The other SDHA variant was identified in a patient with a VHL mutation and PA (patient 21)

Patient 3 A 15-year-old man with a history of occipital headache, slightly elevated blood pressure with normal ophthalmological examination for 2 months was found to have a 4-cm-diameter left adrenal mass at MRI, being the abdominal sonography negative. Medical history was negative except for mild asthma, not necessitating medical treatment. The mass showed inhomogeneous density: hypodensity in T1 and hyperdensity in T2 and after Gadolinium contrast. A 123I-MIBG scintigraphy showed a high tracer uptake in left adrenal gland. Immunometric assays indicated high levels of CgA, 73 UI/L NR 2–18) and catecholamines: noradrenaline levels were 11·775 nmol/day (NR: 90–470), and adrenaline and dopamine levels were in the normal range. The patient underwent surgical removal of the mass, which was 4 × 5 × 4 cm in size. Immunohistochemical assays indicated that the tumour was positive for chromogranin A compatible with a diagnosis of PHEO. Follow-up 4 months after surgery (April 2011) showed normal values for chromogranin A, blood pressure and negative MRI. Family history was negative for hypertension, PHEO or other neuroendocrine tumours.

To elucidate possible effects of the mutation D197N on the structure of VHL and its interaction with co-ligands, we considered the high-resolution (1·85 A) structure of VHL in complex with HIF-1α and elongin B and C, deposited in the Protein Data Bank with identity code 1LM8 and performed a multiple alignment utilizing two different software. Residue D197 participates in a salt bridges network with R120, E134 and R200 (Fig. 4a) that anchors the C-terminal helix to the rest of the protein. The crystallographic structure shows that the bridge networks are in close contact with the first NH2-terminal fluctuating residues. As shown in Fig. 4b, the mutation is located in the COOH-terminal helix and that there are no direct interactions between the C-terminal helix and elongin B, elongin C and HIF-1a, suggesting that D197N probably does not alter directly the complex formation and activity. However, it is likely that the D197N mutation modifies the salt bridges network established by residues 120, 134, 197 and 200. Actually, the important role of the network of salt bridges in the physiological function of the VHL protein is indicated by the multiple alignment of VHL sequences from Swiss-Prot and RefSeq that shows that aminoacid 197 and the other involved in the network are evolutionarily conserved (Fig. 5).

Patient 2 A 17-year-old girl with a history of primitive headache, intense nocturnal sweating and 2-month-long amenorrhoea and elevated blood pressure presented a 7-cm-diameter right adrenal mass at abdominal sonography, confirmed by MRI. The mass showed inhomogeneous density: hypodensity in T1 and hyperdensity in T2 and after Gadolinium contrast. Immunometric assays indicated very high levels of chromogranin A (242 UI/L; NR 2–18) and catecholamines: noradrenaline levels were 14·460 nmol/day (NR: 90–470), and adrenaline and dopamine levels were in the normal range. A 123I-MIBG scintigraphy showed a high tracer uptake in right adrenal gland, as expected. The patient underwent surgical removal of the mass, which was 8 × 6 × 5 cm in size. Immunohistochemical assays indicated that the tumour was positive for chromogranin A and synaptophysin, compatible with a diagnosis of PHEO. Follow-up blood test after 2 years was within normal limits, except for a still moderate elevated CgA (67 UI/L, NR 2–18). The family history was negative for PHEO or other neuroendocrine tumours.