PORD originated CAH was used to be overlooked, since a CAH clinical entity with combined 17α-hydroxylase (CYP17A1) and 21-hydroxylase (CYP21A2) deficiency was first described in 1985. Initially, research suggested that the disorder would be caused by concurrent mutations in both CYP17 and CYP21 genes, mainly based on obvious biochemical evidence for the impair of 17α-hydroyxlase and 21-hydroxylase activities in affected patient. With the progress of gene analysis, coding sequences of CYP17A1 and CYP21A2 in those failed to reveal any mutations. Only until 2004, PORD was first discovered, several individual groups reported that this disease could be due inactivated P450 oxidoreductase, which served as a key electron donor to provide electrons to all microsomal P450 (CYP) enzymes in steroid synthesis, especially, CYP17 and CYP2118 (Fig. 6). This founding strikingly highlights the importance role of P450 oxidoreductase in controlling the redox potential for steroidogenesis, meanwhile uncovered a novel pathogenesis of CAH.
The biochemical phenotype of PORD was similar to partial deficiency of 17-hydroxylase and 21-hydroxylase activities in various combination, since it was required for both enzymes to be functional. The steroidogenesis starts from the substrate of cholesterol toward glucocorticoids, mineralocorticoids and sexual steroids. CYP17 exerts both 17α-hydroxylase and 17,20-lyase activities, the former catalyzes pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, the latter further catalyze them to dehydroepiandrosterone and androstenedione, respectively. As the 17,20-lyase reaction activity is more sensitive to the interaction of P450 oxidoreductase than 17α-hydroxylase reaction, in case of PORD, the formation of dehydroepiandrosterone and androstenedione were blocked, with the accumulation of pregnenolone, progesterone and 17-hydroxyprogesterone. In accordance with our finding that plasma concentration of progesterone, 17-hydroxyprogesterone, pregnenolone were elevated in PORD patient.
In contrast to classical 21-hydroxylase deficiency in CAH, PORD triggered 21-hydroxylase inhibition give rise to mineralocorticoid and glucocorticoid precursors accumulation rather than depletion, due to preferential 17-hydroxylase inhibition over 21-hydroxylase inhibition. Taken our results into condensation, it again supported that the plasma level of 11-deoxycorticosterone, corticosterone, aldosterone and 11-deoxycorticosterone, 11-deoxycortisol and 21-deoxycortisol were ascended.
Converse to classic 21-hydroxylase deficiency form of CAH, PORD affected patients circulating androgen are lower in both sexes. Disordered sex development were found in male and female patients, affected boys grow the symptom of undervirilized, micropenis or malformation of urethra19. Phenotypically, affected girls may present prenatal virilization of external genitalia, hence no progressive virilization ever since, which could be explained by the only existence of alternative androgen synthesis pathway in fetal period contributed to prenatal virilization, but ceasing androgen shortly after birth. Consist to our results, affected boys manifested sharply decreased level of androgen, such as androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone, yet undervirilized biomarkers in female patient was less clear. In addition, the activity of CYP19A1 was also hampered in PORD, leading to the prevention of product estrone and estradiol synthesized from androstenedione and testosterone, which match our results that estradiol was decreased in male PORD patients. In general, sex steroid hormone are lower in PORD patient.
In the case diagnosed patient 6, undervirilized symptoms were depicted by malformation of external genitalia, with unambiguously decreased androgen and increased mineralocorticoid steroid biomarkers20. Apart from that, skeletal abnormalities observed in patient 6 suggested that the patient may also suffered from Antley-Bixler syndrome, whose pathophysiology remains to be expounded21, 22. So far, it suggested that the impairment of P450 oxidoreductase-dependent CYP51A1 in steroid biosynthesis could be accountable for skeletal malformations20. Besides, POR also serves as indispensable electron donor to many hepatic CYP enzymes in metabolism and detoxification, therefore PORD in patient 6 may have impact on hepatic CYP enzyme to some extent and developed fatty liver23. Eventually, gene sequencing proved the diagnosis of PORD, in which pateral 1370G > A (R457H) mutations and materal 1660G > T (R554X) mutations of POR gene were found in patient 6. The R457H was the most frequent disease-causing mutation in Japanese populations24, and has been reported that R457H yields almost no activity for 17,20-lyase and only 1–3% activities for 17-hydroxylase and P450 aromatase. This mutation located in the FAD binding domain of P450 oxidoreductase, guanidinium group of the wild type residue R457 tends to form a salt bridge with FAD, alternatively, mutated H457 weakens the interaction25, 26. R554X mutation located in NADPH binding domain of P450 oxidoreductase, causing the 554th encoding amino acid switched from Arginine to terminator, which may induce protein truncation or activate nonsense-mediated mRNA degradation, thus affecting the function of protein encoded by the gene. In this case report, genotype and biochemical results provided reliable explanation for the impaired activity of P450 oxidoreductase, in turn, elicit the corresponding phenotype.