Analysis of protein structure The same missense substitution may be pathogenic or neutral depending on the location of the amino acid change within the encoded protein. Substitutions at sites that are important for structure or function of the encoded protein are more likely to be pathogenic. In fact, up to three-quarters of the missense variants responsible for human monogenic diseases may destabilize protein structure.20 The VHL protein (pVHL) is the substrate recognition component in a ternary complex with Elongin B and Elongin C, responsible for regulation of hypoxia inducible factor (HIF).21 Studies of the pVHL quaternary structure show that Asp121 is folded into the beta domain hydrophobic core, a relatively buried site that is not directly important for Elongin C binding. However, polar residues in the beta domain core are structurally important and substitutions here have the potential to cause complete unravelling of pVHL.22 Substitutions of Asp121 are not a frequent cause of VHL but there are two that have been demonstrated to be pathogenic: the switch of aspartic acid for asparagine (Asp121Asn) and the switch of aspartic acid for glycine (Asp121Gly).8

Supplemental Table S1

The mother, aged 49, had a history of well-controlled hypertension. Supine plasma normetanephrine was elevated (1382 pmol/l, RR <900 pmol/l) whilst plasma metanephrine was normal (117 pmol/l, RR <500 pmol/l). MRI of the brain, spine and abdomen revealed multiple, asymptomatic VHL-associated lesions. These included a spinal haemangioblastoma, bilateral T2-hyperintense adrenal lesions (18 mm and 12 mm), subsequently confirmed to be PCCs, and two pancreatic lesions (11 mm and 16 mm), subsequently confirmed to be grade 1 neuroendocrine tumours.