Background: P450 oxidoreductase (POR) mutations can present with disordered sexual development (46,XX virilisation as well as 46,XY under-masculinisation), perturbed steroidogenesis and mild to severe skeletal malformations. As POR is an obligate electron donating cofactor to many P450s, and as this interaction may vary from partner to partner, the phenotypic spectrum of PORD is extremely broad. Therefore, to characterize novel POR mutations, specific testing is required.
Case report: A 46,XX patient, second child of consanguineous Kurdish parents, was born at term with ambiguous genitalia (Prader III) and dysmorphic facial features (frontal bossing, low set ears). Newborn screening for 21-hydroxylase deficiency and ACTH-testing were normal. At age 14 days diagnosis of PORD was made by GC-MS urinary steroid metabolome-analysis showing the pathognomonic pattern of combined impaired activities of 17-hydroxylase and 21-hydroxylase. Genetic analysis revealed a novel homozygous mutation P399_E401Dup in POR.
Methods: The novel POR variant was characterized by bioinformatic and functional tests using recombinant proteins produced in bacteria, combined with small molecule and protein substrates. The ability of POR WT and P399_E401Dup variant to reduce ferricyanide (measures intactness of the FAD-binding domain of POR), MTT (as an indicator of electron transfer from the co-factor FMN bound inside the POR to its redox partners), and cytochrome c as well as the activity towards the drug and steroid metabolizing P450s were analysed. Effects of the mutation on cofactor (FAD/FMN) binding and activity under varying substrate and cofactor conditions were also investigated.
Results: The interaction with substrates was altered in the P399_E401Dup. Compared to WT, P399_E401Dup variant showed 51% activity in the cytochrome c reduction assay; in the MTT reduction assay the P399_E401Dup had only 6.2% of WT activity, showing a clear problem in electron transport mechanism. In the ferricyanide reduction assay, a 50% increase in the Michaelis constant (Km) for the FeCN was observed in addition to a 30% loss in maximal velocity. Overall results indicated a structural change by P399_E401Dup mutation in POR, which affects protein conformation and stability.
Conclusion: POR P399_E401Dup leads to alteration in Km and Vmax for multiple substrates, pointing towards an effect on protein conformation and stability. It also affects steroid production as manifested by alterations in the steroid metabolome of the patient. Previously, a P399_E401Del mutation in a Turkish child was reported, which had reduced activities of CYP17A1, CYP21A2 and CYP19A1. P399_E401 seems a sensitive spot for POR mutations.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology