Identification and validation of a novel pathogenic variant in GDF2 (BMP9) responsible for hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multisystemic vascular dysplasia, characterized by arteriovenous malformations (AVMs), mucocutaneous telangiectasia and nosebleeds. HHT is caused by a heterozygous null allele in ACVRL1, ENG, or SMAD4, which encode proteins mediating bone morphogenetic protein (BMP) signaling. Several missense and stop-gain variants identified in GDF2 (encoding BMP9) have been reported to cause a vascular anomaly syndrome similar to HHT, however none of these patients met diagnostic criteria for HHT. HHT families from UK NHS Genomic Medicine Centres were recruited to the Genomics England 100,000 Genomes Project. Whole genome sequencing and tiering protocols identified a novel, heterozygous GDF2 sequence variant in all three affected members of one HHT family who had previously screened negative for ACVRL1, ENG, and SMAD4. All three had nosebleeds and typical HHT telangiectasia, and the proband also had severe pulmonary AVMs from childhood. In vitro studies showed the mutant construct expressed the proprotein but lacked active mature BMP9 dimer, suggesting the mutation disrupts correct cleavage of the protein. Plasma BMP9 levels in the patients were significantly lower than controls. In conclusion, we propose that this heterozygous GDF2 variant is a rare cause of HHT associated with pulmonary AVMs.

Keywords: arteriovenous malformations; bone morphogenetic protein 9; hereditary hemorrhagic telangiectasia.

FIGURE 1

HHT family with GDF2 missense variant and reduced plasma BMP9 levels. (a) Pedigree at the time of family recruitment. The proband (III.1) is arrowed. (b) Sanger sequencing trace from individual III.2 showing the heterozygous GDF2 variant. Nucleotide sequence and protein translation are depicted below, with variants denoted in red font. (c) Circulating BMP9 levels in human plasma from the three individuals carrying the c.1282T>C variant were significantly lower than in five unaffected individuals collected and analyzed under the same conditions (*p = 0.036, Mann–Whitney two-tailed test)

FIGURE 2

GDF2 c.1282T>C disrupts processing of BMP9 protein. (a) Western blot of lysates from 293T cells transfected with wild-type (WT; 500 ng or 1 μg of DNA) or mutant (1282C, clones #3 and #9; 1 μg) GDF2 plasmids, run under reducing conditions and probed with antibody to human BMP9. Full length protein (50 kDa) is expressed from the mutant plasmids, but is not correctly processed, leading to absence of the mature monomer (12 kDa). (b) Conditioned medium from these cells was applied to cultures of human pulmonary artery endothelial cells (PAEC). Medium from the wild-type (WT) but not mutant (c.1282C) plasmid induces mRNA expression of BMP-responsive genes, ID1 and pre-miR-21. Recombinant BMP9 (rBMP9) was used as a positive control, and medium from GFP transfection as a negative control. Expression was normalized to GAPDH, and fold change was calculated relative to untreated PAEC