Biochemical and Biophysical Research Communications
Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency
Highlights
► Mutations in human POR cause congenital adrenal hyperplasia. ► We are reporting a novel 3 amino acid deletion mutation in POR P399_E401del. ► POR mutation P399_E401del decreased P450 activities by 60–85%. ► Impairment of steroid metabolism may be caused by multiple hits. ► Severity of aromatase inhibition is related to degree of in utero virilization.
Introduction
Cytochrome P450 oxidoreductase (POR, CPR; Refseq ID: NP_000932, NM_000941, EC 1.6.2.4) is a flavoprotein that donates electrons to microsomal cytochrome P450s. Mutations in POR result in disorders of steroid metabolism (Fig. 1) [1], [2], [3], [4]. POR was initially identified by Horecker in 1950 as a cytochrome c reductase [5] and later was linked to the microsomal cytochrome P450s involved in drug and steroid metabolism [6], heme oxygenase [7], and other molecules [8]. The requirement of POR in cytochrome P450 reactions was established by Lu and Coon [6] who separated P450 system into POR, cytochrome P450, and lipids (Fig. 2).
POR deficiency (OMIM ID: 201750) was initially known as “mixed oxidase disease” from steroid profiles of patients indicating combined deficiencies of 17α-hydroxylase/17,20 lyase (CYP17A1) and 21-hydroxylase (CYP21A2) activities [9]. More patients were later reported with apparent combined deficiencies of CYP17A1 and CYP21A2 but genetic analysis of CYP17A1 and CYP21A2 revealed no mutations, and genetic basis of mixed oxidase disease remained unclear. Steroidogenic P450s CYP17A1, CYP19A1 and CYP21A2 require electrons form NADPH through POR for their enzymatic activities (Fig. 2) [10], therefore, chances of mutations in POR gene and a malfunctioning POR protein was suggested by Miller [11] but deemed unlikely due to requirement of POR in multiple biochemical processes [11]; embryonic lethality of the POR knock-out mice later seemed to support that theory [12]. However, in 2004 Flück et al. [1], Arlt et al. [13] and Adachi et al. [14] found mutations in POR gene from patients with combined 17α-hydroxylase, 21-hydroxylase deficiency. Patients with POR deficiency manifest with a wide range of clinical features ranging from severe neonatal skeletal malformations with genital ambiguity (Antley–Bixler’s syndrome; OMIM 207410) to phenotypically minor abnormalities in steroid hormone production, manifesting as polycystic ovary syndrome with fertility issues [1], [15], [16], [17], [18]. Our initial report described five missense mutations (A287P, R457H, V492E, C569Y, and V608F) identified in four patients [1] and the later study added the mutants/polymorphisms A115V, T142A, Q153R, P228L, M263V, R316W, G413S, Y459H, A503V, G539R, L565P, R616X, V631I and F646del [15]. Initial POR mutations from ABS patients were found to cause markedly reduced activities of CYP17A1 (17-hydroxylase/17,20 lyase) [1], [15]. Several POR variants identified from patients as well as normal population were initially tested with steroidogenic P450 CYP17A1 [15], [18], [19], but recent studies have also reported effects of POR mutations on other P450s as well as heme oxygenase [2], [16], [20], [21], [22], [23], [24], [25], [26]. A POR allele database site has been established (www.cypalleles.ki.se/por.htm) to list mutations and polymorphisms in POR.
Here we are reporting a novel 3 amino acid deletion mutation in POR identified in two unrelated patients with symptoms of mixed oxidase deficiency causing disorder of sexual development. We produced WT and mutant human POR and CYP17A1, CYP19A1 and CYP21A2 and assayed the enzymatic activities. The novel P399_E401 deletion mutation in POR was found to reduce enzymatic activities of CYP17A1, CYP19A1 and CYP21A2, potentially leading to significantly reduced steroid metabolism.
Section snippets
Genetic analysis
Genomic DNA was extracted from blood leukocytes of patients after obtaining informed consent. The POR gene was analyzed, exon by exon, using specific primers and PCR amplification as described. The PCR-amplified products were purified and sequenced in an automated sequencer (ABI) using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the specifications provided by the manufacturer. Some primers used for sequencing were not similar to those used for PCR. The
Patients and hormonal data
Patient 1 was born to consanguineous parents with a 46,XX disorder of sexual development (DSD) manifesting as genital virilization (Prader V) and was misdiagnosed as 21-hydroxylase deficiency because of elevated serum 17OHP. Because of the discrepancy between the severity of in utero virilization and the mild elevation of 17OHProg with a negative test at neonatal screening, the diagnosis of 21-hydroxylase deficiency was doubted and POR deficiency was suggested when presence of mild ABS features
Discussion
P450 oxidoreductase (POR) is the obligate electron donor to the major steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. Therefore, patients with POR deficiency have a complex pattern of disordered steroidogenesis. Usually mineralocorticoid production remains normal while cortisol and sex steroid production are impaired to variable degrees with different POR mutations. Since reduced CYP17A1 and CYP19A1 activities lead to impaired testosterone and estrogen production, POR mutations may cause
Acknowledgments
This work was supported by the Swiss National Science Foundation Grants 31003A-130710 to C.E.F. and 31003A-134926 to A.V.P. and by Hospices Civils and French Governmental DHOS funds for genetic disease program (D.M. and Y.M.).
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