Mutations in RASA1 and GDF2 identified in patients with clinical features of hereditary hemorrhagic telangiectasia

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder caused by mutations in ENG, ACVRL1 and SMAD4, which function in regulating the transforming growth factor beta and bone morphogenetic protein signaling pathways. Symptoms of HHT can be present in individuals who test negative for mutations in these three genes indicating other genes may be involved. In this study, we tested for mutations in two genes, RASA1 and GDF2, which were recently reported to be involved in vascular disorders. To determine whether RASA1 and GDF2 have phenotypic overlap with HHT and should be included in diagnostic testing, we developed a next-generation sequencing assay to detect mutations in 93 unrelated individuals who previously tested negative for mutations in ENG, ACVRL1 and SMAD4, but were clinically suspected to have HHT. Pathogenic mutations in RASA1 were identified in two samples (2.15%) and a variant of unknown significance in GDF2 was detected in one sample. All three individuals experienced epistaxis with dermal lesions described in medical records as telangiectases. These results indicate that the inclusion of RASA1 and GDF2 screening in individuals suspected to have HHT will increase the detection rate and aid clinicians in making an accurate diagnosis.

Figure 1

Average depth of sequencing coverage for each exon of RASA1 and GDF2 from 93 samples.

Figure 2

Potentially causative variants detected in RASA1 and GDF2 in samples suspected to have hereditary hemorrhagic telangiectasia (HHT). (a) Heterozygous mutations in RASA1 and GDF2 detected by NGS assay and (b) Sanger confirmed.

Figure 3

Protein domain structure of RASA1 depicting the location of the two pathogenic mutations identified in the study.

Figure 4

Structural analysis of the RASA1 PH domain detailing the impact of the identified p.Y528C (c.1583A>G) mutation. (a) The structure of the RASA1 PH domain is shown. The location of the variant p.Y528C is indicated in green, the inositol-phosphate (IP)-binding site is indicated and the C-terminal protein interaction domain is highlighted in yellow. (b). Sequence alignment between PH domain of RASA1 (RASA1_PH: residue 474–577) and PH domain of Pleckstrin (PLEK_PH: residues 235–347). Secondary structure obtained from RASA1_PH model and PLEK_PH (PDB:1XX0) are shown where arrows represent beta-sheet structure and curls represent alpha-helices. Conserved residues are white with red background, residues with similar properties are red with white background and the location of the p.Y528C variant is in a green box. The RASA1_PH domain has overall 30% identity with PLEX_PH domain with most identical residues present in secondary structure elements.

Figure 5

Graphical representation of RASA1 Ras-Gap domain and Ras-p21 crystal structure detailing the impact of the identified p.E1015* (c.3043G>T) mutation. The surface of RASA1 Ras-Gap domain (residues 718–1037) surface is colored based on the Kyte–Doolittle hydrophobicity scale (hydrophobic regions in purple and hydrophilic areas in turquoise) and the Ras-p21 is in shown with gray surface. The p.E1015* variant would remove the C-terminal helix (orange) and would act to destabilize the GTPase-activating protein (GAP) domain by exposing the hydrophobic region to solvent. Images generated with chimera.