P450 Oxidoreductase Deficiency Research Articles

Alternative pathway androgen biosynthesis and human fetal female virilization

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone (DHEA), which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone (DHT) in genital skin, thereby facilitating male external genital differentiation (1). Congenital adrenal hyperplasia (CAH) due to P450 oxidoreductase deficiency (PORD) is an inborn disorder that results in disrupted DHEA biosynthesis, leading to undervirilization of affected boys. However, some affected girls present with severe genital virilization at birth, despite low circulating androgen concentrations (2-4). Here, Reisch et al. hypothesized that this might be explained by a prenatally active, alternative biosynthesis pathway to 5α-DHT. They investigated the presence of an alternative androgen-producing pathway in human fetuses.

Clinical and genetic analysis of a pedigree affected with cytochrome P450 oxidoreductase deficiency

To explore the clinical features and molecular basis of a Chinese pedigree with two siblings affected by cytochrome P450 oxidoreductase deficiency (PORD). Clinical features of the patients were reviewed, and their genomic DNA was subjected to next generation sequencing (NGS). The two siblings presented peculiar facies, genital hypoplasia and skeletal deformity. NGS revealed that both have carried compound heterozygous variants of the POR gene, namely c.1370G>A and c.517-19_517-10delGGCCCCTGTGinsC, which were respectively inherited from their parents. Both siblings were diagnosed with PORD based on sequencing of the POR gene. The newly discovered POR c.517-19_517-10delGGCCCCTGTGinsC has enriched the spectrum of PORD-related genetic variants.

Clinical, endocrinological, and molecular features of four Korean cases of cytochrome P450 oxidoreductase deficiency

PurposeCytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder caused by mutations in the POR gene encoding an electron donor for all microsomal P450 enzymes. It is characterized by adrenal insufficiency, ambiguous genitalia, maternal virilization during pregnancy, and skeletal dysplasia. In this study, we investigated the clinical, hormonal, and molecular characteristics of patients with POR deficiency in Korea.MethodsThis study included four patients with POR deficiency confirmed by biochemical and molecular analysis of POR. Clinical and biochemical findings were reviewed retrospectively. Mutation analysis of POR was performed by Sanger sequencing after polymerase chain reaction amplification of all coding exons and the exon-intron boundaries.ResultsAll patients presented with adrenal insufficiency and ambiguous genitalia regardless of their genetic sex. Two patients harbored homozygous p.R457H mutations in POR and presented with adrenal insufficiency and genital ambiguity without skeletal phenotypes. The other two patients with compound heterozygous mutations of c.[1329_1330insC];[1370G>A] (p.[I444Hfs*6];[R457H]) manifested skeletal abnormalities, such as craniosynostosis and radiohumeral synostosis, suggesting Antley-Bixler syndrome. They also had multiple congenital anomalies involving heart, kidney, and hearing ability. All patients were treated with physiologic doses of oral hydrocortisone.ConclusionsWe report the cases of 4 patients with POR deficiency identified by mutation analysis of POR. Although the study involved a small number of patients, the POR p.R457H mutation was the most common, suggesting founder effect in Korea. POR deficiency is rare and can be misdiagnosed as 21-hydroxylase or 17α-hydroxylase/17,20-lyase deficiency. Therefore, molecular analysis is critical for confirmatory diagnosis.

MON-062 46 XX DSD Due to POR Deficiency

BackgroundPORD (P450 oxidoreductase deficiency) is a rare form of CAH with marked phenotypic variations due to differences in the degree of steroid hormone excess/deficiency. PORD results in17αhydroxylase/17,20lyase- CYP17, 21αhydroxylase- CYP21, and aromatase- CYP19A1 inhibition. In the absence of characteristic skeletal features of Antley Bixler phenotype, differentiating PORD from other types of CAH is challenging.Case details04 day child, second of the non-identical twins, product of 2nd degree consanguinity, third in birth order was brought with abnormal genitalia. The other male twin had no genital ambiguity but had pigmented scrotum. There was no adrenal crisis in index case or maternal virilization. First child is normal female. Child was hemodynamically stable weighed 02 kgs and measured 51 cms, had no hyperpigmentation, skeletal deformities or dysmorphic features. Phallus was 10mm, clitoral index 40mm2 with single urogenital opening and posteriorly fused labia (anogenital ratio 0.6).Gonads were not palpable. Karyotype was 46XX with normal Mullerian structures and non-visualized gonads on ultrasonography. Biochemical workup showed random plasma glucose level of 99mg/dl and normal electrolytes. Baseline serum 8am Cortisol was 1.15 mcg/dl (normal 5-18 mcg/dl) and 17OHP was 20 ng/ml (normal < 02 ng/ml). Serum Androstenedione level was 0.39 nmol/L (normal 0.5-3.4 nmol/L).In view of clinical, laboratory and imaging findings, presumptive diagnosis of 46 XX DSD due to 21 alpha hydroxylase deficiency CAH was made. Oral Hydrocortisone 15mg/m2 and Fludrocortisone 0.05 mg were initiated. Therapy was monitored with serum 17(OH)P levels and Androstenedione. Genetic testing for 21 alpha hydroxylase gene however came negative.This led to further genetic mutational analysis, and there was a homozygous R457H POR gene mutation at exon 12 leading to protein change p.Arg457His (nucleotide change c.1370G>A), confirming PORD. Retrospectively, maternal triple test done during the candidate pregnancy showed low serum estriol, suggestive of placental aromatase deficiency. Mineralocorticoid supplements were stopped thereafter. Conclusions PORD needs to be considered and differentiated from garden variety of CAH in 46 XX DSD when elevation of 17(OH)P is modest with isolated cortisol deficiency and aromatase deficiency (evidenced by low maternal estriol levels and/or virilization), as its course and follow up are different.

SUN-091 Change in Sex of Rearing in Individuals with Disorders of Sex Development

Introduction: Differences of Sex Development (DSDs) encompass variations in formation of internal or external sex characteristics. Historically, sex assignment in children with DSDs depended on phenotype, and gender was thought to be malleable. Attention in DSD has recently shifted toward reducing gender dysphoria and preserving fertility potential. Our multidisciplinary DSD clinic was formed in 2008 with these goals and includes specialists from Endocrinology, Gynecology, Urology, Psychology, Social Work, Genetics, and Chaplaincy. Subjects: A chart review was done on all patients seen in our DSD clinic between April 2008 and June 2019 to determine rates of sex reassignment and gender dysphoria. Two hundred patients were seen: 23 were found to not have DSDs; 61 were 46,XX; 66 were 46,XY; 31 had a sex chromosome DSD; 5 had gonadal dysgenesis without known chromosome mosaicism; and 14 had syndromic genital atypia. Mean age of follow-up is 8.77 years. Results: Only 2 patients underwent sex reassignment at our institution. One was assigned male at birth, but was found to be 46,XX with 21-hydroxylase deficiency and was reassigned female at 1 month of age. The second was assigned male at birth and was found to be 46,XY with an NR5A1 variant. Sex was reassigned female at two months of age. Two additional patients had a sex reassignment outside our institution. One was born abroad and assigned male at birth. The patient was found to be 46,XY with an NR5A1 variant, and was reassigned female. The second patient was assigned male at birth, but was found to be 46,XX with P450 oxidoreductase deficiency. The parents changed the sex of rearing to female at 19 months of age. To date, none has signs of gender dysphoria.A total of three patients experienced gender dysphoria and underwent transition. The first had genital ambiguity with sex chromosome mosaicism in the gonads and was assigned female at birth. We held care conferences with the family and discussed the possibility of gender dysphoria. At age 3, the patient declared that he was male, and parents socially transitioned him at that time. Two were assigned female after receiving the diagnosis of 46,XX 21-hydroxylase deficiency. Both declared male gender identity later, one at 12.5 years of age, and one at 20.5 years of age. Conclusion: Whereas our patient population is still relatively young, it is reassuring that the overall rate of gender dysphoria is low. The rate in patients with CAH is similar to previous reports in the literature. Careful attention to sex assignment in early childhood may reduce the rates of gender dysphoria in children with DSDs.

MON-060 P-450 Oxidoreductase Deficiency with Antley Bixler Phenotype: A Novel Mutation

IntroductionWe present first case of 46 XY Disorder of Sex Development (DSD) from India due to P-450 Oxidoreductase Deficiency with Novel variant (p.Ala541Thr) in a heterozygous state.Case Discussion6 months old boy presented with ambiguous genitalia since birth. No history of neonatal crisis, failure to thrive and pigmentation of skin, maternal virilisation or drug ingestion during pregnancy. On examination: weight 6.2 Kg (3rd centile), height 64 cm (3rd centile), MPH-170 (25-50th centile), head circumference 38 cm (−2.7 SD), vitals stable, trigonocephaly with fused anterior and posterior fontanelle, prominent pointed forehead, midfacial hypoplasia, up slanting eyes, hypertelorism and low set ears were present. Genitalia: 1.5 cm phallus like structure with foreskin, chordee, single perineal opening in form of peno-scrotal hypospadias, bifid scrotum with poor rugosity and poor pigmentation and both gonads (1 ml) were palpable in labio-scrotal fold with external masculinization score (EMS), 6/12 and Prader stage 4.Investigations showed normal electrolytes and blood sugar, High basal ACTH, post stimulation cortisol 14mcg/dl, Basal 17-OHP was 8.6 ng/ml and post stimulation 12ng/ml, with low DHEAS 36.4 mcg/dl and androstenedione 0.42 ng/ml, LH 16.09 mIU/ml (elevated), FSH 2.97 mIU/ml (normal) and low Testosterone for his age. T/DHT 9.6 (normal<10) and Testosterone /Androstenedione ratio 0.95 (normal >0.8). Abdominal and Pelvic imaging showed normal adrenal glands and absent female internal genitalia, bilateral testis in labio-scrotal fold (right testis-6x6.5x11 mm, left testis-6.6x7x10 mm), corpora cavernosa and bifid scrotum. NCCT Head showed metopic craniosynostosis with trigonocephaly and hypotelorism. Skeletal survey showed bowing of femora. 20 cell Karyotype of peripheral blood lymphocyte was 46 XY. NGS was done of the POR gene, which revealed a heterozygous missense variation in exon 13 of the POR gene variant (p.Ala541Thr) which has not been reported yet.The patient was initiated hydrocortisone, fludrocortisone, DHT gel and corrective surgery is planned.Clinical learningAlthough in PORD classically the inheritance is generally autosomal recessive, but manifesting heterozygotes are not uncommon1. This case also shows the value of Next gen sequencing and the role it can play in DSD1. Scott RR, Gomes LG, Huang N, Van Vliet G, Miller WL. Apparent manifesting heterozygosity in P450 oxidoreductase deficiency and its effect on coexisting 21-hydroxylase deficiency. J Clin Endocrinol Metab. 2007;92:2318–2322

SAT-010 Non-Classic POR Deficiency as a Cause of Menstrual Disorders &amp; Infertility

P450 oxidoreductase deficiency (PORD) is an autosomal recessive disease caused by bi-allelic mutations of the POR gene. It is responsible for decreased activity of several P450 enzymes including CYP21A2, CYP17A1 and CYP19A1 that are involved in adrenal and/or gonadal steroidogenesis. PORD is typically diagnosed in neonates and children with ambiguous genitalia and/or skeletal abnormalities. Adult-onset PORD has been very seldom reported and little is known about the optimal way to investigate and treat such patients. In this series, we report five women aged 19-38 years, who were referred for unexplained oligo-/amenorrhea and/or infertility. Genetic testing excluded 21-hydroxylase deficiency (21OH-D), initially suspected due to increased 17-hydroxyprogesterone (17-OHP) levels. Extensive phenotyping, steroid profile by mass spectrometry, pelvic imaging and next-generation sequencing of 84 genes involved in gonadal and adrenal disorders were performed in all patients. In Vitro Fertilization (IVF) followed by frozen embryo transfer under glucocorticoid suppression therapy was performed in two patients. All patients had oligomenorrhea or amenorrhea. None had hyperandrogenism. Low-normal serum estradiol (E2) and testosterone levels contrasted with chronically increased serum progesterone (P) and 17-OHP levels, which further increased after ACTH administration. Despite excessive P, 17OH-P and 21-deoxycortisol rises after ACTH stimulation suggesting non-classic 21-hydroxylase deficiency, CYP21A2 sequencing did not support this hypothesis. Basal serum cortisol levels were low to normal, with inadequate response to ACTH in some women, suggesting partial adrenal insufficiency. Pelvic imaging revealed bilateral ovarian macrocysts in all women. All patients were found to harbor rare bi-allelic POR mutations classified as pathogenic according to American College of Medical Genetics standards. IVF was performed in two women after retrieval of a normal oocyte number despite very low E2 levels during controlled ovarian hyperstimulation. Frozen embryo transfer under glucorticoid suppression therapy led to successful pregnancies. These observations suggest that diagnosis of PORD must be considered in infertile women with chronically elevated P and 17OH-P levels and ovarian macrocysts. Differentiation of this entity from non-classic 21-hydroxylase deficiency is important, as the multiple enzyme deficiency requires a specific management. Successful fertility induction is possible by IVF, providing that P levels be sufficiently suppressed by glucocorticoid therapy prior to implantation.

Non-classic cytochrome P450 oxidoreductase deficiency strongly linked with menstrual cycle disorders and female infertility as primary manifestations.

Can cytochrome P450 oxidoreductase deficiency (PORD) be revealed in adult women with menstrual disorders and/or infertility? PORD was biologically and genetically confirmed in five adult women with chronically elevated serum progesterone (P) who were referred for oligo-/amenorrhea and/or infertility. PORD is an autosomal recessive disease typically diagnosed in neonates and children with ambiguous genitalia and/or skeletal abnormalities. It is responsible for the decreased activity of several P450 enzymes, including CYP21A2, CYP17A1 and CYP19A1, that are involved in adrenal and/or gonadal steroidogenesis. Little is known about the optimal way to investigate and treat patients with adult-onset PORD. In this series, we report five adult females who were evaluated in three tertiary endocrine reproductive departments between March 2015 and September 2018. Five women aged 19-38years were referred for unexplained oligo-/amenorrhea and/or infertility. Genetic testing excluded 21-hydroxylase deficiency (21OH-D), initially suspected due to the increased 17-hydroxyprogesterone (17-OHP) levels. Extensive phenotyping, steroid profiling by mass spectrometry, pelvic imaging and next-generation sequencing of 84 genes involved in gonadal and adrenal disorders were performed in all patients. IVF followed by frozen embryo transfer (ET) under glucocorticoid suppression therapy was performed for two patients. All patients had oligomenorrhea or amenorrhea. None had hyperandrogenism. Low-normal serum estradiol (E2) and testosterone levels contrasted with chronically increased serum P and 17-OHP levels, which further increased after adrenocorticotrophic hormone (ACTH) administration. Despite excessive P, 17OH-P and 21-deoxycortisol rise after ACTH stimulation suggesting non-classic 21OH-D, CYP21A2 sequencing did not support this hypothesis. Basal serum cortisol levels were low to normal, with inadequate response to ACTH in some women, suggesting partial adrenal insufficiency. All patients harbored rare biallelic POR mutations classified as pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics standards. Pelvic imaging revealed bilateral ovarian macrocysts in all women. IVF was performed for two women after retrieval of a normal oocyte number despite very low E2 levels during ovarian stimulation. Frozen ET under glucocorticoid suppression therapy led to successful pregnancies. The number of patients described here is limited and these data need to be confirmed on a larger number of women with non-classic PORD. The diagnosis of PORD must be considered in infertile women with chronically elevated P and 17OH-P levels and ovarian macrocysts. Differentiation of this entity from non-classic 21OH-D is important, as the multiple enzyme deficiency requires a specific management. Successful fertility induction is possible by IVF, providing that P levels be sufficiently suppressed by glucocorticoid therapy prior to implantation. No specific funding was used for this study. There are no potential conflicts of interest. N/A.

Impact of Common Polymorphic Variant A503V in POR on Drug Metabolism: Implications for POR Deficiency

A broad spectrum of human diseases, including abnormalities in steroidogenesis, is caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR) [1, 2]. Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. Therefore, genetic variations in POR can impact many different metabolic pathways by changing the activities of cytochromes P450. In 2004 the first human patients with defects in POR were reported with symptoms of ambiguous genitalia and bone malformations, and over 200 variations in POR are known.By analyzing the POR sequences from genomics databases, we focused on the common variant in POR A503V, which is often present in heterozygous form in many PORD patients. We expressed the A503V variant of POR in bacteria and characterized it by functional studies using purified recombinant proteins. The POR, as well as P450 Proteins, were expressed in E. coli BL21(DE3) and purified by ion‐exchange, affinity, and metal chelate chromatography for activity assays as described in our earlier publications [3, 4]. Activities of cytochrome P450 enzymes were tested with lipids into which P450 and P450 reductase proteins were embedded and assayed using fluorogenic substrates on a microplate spectrofluorometer.We found a positive effect on many drug‐metabolizing enzyme activities due to the A503V variant in POR. The activity of CYP2C9 was increased to 180% of the WT POR, and CYP2C19 showed 243% of WT activity with the A503V variant of POR. Most remarkable was the impact of the A503V variant on CYP3A5 activity, which was increased by 421% compared to WT POR.Effect on drug metabolism in patients with PORD requires careful considerations of cytochrome P450 mediated activities. While many PORD patients have low or reduced drug metabolism due to defects in POR, the presence of the A503V allele can alter the overall effect on drug metabolism. Significantly, tacrolimus, a drug given during organ transplant, can be metabolized several folds faster due to the presence of the A503V variant of POR. The presence of the A503V allele is likely beneficial in patients with POR deficiency.Support or Funding InformationThis work was supported by the Swiss National Science Foundation (31003A‐134926), the Novartis Foundation for Medical‐Biological Research (18A053), and a grant from Burgergemeiende Bern to AP.

Clinical characteristics of cytochrome P450 oxidoreductase deficiency: a nationwide survey in Japan.

Cytochrome P450 oxidoreductase deficiency (PORD) is a disorder of steroidogenesis that causes various symptoms such as skeletal malformations, disorders of sex development, and adrenal insufficiency. The aim of this study was to elucidate the clinical characteristics, especially age at diagnosis and treatment, of PORD from the perinatal period to adulthood in Japan. The first questionnaire was sent to 183 council members of the Japanese Society for Pediatric Endocrinology on 1 September 2018. The response rate was 65%, and a total of 39 patients with PORD were examined at 20 hospitals. The second questionnaire was sent in November 2018 to the council members examining these 39 patients with PORD. The response rate was 77%, and we received clinical information on 30 of the 39 patients. The two novel clinical findings were the age at diagnosis and the treatment of Japanese patients with PORD. In many cases, PORD can be diagnosed at <3 months of age. Hydrocortisone as the primary treatment during infancy can be used daily or in stressful situations; however, because patients with PORD generally have mild to moderate adrenal insufficiency, some might be able to avoid hydrocortisone treatment. Patients with PORD should be carefully followed up, and treatment should be optimized as for patients with other types of adrenal insufficiency. Other characteristics in the present study were similar to those described in previous reports.

P450 Oxidoreductase Deficiency: A Systematic Review and Meta-analysis of Genotypes, Phenotypes, and Their Relationships

P450 oxidoreductase deficiency (PORD) is a rare genetic disorder that is associated with significant morbidity. However there has been limited analysis of reported PORD cases. To determine, based on the cohort of reported PORD cases, genotype-phenotype relationships for skeletal malformations, maternal virilisation in pregnancy, adrenal insufficiency, and disorders of sexual development (DSD). PubMed and Web of Science from January 2004 to February 2018. Published case reports/series of patients with PORD. Eligible patients were unique, had biallelic mutations, and their clinical features were reported. Patient data were manually extracted from the text of case reports/series. A malformation score, representing the severity of skeletal malformations, was calculated for each patient. Of the 211 patients published in the literature, 90 were eligible for inclusion. More than 60 unique mutations were identified in this cohort. Four groups of mutations were identified, through regression modeling, as having significantly different skeletal malformation scores. Maternal virilization in pregnancy, reported for 21% of patients, was most common for R457H mutations. Adrenal insufficiency occurred for the majority of patients (78%) and was typically mild, with homozygous R457H mutations being the least deficient. DSD affected most patients (72%), but were less common for males (46XY) with homozygous R457H mutations. PORD is a complex disorder with many possible mutations affecting a large number of enzymes. By analyzing the cohort of reported PORD cases, this study identified clear relationships between genotype and several important phenotypic features.

Novel phenotypes and genotypes in Antley-Bixler syndrome caused by cytochrome P450 oxidoreductase deficiency: based on the first cohort of Chinese children

BackgroundAntley-Bixler syndrome (ABS) caused by P450 oxidoreductase deficiency (PORD) is a congenital adrenal hyperplasia with skeletal malformations and disordered sex development in both sexes. There have been no reports of ABS caused by PORD in Chinese children.MethodsWe described the clinical and genetic characteristics of eight Chinese children with ABS caused by PORD and compared them with those of subjects in previous studies.ResultsEight patients, aged 6 months–17.8 years, showed strikingly similar craniofacial malformations. We first described four unreported features: lower eyelid fat pads (4/8), prominent lower eyelid-zygoma transverse line (4/8), underdeveloped or absent antihelix (5/8) and single earlobe crease (5/8). Five 46, XY patients presented various degrees of undervirilization, while three 46, XX cases showed masculinization. Basal endocrine measurements revealed the following consistent results: normal cortisol; elevated adrenocorticotropic hormone, progesterone, pregnenolone, 17-hydroxypropgesterone, and corticosterone; and decreased or normal testosterone/oestradiol. We identified three previously reported variants and four novel variants (c.51719_51710delGGCCCCTGTGinsC, p.D210G, p.Y248X and p.R554X) of POR. The most prevalent variant was p.R457H (8/16). The hydrocortisone dosages of patients differed because of variable degrees of adrenal insufficiency.ConclusionsWe described novel phenotypes and genotypes of ABS caused by PORD. The variant p.R457H was the most prevalent in this cohort. All subjects had combined characteristics of 17-hydroxylase and 21-hydroxylase deficiency. Steroid replacement therapy for patients with PORD requires individually tailored dosing.

Alternative pathway androgen biosynthesis and human fetal female virilization

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

The mechanism of Psoralen and Isopsoralen hepatotoxicity as revealed by hepatic gene expression profiling in SD rats.

The main bioactive components of Fructus psoraleae, such as psoralen and isopsoralen, are known to be hepatotoxic. However, its underlying mechanism is to be elucidated. To address this, SD rats were randomly divided into control group, 60mg/kg psoralen group and 60mg/kg isopsoralen group. Blood was collected to detect serum biochemical indices. RNA was extracted from liver samples, and then, cDNA gene expression profiles were analysed. Psoralen administration significantly up-regulated serum AST (aspartate aminotransferase) while addition of isopsoralen increased serum ALT (alanine aminotransferase), AST, TBA (total bile acid) and TG (total triglyceride) levels. A total of 172 differentially expressed genes (DEGs) were acquired between psoralen group and control group while 884 DEGs were screened between isopsoralen group and control group. Chemical Carcinogenesis and Metabolism of Xenobiotics by Cytochrome P450 were the two most significantly enriched pathways as revealed by DEGs. Liver was the most impacted organ, and endoplasmic reticulum was the most impacted organelle in subcellular level. Finally, some kinds of cancers and cytochrome p450 oxidoreductase deficiency were predicted. Taken together, psoralen and isopsoralen might cause hepatotoxicity mainly through cytochrome P450 metabolism of xenobiotics. Furthermore, Cyp1a1, Cyp1a2, Gstm1 and Akr7a3 worked as key genes in hepatotoxicity. Moreover, endoplasmic reticulum was the main target subcellular structure in hepatotoxicity induced by psoralen and isopsoralen.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20–50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

An unusual association of P450 oxidoreductase Deficiency and Argininosuccinatelyase Deficiency

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Genetic progress and future research directions of congenital adrenal hyperplasia

Congenital adrenal hyperplasia(CAH) is an umbrella term for inherited enzymatic deficiencies in cortisol synthesis.It′s a monogenic, autosomal-recessive genetic disease.It is mainly divided into 21-hydroxylase deficiency, 11β-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase type 2, 17α-hydroxylase deficiency, lipoid CAH and P450-oxidoreductase deficiency, and the clinical phenotype is complicated.Further study of the relation of the genotypes to the clinical symptoms of various types of CAH contributes to the understanding, diagnosis, individualized treatment and genetic counseling of the disease.The influence of CAH related early prenatal diagnosis, modified genes, environmental factors on phenotype and the value of total exon sequencing are also worthy of further study.This article reviews genetic development in recent years and future research directions of the disease. Key words: Congenital adrenal hyperplasia; Inheritance; Gene

Pitfalls in the diagnosis of an infant with 46,XX DSD with Congenital Adrenal Hyperplasia due to Cytochrome P450 Oxidoreductase deficiency - the value of simultaneous genetic analysis to the diagnosis in DSD

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Compound heterozygous variants in POR gene identified by whole-exome sequencing in a Chinese pedigree with cytochrome P450 oxidoreductase deficiency.

ImportanceCytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. This condition specifically leads to disordered steroidogenesis, which can affect the development of the reproductive system, skeleton, and other parts of the body. The severe form of PORD is difficult to differentiate with Antley‐Bixler syndrome (ABS). The genetic characters and clinical evaluation of PORD are still unclear in China.ObjectiveTo perform an exome analysis and identify the pathogenic cause in order to assist clinicians to obtain a proper evaluation on the genetic condition.MethodsThe proband underwent detailed physical evaluations. DNA of the proband and his parents was isolated and whole‐exome sequencing (WES) was performed. Variants were analyzed and evaluation according to the ACMG guideline.ResultsA 1‐year‐old Chinese boy with midface hypoplasia, choanal stenosis, multiple joint contractures, micropenis and right cryptorchidism was misdiagnosed with Crouzon syndrome. By trio‐whole‐exome sequencing, we identified an unreported compound heterozygous mutation (c.667C>T, p.R223* and c.1370G>A, p.R457H) in POR in the proband. This mutation was inherited from healthy heterozygous parents, supporting the diagnosis of PORD, which was further confirmed by biochemical characteristics.InterpretationWe have identified a pathogenic variant with an unreported compound heterozygous POR mutation, which expands the clinical and genetic spectra of PORD and emphasizes the usefulness of WES for genetic diagnosis.

Congenital adrenal hyperplasia: clinical symptoms and diagnostic methods.

The aim of this paper is a straightforward presentation of the steroidogenesis process and the most common type of congenital adrenal hyperplasia (CAH) - 21-hydroxylase deficiency - as well as the analytical diagnostic methods that are used to recognize this disease. CAH is a family of common autosomal recessive disorders characterized by impaired adrenal cortisol biosynthesis with associated androgen excess due to a deficiency of one or more enzymes in the steroidogenesis process within the adrenal cortex. The most common and prototypical example of the CAH disorders group (90-95%) is caused by 21-hydroxylase deficiency. Less frequent types of CAH are 11β-hydroxylase deficiency (up to 8% of cases), 17α-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase deficiency, P450 oxidoreductase deficiency and StAR deficiencies. In the 21-hydroxylase and 11β-hydroxylase deficiency, only adrenal steroidogenesis is affected, whereas a defect in 3β-hydroxysteroid dehydrogenase or 17α-hydroxylase also involves gonadal steroid biosynthesis. Many countries have introduced newborn screening programs based on immunoassays measuring 17-hydroxyprogesterone from blood spots used for other neonatal screening tests which enable faster diagnosis and treatment of CAH. Currently, chromatographic techniques coupled with mass spectrometry are gaining popularity due to an increase in the reliability of the test results.