Molecular characterization of sequence variants. (A) Mapping of ENG missense substitutions in cohorts 1 and 2 onto the crystal structures of ENG and its complex with BMP9. Proteins are shown in cartoon representation (BMP9, yellow), with specific ENG amino acids depicted as sticks and carbon atoms colored dark magenta and N-glycosylation site N307 colored cyan. (i) The relative position of 6 pathogenic or likely pathogenic variants described in the present report (Cys[C]207Tyr, Leu[L]300Pro, Leu[L]299Arg, Ile[I]220Asn, Leu[L]221Gln, and Asn[N]307Leu) defines a hotspot for pathogenic or likely pathogenic missense variants, which includes the α-helix 2 of the ENG OR1 domain, the C-terminal end of which lies close to the BMP9 binding site.³⁷  (ii) A second hotspot for pathogenic or likely pathogenic variants (Lys[K]216Glu and Glu[E]217delinsGluAla) affects residues at the N-terminal end of OR1 β-strand 16, including Lys216, which connects the C-terminal end of α-helix 2 to Gln270 at the ENG/BMP9 interface via hydrogen bond interactions. This view, which depicts amino acid contacts as observed in the high-resolution structure of ENG OR,³⁷  also shows the location of the Cys(c)207Tyr and Ile(I)220Asn mutations from a different perspective. (iii) The duplication of Leu(L)170 affects residues in the core of ENG OR2, where Leu170 is involved in a number of hydrophobic interactions. The duplication most likely affects the folding of ENG, rather than directly affecting its function; the insertion could either disrupt the register of the N-terminal part of OR2 β-strand 12 or, more likely, cause an additional residue to be accommodated in the loop that follows the same β-strand. In the latter case, the extra Leu would take the place of Arg(R)171, disrupting a hydrogen bond with Glu(E)195 as well as a stacking interaction with Arg(R)192. Moreover, by shifting Arg(R)171 to take the place of Leu(L)172, the duplication would replace a hydrophobic residue with a charged residue at the bottom of the hydrophobic core of the OR2 β-sandwich. (iv) Three variants that are benign in terms of HHT pathogenesis (Gly413Asp, Asp446Gly, and Arg571His) affect residues that are all exposed on the same face of the ENG ZP module.³⁷  The 3 variants were independently assigned as benign without reference to the tertiary structure because of their presence in the same HHT DNA as an ENG nonsense (stop), splice, or frameshift variant, respectively. (B) Bar plot of the number of pathogenic or likely pathogenic variants in the HHT Mutation Database (DB) and in cohort 1, broken down by sequence ontology (SO) term in ENG, ACVRL1, and SMAD4. The upper bars give the number in the HHT Mutation DB in 2018⁴⁰  (620 variants in total); the lower bars give the number in cohort 1, with novel variants highlighted in dark blue (106 variants in total). TG, ThromboGenomics; UTR, untranslated region.