Mutation in Vascular Endothelial Growth Factor-C, a Ligand for Vascular Endothelial Growth Factor Receptor-3, Is Associated With Autosomal Dominant Milroy-Like Primary LymphedemaNovelty and Significance
Rationale: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity.
Objective: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation.
Methods and Results: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC.
Conclusions: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.
- lymphatic capillary
- Milroy disease
- primary lymphedema
- vascular endothelial growth factor
- vascular endothelial growth factor receptor
Primary lymphedema (PL) is clinically and genetically heterogeneous.1 PL is caused by anatomic or functional defects in the lymphatic system, leading to chronic swelling of ≥1 body parts. To date, mutations in 7 genes, CCBE1 (MIM 235510), FOXC2 (MIM 153400), GATA2 (MIM 614038), GJC2 (MIM 613480), KIF11 (MIM 152950), SOX18 (MIM 607823), and vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3; MIM 153100) have been identified as causative for disorders in which PL is a major feature. Still, there are a substantial number of PL patients, where the underlying cause has yet to be identified.
In This Issue, see p 875
Milroy disease (MD) is an autosomal dominant, congenital form of PL with reduced penetrance. The edema is usually bilateral, painless, and chronic, presenting most often at birth and predominantly at the dorsum of the feet, but may extend further up the lower limb. Variability of expression has been reported. Other clinical features of MD include prominent veins, hydroceles, and abnormal lymphoscintigraphy with functional aplasia.2 Mutations in VEGFR3 are known to be causative for ≈70% of MD3 cases, and these have recently been summarized.4 To date, despite several candidate gene screening efforts, no other genes have been associated with MD or Milroy-like disorders.
An expanded Methods section including a detailed clinical description of the whole family is available in the online-only Data Supplement.
We present a multigenerational pedigree in which MD-like lymphedema segregates in a pattern consistent with autosomal dominant inheritance (Figure 1A). The index patient (Patient II:4) is a male who on examination revealed moderate lymphedema affecting the left below-knee region, with less severe changes in the right below-knee region (Figure 1B). His mother (Patient I:2) had suffered with bilateral below-knee lymphedema since childhood (Figure 1C). The proband’s sister (Patient II:3) presented with congenital lymphedema of both feet and ankles and had prominent veins around the ankles and dorsum of the feet (Figure 1D). Patient II:3 had a son (Patient III:2) with congenital lymphedema and prominent veins of both feet and ankles (Figure 1D). His swelling spontaneously improved in the third year of life. The affected family members show variable clinical signs, which is common in MD2 and are described in greater detail in the online-only Data Supplement.
Although the index patient’s presentation was suggestive of MD, he was found to be negative for VEGFR3 mutations. Whole exome sequencing was performed, and we identified 1 heterozygous frameshift variant (c.571_572insTT; p.Pro191Leufs*10) in VEGFC (Figure 2A). The change is predicted to be disease-causing and cosegregated with the disease status in the rest of the family on Sanger sequencing (log10 of odds score 2.1).
The 4 affected adults underwent lymphoscintigraphy, which showed reduced uptake with tortuous lymphatic tracts and evidence of rerouting (Figure 1F). This contrasts with the lymphoscintigrams seen in MD patients with proven mutations in VEGFR3, which show no uptake within the main lymphatic tracts after 2 hours, suggestive of initial lymphatic vessel dysfunction (Figure 1G).
VEGFC codes for one of the ligands of the tyrosine kinase receptor VEGFR3. The c.571_572insTT mutation lies within exon 4, encoding part of the VEGF homology domain of VEGFC, leading to a predicted frameshift from codon 191 and a stop codon 10 amino acids further downstream (Figure 2B). We analyzed stability and secretion of the c.571_572insTT variant (VEGFCinsTT) by Western blotting of lysates and supernatants of 293T cells transiently transfected with wild-type human VEGFC (hVEGFC) and the human mutant VEGFC (hVEGFCinsTT) variant. In hVEGFC transfected cell lysates and supernatants, we detected bands corresponding in size to the differentially processed isoforms of VEGFC (58 kD, 31 kD, 21 kD, and 15 kD; Figure 2C). In lysates of cells transfected with the hVEGFCinsTT variant, we detected a band of ≈22 kD, the predicted molecular weight of the variant but did not detect any protein in the supernatant (Figure 2C). Thus, although VEGFCinsTT encodes a stable protein in cell lysates, secretion of this mutant variant is strongly impaired as compared with wild-type VEGFC.
To analyze the activity of the c.571_572insTT variant in vivo, we established an assay in zebrafish by overexpressing hVEGFC and hVEGFCinsTT in the floorplate, while monitoring expression by an internal ribosome entry site tagRFP cassette. Zebrafish Vegfc shares 57% identity with human VEGFC, and in zebrafish, Vegfc signaling via Vegfr3 is required for venous angiogenic sprouting5 and the development of the lymphatic system.6 Simultaneous overexpression of hVEGFC and tagRFP in the floorplate of zebrafish embryos led to excessive vessel sprouting at 56 hpf (Figure 3D), whereas overexpression of tagRFP alone had no effect on vessel growth (Figure 3C). hVEGFC overexpression promoted hypersprouting of venous and lymphatic vessels, but not of arterial vessels (Online Figure II). We used this model for testing pathogenicity of the c.571_572insTT mutation in vivo. In contrast to the expression of hVEGFC, expression of hVEGFCinsTT and tagRFP in the floorplate had no detectable effect (Figure 3E), indicating that the mutation significantly reduces, or possibly completely abolishes, the biological activity of the protein.
We also considered the possibility of the mutation resulting in a truncated protein with dominant negative properties. However, expressing the c.571_572insTT allele in the zebrafish floorplate assay did not interfere with normal vasculogenesis or angiogenesis (Figure 3E), rendering the possibility of a dominant negative effect unlikely. Furthermore, we co-overexpressed hVEGFC and hVEGFCinsTT in the zebrafish floorplate, monitoring levels of hVEGFC and hVEGFCinsTT, using IRES tagRFP and IRES mTurquoise cassettes, respectively. Co-overexpression led to hypersprouting at a similar level to embryos expressing only hVEGFC in the floorplate (Figure 4). Note that the 2 transgenes were expressed in the same cells (Online Figure III). We conclude that the VEGFC insTT variant does not have dominant negative activity but most likely leads to a haplo-insufficient phenotype.
VEGFC belongs to the family of VEGFs, which act as ligands for transmembrane tyrosine kinase receptors of the VEGF-receptor family. The c.571_572insTT variant reported here encodes a truncating mutation predicted to be pathogenic using protein structure prediction and a functional assay. In vitro data suggest that the protein is indeed truncated, stable but does not get secreted efficiently. In patients, this would be predicted to result in reduced VEGFC protein levels, thus leading to mild edema formation. Interestingly, haplo-insufficient Vegfc mice and Chy3 mice (hemizygous for Vegfc) survive to adulthood with paw edema and a hypoplastic dermal lymphatic network,7,8 and the 2 mouse models are good representatives of the human phenotype. As we have found no evidence for a dominant negative effect of the protein variant, the human patients are likely to present haplo-insufficient scenarios.
To date, only 1 VEGFC mutation positive patient and 6 affected family members have been identified. Screening of a small selection (n=16) of patients with a similar phenotype did not identify additional mutations in the ligand. Other unknown ligands and ligand-independent signaling via VEGFR39 may explain why mutations in VEGFC are only responsible for a small fraction of MD-like cases. It is also possible that proband and his family are genetically protected and can compensate for the presumed loss of VEGFC activity by other factors. Further studies are required to fully elucidate this.
In conclusion, we have identified a VEGFC mutation that causes a Milroy-like primary lymphedema. Our findings demonstrate that mutations in VEGFC can cause a phenotype similar to that found in patients with mutations in the VEGFC receptor, VEGFR3. We propose that VEGFC screening should be considered in patients presenting with a Milroy-like phenotype, but not identifiable VEGFR3 mutation, particularly, if the lymphoscintigram demonstrates poor uptake with tortuous lymphatics and rerouting. This is the first report in the literature of a human phenotype associated with a VEGFC mutation.
Sources of Funding
This work was supported by the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre (BRC) award to Guy's and St Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. Supported by the Biomics Centre, St. George’s University of London. K. Gordon and P. Ostergaard received support from the British Heart Foundation (FS/11/40/28739 and PG/10/58/28477); S. Schulte-Merker was supportd by KNAW; D. Schulte and A.v. Impel were supported by MC IEF awards; and M.G. Roukens was supported by a VENI grant from the Netherlands Organization for Scientific Research (NWO).
In January 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.2 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.300350/-/DC1.
- human wild-type VEGFC
- human mutant VEGFC
- Milroy disease
- primary lymphedema
- vascular endothelial growth factor–C
- vascular endothelial growth factor receptor-3
- Received February 1, 2013.
- Revision received February 13, 2013.
- Accepted February 13, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Primary lymphedema is a clinically heterogeneous disease with significant inter- and intrafamilial variation.
Milroy disease is an autosomal dominant congenital form of lymphedema. Only 70% of patients with this disease have mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), suggesting genetic heterogeneity.
VEGFC encodes a key ligand for VEGF receptor 3 (VEGFR3) and is important in lymphangiogenesis.
What New Information Does This Article Contribute?
Mutations in VEGFC can lead to a Milroy-like phenotype.
Mutant VEGFC protein is produced but not secreted, suggesting haploinsufficiency as a likely mechanism.
Forced expression of wild-type, but not mutant VEGFC, in the floorplate of zebrafish embryos lead to hypersprouting of venous and lymphatic vessels.
We have identified a cohort of patients with congenital primary lymphedema resembling Milroy disease, but without mutations in VEGFR3. Whole exome sequencing identified a frameshift variant in VEGFC in 1 Milroy-like family, which was predicted to be pathogenic. VEGFC is known to be a ligand for VEGFR3 and to control lymphatic sprouting during embryonic development. Using a novel in vivo overexpression assay, we show that wild-type human VEGFC, but not mutant VEGFC, leads to excessive sprouting of venous-lymphatic vessels in zebrafish. This indicates that the VEGFC frameshift mutation is causal for the Milroy-like phenotype seen in this family. VEGFC screening should therefore be considered in patients with Milroy disease, particularly, in those who do carry a mutation in the VEGFR3 gene.