Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase

Abstract

Hereditary lymphedema is a chronic swelling of limbs due to dysfunction of lymphatic vessels. An autosomal dominant, congenital form of the disease, also known as "Milroy disease," has been mapped to the telomeric part of chromosome 5q, in the region 5q34-q35. This region contains a good candidate gene for the disease, VEGFR3 (FLT4), that encodes a receptor tyrosine kinase specific for lymphatic vessels. To clarify the role of VEGFR3 in the etiology of the disease, we have analyzed a family with hereditary lymphedema. We show linkage of the disease with markers in 5q34-q35, including a VEGFR3 intragenic polymorphism, and we describe an A-->G transition that cosegregates with the disease, corresponding to a histidine-to-arginine substitution in the catalytic loop of the protein. In addition, we show, by in vitro expression, that this mutation inhibits the autophosphorylation of the receptor. Thus, defective VEGFR3 signaling seems to be the cause of congenital hereditary lymphedema linked to 5q34-q35.

Figure 1

A, Pedigree of the family with inherited congenital lymphedema. Blackened symbols represent affected individuals. B, Deduced haplotypes for three markers in 5q34-35. FLT4I, VEGFR3 intragenic polymorphism. Haplotype transmitted with disorder is boxed. C, Autoradiogram of the intragenic FLT4I marker. D, Allele-specific PCR for the A3123G mutation. Lower band, mutated allele. Upper band, internal control for PCR.

Figure 2

Autoradiogram showing sequences of two clones corresponding to wild-type (W) and mutant (M) alleles in individual I.1. Small letters, intronic sequence; capital letters, exonic sequence. * = mutation.

Figure 3

Effect of H1035R mutation on tyrosyl autophosphorylation of VEGFR3. Expression plasmids for WT and mutant VEGFR3 were transfected into 293T human embryonic kidney cells (lanes 1:0 and 0:1, respectively) or cotransfected using 3:1, 1:1, and 1:3 ratios of WT to mutant plasmid. Receptor phosphorylation was analyzed from VEGFR3 immunoprecipitates by western blotting using antiphosphotyrosine antibodies (P-Tyr, upper part). Total amount of receptor protein is shown in VEGFR3 western blotting (lower part). Sizes on the right correspond to four different forms of VEGFR3 (see text).

Figure 4

A, Amino acid sequence alignment for seven different human tyrosine-kinase receptors. Conserved catalytic loop residues are highlighted with gray background. ↑ = mutated histidine (H1035R). Δ = residue mutated in KIT causing piebaldism (R→G). * = catalytic aspartate. B, Crystal structure of VEGFR2, a tyrosine kinase receptor homologous to VEGFR3. Histidine residue mutated in VEGFR3 receptor of patients with lymphedema shown in black. The kinase activation loop was disordered in the crystal and is not shown.