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Lymphedema Gene FOXC2

Perhaps the most well known of genes responsible for hereditary lymphedema is the FOXC2 gene. Identified by University of Michigan scientists and collaborators from the University of Arizona, FOXC2 is the genetic cause of Lymphedema Praecox Meige Syndrome (Hereditary Lymphedema II).

It has also been identified as a causative factor in lymphedema-distichiasis syndrome, lymphedema ptosis and lymphedema yellow nail syndrome. In addition to the information presented on this page, we have a significant links section for further study.

Pat

FORKHEAD BOX C2; FOXC2

Alternative titles; symbols FORKHEAD, DROSOPHILA, HOMOLOG-LIKE 14; FKHL14 MESENCHYME FORKHEAD 1; MFH1 LYMPHEDEMA-DISTICHIASIS SYNDROME WITH RENAL DISEASE AND DIABETES MELLITUS, INCLUDED

Gene map locus 16q24.3

TEXT

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

CLONING

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

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

GENE FUNCTION

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

Using gene expression profiling, Mani et al. (2007) found that FOXC2, a gene involved in specifying mesenchymal cell fate during embryogenesis, was associated with metastatic capabilities of cancer cells. Foxc2 expression was required for murine mammary carcinoma cells to metastasize to lung, and overexpression of Foxc2 enhanced their metastatic potential. FOXC2 was induced in human and mouse cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals. FOXC2 specifically promoted mesenchymal differentiation during an EMT, suggesting that FOXC2 orchestrates the mesenchymal component of the EMT program. FOXC2 was significantly overexpressed in the highly aggressive basal-like subtype of human invasive ductal breast cancer.

MAPPING

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

GENE STRUCTURE

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

MOLECULAR GENETICS

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

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.

Yildirim-Toruner et al. (2004) reported a family of German-Irish descent with lymphedema-distichiasis syndrome in 6 members over 3 generations. In addition to LD, 4 had renal disease and 3 had type II diabetes. All affected members were found to have a frameshift mutation in the FOXC2 gene (1006insA; 602402.0010); all affected and unaffected members of the family were homozygous for the T allele of the 512C-T polymorphism in the 5-prime UTR of the FOXC2 gene. This polymorphism had been found to be associated with insulin sensitivity in Swedish persons (Ridderstrale et al., 2002) but not in Japanese (Osawa et al., 2003) or Pima Indians (Kovacs et al., 2003). Yildirim-Toruner et al. (2004) suggested that the phenotype of LD, renal disease, and diabetes might be the result of a combination of the mutation and the polymorphism.

In 900 dizygotic female twin pairs who had responded to a self-administered questionnaire regarding varicose veins (192200), Ng et al. (2005) found significant linkage between D16S520, located about 80 kb from FOXC2, and varicose veins, but found no association. Ng et al. (2005) suggested that FOXC2 is implicated in the development of varicose veins in the general population.

Sholto-Douglas-Vernon et al. (2005) reported the ascertainment of 34 families and 11 sporadic cases of lymphedema-distichiasis syndrome in the United Kingdom. In 2 families linked to the FOXC2 locus, no mutations or deletions were identified, leaving promoter mutations as the most likely cause of disease. Sixteen previously unpublished mutations were reported, including 2 missense mutations.

ANIMAL MODEL

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

Kriederman et al. (2003) performed dynamic lymphatic imaging and immunohistochemical examination of lymphatic tissues in mice heterozygous for a targeted disruption of Foxc2. Adult heterozygous mice characteristically exhibited a generalized lymphatic vessel and lymph node hyperplasia and rarely exhibited hindlimb swelling. Retrograde lymph flow through apparently incompetent interlymphangion valves into the mesenteric nodes, intestinal wall, and liver was also observed. In addition, Foxc2 heterozygous mice uniformly displayed distichiasis. Kriederman et al. (2003) noted that the craniofacial, cardiovascular, and skeletal abnormalities sometimes associated with lymphedema-distichiasis syndrome had previously been shown to be fully penetrant in homozygous Foxc2-null mice (Iida et al., 1997; Winnier et al., 1997). They concluded that Foxc2 haploinsufficient mice mimic closely the distinctive lymphatic and ocular phenotype of patients with lymphedema-distichiasis syndrome.

ALLELIC VARIANTS

(selected examples)

.0001 LYMPHEDEMA-DISTICHIASIS SYNDROME FOXC2, TYR99TER

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

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

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

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

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

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

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

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

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

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

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

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

YELLOW NAIL SYNDROME, INCLUDED

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

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

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

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

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

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

.0010 LYMPHEDEMA-DISTICHIASIS SYNDROME WITH RENAL DISEASE AND DIABETES MELLITUS FOXC2, 1-BP INS, 1006A

In 6 affected members spanning 3 generations of a German-Irish family with lymphedema-distichiasis syndrome (see 153400), Yildirim-Toruner et al. (2004) identified a 1-bp insertion (1006insA) in the FOXC2 gene, resulting in a frameshift mutation that predicted a premature stop at codon 462. In addition to LD, 4 of the affected members had renal disease and 3 had type II diabetes mellitus (see 125853), features not usually seen in LD. Sequence analysis of the 5-prime untranslated region for the 512C-T polymorphism showed the homozygous T allele in all family members tested. The earliest affected member of the family was 73 years old at the time of report and was on chronic renal dialysis. One of her sons, aged 45 years, had developed proteinuria at age 32 years. Renal biopsy showed chronic sclerosing glomerulopathy and chronic tubulointerstitial nephritis. One member of the family underwent renal transplantation and, shortly thereafter, pancreatic transplantation, both with excellent results. She was 36 years old at the time of report and had distichiasis but no lymphedema. Yildirim-Toruner et al. (2004) concluded that the novel phenotype of LD with renal disease and type II diabetes might be the result of a combination of the 1-bp coding region insertion and homozygosity for the T allele of the upstream UTR 512C-T polymorphism [http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602402&rn=1|Online Mendelian Inheritance in Man]]

LYMPHEDEMA, HEREDITARY, II

Alternative titles; symbols MEIGE LYMPHEDEMA LYMPHEDEMA, LATE-ONSET LYMPHEDEMA PRAECOX Gene map locus 16q24.3

TEXT

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

CLINICAL FEATURES

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

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.

MOLECULAR GENETICS

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

HETEROGENEITY

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

[http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=136352|PubMed]]

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.

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

FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance

Cederberg A, Gronning LM, Ahren B, Tasken K, Carlsson P, Enerback S.

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

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

PMID: 11551504 [PubMed - indexed for MEDLINE]

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

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. © 2006 Wiley-Liss, Inc.

Wiley InterScience

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

Gina B. Di Gregorio1*, Rickard Westergren2, Sven Enerback2, Tong Lu1, and Philip A. Kern1 1 The Central Arkansas Veterans Healthcare System, Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2 Medical Genetics, Department of Medical Biochemistry, Goteborg University, Goteborg, Sweden To whom correspondence should be addressed. E-mail: Digregorioginab@uams.edu.

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

External Links

Lymphedema Family Study - Genetics

This is perhaps one of the most important centers for us as lymphedema patients. The pace of genetic research is exploding and it will be through this type of research that we will come to know and understand:

1.) What causes lymphedema - the genetics involved.

2.) The path to a genetic cure

3,) The ability to provide treatment.

How many times have we said, “we wished someone could find out what causes lymphedema?” Countless, countless times.

The serious problem we face now is that due to the economy funding programs for this research has almost dried up and the program in very much in danger of being shutdown early next year.

Friends, we absolutely must save this program. Should it be closed, we would loose just about our only hope of a cure. We would also loose some of the most committed doctors, and support staff.

Please visit the site and go through their information. Support them as much as it is possible.

Pat O'Connor

University of Pittsburg

Mutations in FOXC2 are strongly associated with primary valve failure in veins of the lower limb.

Foxc2 induces expression of MyoD and differentiation of the mouse myoblast cell line C2C12.

Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers.

Spinal extradural arachnoid cysts associated with distichiasis and lymphedema.

Evaluation of FOXC2 as a candidate gene for chronic progressive lymphedema in draft horses.

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

Lymphedema People Internal Links FOXC2

Lymphedema Genetics

Yellow nail syndrome: not a genetic disorder? Eleven new cases and a review of the literature

The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development

Forkhead transcription factors, Foxc1 and Foxc2, are required for the morphogenesis of the cardiac outflow tract.

Lymphedema People Related Internal Links

Lymphedema People Resources

Lymphedema Family Study

Please support this important work

Welcome to the Lymphedema Family Study at the University of Pittsburgh! The goal of this project is to identify genes responsible for primary lymphedema. It is our hope that a new understanding of the genetic basis of inherited lymphedema will provide insight into its treatment and contribute to early identification of individuals at risk. Click on the links to the left for frequently asked questions about this condition, information about the inheritance of primary lymphedema, previous investigations into the genetic aspects of lymphedema, an update of our research, and listings of our references and other lymphedema websites.

This study does not involve diagnosis or treatment of lymphedema, and it was not designed to provide any direct benefit to the participants. However, it is our hope that it will benefit many lymphedema patients in the future. If there are at least two people (including you) in your family with primary lymphedema, and you would like more information on how to become involved in the Lymphedema Family Study, please contact the coordinator of this study, Kelly Knickelbein, M.S., at the address or phone number below: Lymphedema Family Study University of Pittsburgh Department of Human Genetics A300 Crabtree Hall, GSPH Pittsburgh, PA 15261 Phone: (412) 624-4657 or (800) 263-2152 e-mail: genetics@pitt.edu

Please be sure to include the words LYMPHEDEMA or GENETIC in your subject line.

http://www.pitt.edu/~genetics/lymph/

The Lymphatic Research Foundation

The Lymphatic Research Foundation is a 501©(3) not-for profit organization whose mission is to advance research of the lymphatic system and to find the cause of and cure for lymphatic diseases, lymphedema, and related disorders.

http://www.lymphaticresearch.org/main.php?content=home

Updated Nov. 3, 2011

lymphedema_gene_foxc2.txt · Last modified: 2012/10/16 14:40 (external edit)