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Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

ABSTRACT: Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (K_s) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b₅, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d₃-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 10⁴-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.

SUBMITTER: Wang C

PROVIDER: S-EPMC5491764 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

Wang Chunxue C Pallan Pradeep S PS Zhang Wei W Lei Li L Yoshimoto Francis K FK Waterman Michael R MR Egli Martin M Guengerich F Peter FP

The Journal of biological chemistry 20170524 26

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 <i>CYP21A2</i> variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bou ...[more]

PMID: 28539365

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Project description:ObjectiveTo raise awareness of Cytochrome P450 Oxidoreductase Deficiency (PORD, a rare form of congenital adrenal hyperplasia (CAH), through a case of pregnant woman with virilization symptoms.Case descriptionA 30-year-old Chinese woman was referred to hospital after 7 years of presenting signs of virilization, including voice deepening, acromegaly, hirsutism, clitoromegaly, and acne. These symptoms appeared since her third gestation. Her second birth died 9 hours after birth and had signs of clitoris hypertrophy. Her third born was a son who presented with flat nose, radius and humerus bone malformation, and small penis at birth. Panel of POR-related genetic tests revealed that the patient carried c.1370 G>A (p.R457H), which is a POR heterozygous gene, while her husband carried a POR heterozygous gene as well, c.1379 C>A (p.S460Y). Two heterozygous mutations of the POR were found in her son: c.1370 G>A and c.1379 C>A. In PORD, c.1370 G>A (p.R457H) was reported as a susceptible gene, while c.1379 C>A (p.S460Y) has not been reported as responsible for the disease so far.Discussion and literature reviewPORD is a rare form of CAH and caused by POR gene mutations. Most PORD patients are identified and diagnosed in pediatrics department. Internal medicine and obstetrics physicians are unfamiliar with the disease. As clinical manifestations are diverse, PORD could be easy to miss or to be misdiagnosed. Typical clinical manifestation includes adrenal insufficiency-related symptoms, such as bone malformations and sexual development disorders. PORD is diagnosed through genetic testing. Investigations of steroid metabolic products in urine through gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry are also helpful for the diagnosis, but neither of them are widely available in China. In this case, the patient had a history of infertility, and her third child was born with congenital defect and carried a PORD-related gene. In general clinical practice, if a pregnant woman presents with abnormal virilization symptoms, CAH possibilities should be considered, including rare causes such as PORD.ConclusionPORD is a rare autosomal recessive genetic disease. We summarised the clinical characteristics and genotypes that were previously reported in the Chinese population and identified a novel mutation.

Project description:Cytochrome P450 oxidoreductase deficiency (PORD) is a rare form of congenital adrenal hyperplasia that can manifest with skeletal malformations, ambiguous genitalia, and menstrual disorders caused by cytochrome P450 oxidoreductase (POR) mutations affecting electron transfer to all microsomal cytochrome P450 and some non-P450 enzymes involved in cholesterol, sterol, and drug metabolism. With the advancement of molecular biology and medical genetics, increasing numbers of PORD cases were reported, and the clinical spectrum of PORD was extended with studies on underlying mechanisms of phenotype-genotype correlations and optimum treatment. However, diagnostic challenges and management dilemma still exists because of unawareness of the condition, the overlapping manifestations with other disorders, and no clear guidelines for treatment. Delayed diagnosis and management may result in improper sex assignment, loss of reproductive capacity because of surgical removal of ruptured ovarian macro-cysts, and life-threatening conditions such as airway obstruction and adrenal crisis. The clinical outcomes and prognosis, which are influenced by specific POR mutations, the presence of additional genetic or environmental factors, and management, include early death due to developmental malformations or adrenal crisis, bilateral oophorectomies after spontaneous rupture of ovarian macro-cysts, genital ambiguity, abnormal pubertal development, and nearly normal phenotype with successful pregnancy outcomes by assisted reproduction. Thus, timely diagnosis including prenatal diagnosis with invasive and non-invasive techniques and appropriate management is essential to improve patients' outcomes. However, even in cases with conclusive diagnosis, comprehensive assessment is needed to avoid severe complications, such as chromosomal test to help sex assignment and evaluation of adrenal function to detect partial adrenal insufficiency. In recent years, it has been noted that proper hormone replacement therapy can lead to decrease or resolve of ovarian macro-cysts, and healthy babies can be delivered by in vitro fertilization and frozen embryo transfer following adequate control of multiple hormonal imbalances. Treatment may be complicated with adverse effects on drug metabolism caused by POR mutations. Unique challenges occur in female PORD patients such as ovarian macro-cysts prone to spontaneous rupture, masculinized genitalia without progression after birth, more frequently affected pubertal development, and impaired fertility. Thus, this review focuses only on 46, XX PORD patients to summarize the potential molecular pathogenesis, differential diagnosis of classic and non-classic PORD, and tailoring therapy to maintain health, avoid severe complications, and promote fertility.

Dataset Information

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

Publications

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

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