, as well as the other observed
anomalies-such as thinness and transparency of the skin, hydrocele,
and small cutaneous papular vascular lesions-were observed only in
some patients. The phenotypic heterogeneity is reminiscent of that seen
in lymphedema-distichiasis (Finegold et al. 2001) and could be
explained by the presence of modifier genes. In fact, the influence of
modifier genes has been demonstrated in the Sox18-deficient ragged
mouse, in which the accumulation of chyle in the peritoneum depends on
the genetic background (Wallace 1979). Thus, SOX18 mutations are
expected to present as isolated alopecia in some patients-especially
young children, before the onset of lymphedema-and as nonimmune
hydrops fetalis of unknown etiology in others. ... id=1180307


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