Pathogenesis of disorders caused by human P450 oxidoreductase mutations
Patients with POR deficiency have disordered steroidogenesis due to malfunctioning of several key enzymes in the steroidogenesis pathway. Patients with genital abnormalities and an abnormal urinary steroid profile that suggested combined defects in CYP17A1 (17a-hydroxylase/17,20 lyase) and CYP21A2 (21-hydroxylase) were reported that suggested a role of POR, however, when the POR knockout was found to be embryonically lethal in mice POR was excluded as a candidate gene. Almost two decades later in a 2004 we reported (Flück et al, Nature Genetics, March 2004) the first four patients with mutations in POR gene. Subsequent studies confirmed the presence of POR mutations in patients with similar patterns of steroid abnormalities with and without ABS (Huang, Pandey et al. Am. J. Hum. Genetics 2005). In the patients of European descent the A287P mutation is most common; while Japanese patients often have the R457H mutation.
Role of POR in steroid metabolism:
In the initial report the POR mutants A287P, R457H, V492E, C569Y and V608F were tested with 17a-hydroxylase and 17,20 lyase activities of CYP17A1. A good correlation between the clinical features and the CYP17A1 activities was observed. Later on we tested the effects of some POR variants on CYP19A1 (aromatase) activity. Mutations R457H and V492E located at FAD binding site of POR caused a complete loss of CYP19A1 activity, confirming that POR mutations disrupting the FAD binding and electron transfer will severely affect all P450s.
We have recently reported a novel POR mutation P399_E401del found in two unrelated Turkish families. The novel POR mutation P399_E401del had a clinical phenotype of ABS and DSD but only subclinical cortisol deficiency. In vitro functional testing of mutant POR P399_E401del on single enzymes showed a loss 68-85% activity for different P450s.
PORD can be diagnosed prenatally from the amniotic fluid or urine of the mother. The urine steroid profile of a mother carrying a fetus with PORD is characteristic for low levels of estriol but increased epiallopregnanediol disulfate (metabolite of pregnenolone), and increased androsterone and its precursor 17a-hydroxyallopregnanolone (5a reduced products coming from the alternative backdoor pathway leading to dihydrotestosterone production. The diagnosis of POR deficiency may be suggested from clinical and hormonal analysis but it requires molecular genetic analysis for confirmation and to know the specific defect as treatment may depend on severity of the effect on different interaction partners of POR.
Role in Drug metabolism:
We have found that POR mutants Y181D, A457H, Y459H, V492E and R616X lost more than 99% of drug metabolizing CYP3A4 activity, while 60-85% activity loss was observed for POR mutations A287P, C569Y and V608F . Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with severe POR mutations. Among non P450 partner proteins we have observed that POR mutants Y181D, A457H, Y459H, V492E and R616X result in total loss of heme oxygenase 1 (HO1) activity, while POR mutations A287P, C569Y and V608F lost 50-70% of HO1 activity
Treatment may include replacement of glucocorticoids and sex steroids (and rarely mineralocorticoids) as assessed by low serum hormone levels. The skeletal malformations caused by POR deficiency require orthopedic management, and often reported mortality is due to skeletal abnormalities leading to respiratory problems (e.g. choanal obstruction). Computational docking studies and functional assays suggest that the activity loss of some POR variants may be rescued by flavin supplementation
We were able to restore activities of POR Y181D and A287P which is not near the cofactor binding sites. However, which POR variant with what partner P450 may respond to external FMN, and whether flavin can be used as a treatment for affected patients remains to be tested.
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