Expression analysis of four endoglin missense mutations suggests that haploinsufficiency is the predominant mechanism for hereditary hemorrhagic telangiectasia type 1

ENDOGLIN codes for a homodimeric membrane glycoprotein that interacts with receptors for members of the TGF-beta superfamily and is the gene mutated i n the autosomal dominant vascular disorder hereditary hemorrhagic telangiec tasia type 1 (HHT1), We recently demonstrated that functional endoglin was expressed at half levels on human umbilical vein endothelial cells (HUVECs) and peripheral blood activated monocytes from HHT1 patients. Two types of mutant protein were previously analyzed, the product of an exon 3 skip whic h was expressed as a transient intracellular species and prematurely trunca ted proteins that were undetectable in patient samples. Here we report the analysis of four proteins resulting from point mutations, with missense cod ons G52V and C53R in exon 2, W149C in exon 4 and L221P in exon 5, Metabolic labeling of activated monocytes from confirmed, clinically affected patien ts revealed reduced expression of fully processed normal endoglin in all ca ses. Pulse-chase analysis with HUVECs from a newborn with the C53R substitu tion indicated that mutant endoglin remained intracellular as a precursor f orm and did not impair processing of the normal protein. Biotinylation of c ell surface proteins, metabolic labeling and pulse-chase analysis revealed that none of the engineered missense mutants was significantly expressed at the surface of COS-1 transfectants, Thus, these four HHT1 missense mutatio ns lead to transient intracellular species which cannot interfere with norm al endoglin function. These data suggest that haploinsufficiency, leading t o reduced levels of one of the major surface glycoproteins of vascular endo thelium, is the predominant mechanism underlying the HHT1 phenotype.