Anti-CYB5R3 monoclonal antibody, clone 2F9

We report a case of prophylactic management with methylene blue (MB) in an almost 4-year-old male with congenital methemoglobinemia type II. He has a CYB5R3 compound heterozygote mutation, causing a cytochrome-b(5) reductase deficiency. Since the MB treatment regimen has commenced, his methemoglobin level has been significantly lower. He has shown modest behavioral improvements (as assessed on the Achenbach behavior report scales). There have been no iatrogenic side effects. These findings are encouraging for symptomatic improvement with regular prophylactic MB treatment but represent a single case report, which must be interpreted with caution.

Cytochrome P450 17A1 (CYP17A1) catalyses the 17 alpha-hydroxylation and 17,20 lyase reactions to convert pregnenolone to 17 alpha-hydroxypregnenolone (17OHP) and subsequently the androgen dehydroepiandrosterone (DHEA). In pigs and humans, CYP17A1 also catalyses the delta-16-synthase reaction to produce the 16-androstene steroid 5,16-androstadien-3 beta-ol (16A), which is then further metabolised to the sex pheromone androstenone. Cytochrome b5A (CYB5A) stimulates the 17,20 lyase reaction and is required for the delta 16-synthase reaction. We have identified and mutated residues in porcine CYP17A1 and CYBSA that may alter the synthesis of DHEA and 16A. This included residues in the steroid binding pocket of CYP17A1 and residues on the surface of CYP17A1 and CYBSA that are involved in binding of CYP17A1 to CYB5A. We then expressed the various mutations of CYBSA and CYP17A1 along with porcine cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase (CYB5R3) in HEK293 cells and measured the formation of metabolites 16A, 17011P and DHEA from radiolabelled pregnenolone by high performance liquid chromatography (HPLC). Mutations were identified in both CYP17A1 and CYBSA that affected the production of the different metabolites and also affected the overall production of metabolites. Several combinations of mutations decreased the production of both 16A and DHEA and increased production of 17OHP, while the N62S mutation of CYB5A with wild type CYP17A1 increased production of both 16A and DHEA. The best combination of mutations to reduce the production of 16A, while maintaining the production of DHEA and the overall conversion similar to wild type are the N21K, L28V, N21K/L28V and the R52 M/N62S mutations of CYBSA with the D103S mutation of CYP17A1.