Establishment of Flp-In CHO recombinant cell lines stably expressing CYP2A13 and CYP2A13/POR / 中国药理学通报

Aim To construct Flp-In CHO cell line(CYP2A13-CHO)stably expressing cytochrome P450 family 2 subfamily A member 13(CYP2A13)and Flp-In CHO cell line(CYP2A13-POR-CHO)stably co-expressing CYP2A13 and cytochrome P450 oxidoreductase(POR), from which a cell line with better metabolic activity is selected.Method In our previous study, we had constructed a Flp-In CHO cell line(POR-Flp-In CHO)stably expressing POR using lentiviral vector.The recombinant plasmids of pcDNA5/FRT-CYP2A13 were constructed and transfected into Flp-In CHO cells and POR-Flp-In CHO cells through LipofectamineTM 2000.The expression and activity of CYP2A13 were detected by real-time quantitative PCR(qRT-PCR), Western blot and Aflatoxin B1(AFB1)/4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)cytotoxicity assay and the metabolic activity was compared between CYP2A13-CHO and CYP2A13-POR-CHO.Results Compared with non-transfected cells, the mRNA and protein expression of CYP2A13 in CYP2A13-CHO and CYP2A13-POR-CHO cells both increased significantly.Besides, compared with CYP2A13-POR-CHO, CYP2A13-CHO cells were more sensitive to AFB1 and NNK.Conclusions The Flp-In CHO cell line stably expressing CYP2A13 and with better metabolic activity has been established successfully, which provides a tool for screening of pre-carcinogens that can be metabolically activated by CYP2A13.

A case of Antley-Bixler syndrome caused by novel POR mutations / 中华医学遗传学杂志

Objective@#To explore the genetic basis for a child affected with multiple malformations.@*Methods@#Genomic DNA was extracted from peripheral blood samples from the child and her parents. Tro whole exome sequencing and bioinformatics analysis were carried out. Suspicted mutations were verified by PCR and Sanger sequencing.@*Results@#The patient, a 2-year-old girl, presented with multiple malformations including dysmorphism, skeletal malformations and ambigulous genitalia. Through genetic testing, she was diagnosed with Antley-Bixler syndrome caused by compound heterozygous mutations of the POR gene (c.919G>T and c. 1615G>A), which were derived from her mother and father, respectively.@*Conclusion@#The compound heterozygous mutations of the POR gene probably underlie the Antley-Bixler syndrome in this patient.

Clinical characteristics and molecular diagnosis in 2 cases of P450 oxidoreductase deficiency / 中华内分泌代谢杂志

Objective To analyze clinical characteristics and gene mutation of two patients diagnosed with P450 oxidoreductase deficiency(PORD). Methods Clinical data of 2 patients with PORD was collected from Ruijin hospital. POR gene mutation was analyzed by PCR-Sanger sequencing. A retrospective analysis of literatures concerning PORD was performed. Results Patient 1, female, 16 years old, with 46,XX karyotype, presented with anorectal anomalies, clitoral hypertrophy at birth and irregular menstruation; Patient 2, female, 32 years old, with 46,XX karyotype, showed irregular menstruation and infertility, both without obvious skeletal deformity. Genetic test of POR gene mutation revealed that patient 1 carried a homozygous missense mutation (R457H) and patient 2 carried a heterozygous mutation (R223X/ Y607C). The two mutations (R223X and Y607C) are reported for the first time in China. Conclusion P450 oxidoreductase deficiency which caused by mutations in POR gene has a variety of clinical manifestations, including abnormal steroid hormone synthesis with or without Antley-Bixler syndrome. The affirmative diagnosis should rely on steroid hormone measurement and POR gene analysis.

Approach to the patient with cytochrome P450 oxidoreductase deficiency / 中华内分泌代谢杂志

[Summary] Cytochrome P450 oxidoreductase deficiency ( PORD) is a rare disease, which is a subtype of congenital adrenal hyperplasia. The predominant signs include no puberty development, infantile reproductive organs, ear deformities, and bone synostosis in skull or limbs. Here, we analyzed the clinical features of a case with PORD confirmed by gene sequencing. The pathology, genetic features, clinical manifestations, diagnosis and treatment for PORD were reviewed.

A Case of Antley-Bixler Syndrome with New Genetic Study Result.

Aims: Antley-Bixler syndrome (ABS) is a rare disease which is a complex of skeletal, visceral, extremity and genital anomalies and occasionally is associated with adrenal insufficiency due to P450-oxidoreductase deficiency. In this article we report a patient, a suspicious case of ABS with different phenotypic and genotypic characteristics. Presentation of Case: The patient is a male infant with facial dysmorphism, syndactyly, multiple joint contractures, and ambiguous genitalia. He had hyponatremia, hyperkalemia and elevated 17.OH.progestrone level of serum. In genetic analysis, no mutation was found in POR gene. Discussion: This patient has clinical and paraclinilical manifestations of ABS. Although different mutations have been reported as the cause of this syndrome, all reported patients who suffered from adrenal insufficiencies, had mutations in POR gene. Conclusion: According to our search in literature, this is the first case of ABS associated with adrenal insufficiency who does not have any mutation in POR gene. More genetic studies are needed to determine new mutations in such patients.

Male pseudohermaphroditism and gene mutation analysis:one case report / 中华内分泌代谢杂志

[Summary] The clinical and genetic characteristics of a patient with male pseudohermaphroditism, being considered as an isolated 17, 20-lyase deficiency case, were analyzed. The social gender of the patient aged 30-year-old was female. The patient presented with 46, XY karyotype, unclosed epiphysis, after perineal block resection, hypergonadotropic hypogonadism, while the production of mineralocorticoids and glucocorticoids hormone was intact. A503V heterozygous mutation in exon 13 and a deletion in intron 11 of POR gene were detected. The gene mutations may lead to the occurrence of the isolated 17,20-lyase deficiency.

Associations of POR polymorphisms and warfarin stable maintenance dose in Han Chinese patients / 中国药理学通报

Aim To explore the effect of genetic poly-morphisms of POR on the stable warfarin maintenance doses in Han Chinese patients receiving mechanical heart valve replacement. Methods The association between POR gene polymorphisms and warfarin doses of 185 Han Chinese patients were investigated through ANOVA or t test. SNPs of POR and VKORC1 were de-tected by Sequenom? DNA MassArray genotyping method. CYP2C9*3 was genotyped by polymerase chain reaction-restriction fragment length polymorphism method ( PCR-RFLP ) . Patients ’ clinical characteris-tics, INR value and daily dose were obtained from their medical records. Statistical analysis was performed by SPSS 21. 0 software. Results No mutant carriers of POR rs17148944 , POR rs56256515 and rs72553971 were found in this study. The genotype frequencies of other SNPs were in accordance with Hardy-Weinberg e-quilibrium. In the group of patients with CYP2C9*1*1 , the mutant type carriers ( T carriers ) of POR rs17685 had a significantly higher dose than CC carri-ers(3. 50 ± 1. 07) mg·d-1 vs (3. 14 ± 0. 94) mg· d-1,P =0. 03. Also, in the group of patients with CYP2 C9*1*1 and VKORC1 rs9934438 G allele carri-ers, the mutant type carriers ( T carriers ) of POR rs17685 had a significantly higher dose than CC carri-ers(4. 76 ± 0. 90) mg·d-1 vs (4. 08 ± 1. 03) mg· d-1 ,P=0. 04. No significant difference was found in different genotypes of POR rs2868177 . Conclusion These results illustrate that POR rs17685 T carrier is closely associated with a higher warfarin maintenance dose, suggesting that this SNP is useful for clinical guidance of warfarin.

Estudo da proteína P450 óxido-redutase e dos citocromos hepáticos 2C19 e 3A4 como possíveis moduladores do fenótipo da deficiência da 21-hidroxilase / Study of P450 oxidoreductase protein and hepatic cytochromes 2C19 and 3A4 as a potential modulatory factors in 21-hydroxylase deficiency phenotype

A deficiência da 21-hidroxilase é uma doença genética comum, causada por mutações no gene CYP21A2, que codifica a enzima 21-hidroxilase (P450c21). A deficiência da 21-hidroxilase afeta a síntese de cortisol e aldosterona e promove acúmulo de precursores, que são desviados para a síntese de andrógenos. Observa-se três principais fenótipos: a forma clássica virilizante simples (VS), na qual as meninas nascem com virilização da genitália externa e ambos os sexos apresentam virilização pós-natal; a forma perdedora de sal (PS), na qual além da virilização, ambos os sexos apresentam crise de perda de sal no período neonatal; e a forma não clássica (NC), na qual os sintomas de hiperandrogenismo iniciam-se mais tardiamente, na infância, adolescência ou idade adulta. Os estudos in vitro das mutações do CYP21A2 demonstram que existe uma boa correlação do grau de comprometimento da atividade enzimática conferido pelo genótipo com o fenótipo. Entretanto, existem algumas divergências como: pacientes que apresentam quadro clínico e hormonal de forma não clássica, nos quais mutações não são identificadas em um ou em ambos os alelos do CYP21A2, e pacientes que apresentam o fenótipo mais leve do que o predito pelo genótipo. Essas divergências sugerem a presença de fatores moduladores do fenótipo na deficiência da 21-hidroxilase. A primeira hipótese foi de que houvesse mutações no gene P450 óxido-redutase (POR), que codifica uma flavoproteína que doa elétrons para as enzimas microssomais P450, inclusive a P450c21, passo fundamental para a atividade enzimática das mesmas. A segunda hipótese foi de que outras enzimas P450, que não a P450c21, tivessem a capacidade de realizar a 21-hidroxilação extra-adrenal da progesterona e 17OH-progesterona (17OHP), sendo então capazes de modular o fenótipo da perda de sal e/ou virilização. Os citocromos P450 hepáticos CYP2C19 e CYP3A4, responsáveis pelo metabolismo de drogas, são capazes de realizar 21-hidroxilação da progesterona...

Adrenal 21-hydroxylase deficiency is a common genetic disorder, caused by mutations in the CYP21A2 gene, which encodes the 21-hydroxylase P450c21. The 21-hydroxylase deficiency disrupts cortisol and aldosterone biosynthesis and leads to accumulation of androgen precursors. There are 3 main phenotypes: the simple virilizing (SV) form, in which girls present with virilized external genitalia at birth and both sexes present with precocious pseudopuberty; the salt wasting (SW) form, characterized by additional salt-wasting crisis in the neonatal period in both sexes; and the nonclassic (NC) form, in which the hyperandrogenic signs occur later in life, during childhood, adolescence or adulthood. In vitro studies show a good correlation between the degree of enzymatic impairment determined by genotype and phenotype. However, there are some discrepancies as: patients with clinical and hormonal profiles of nonclassic form in whom mutations are not found in one or both alleles, and patients with milder phenotype than the ones predicted by genotyping. These discrepancies suggest the existence of modulatory factors in 21- hydroxylase deficiency phenotype. The first hypothesis was that there were mutations in P450 oxidoreductase (POR), a gene which encodes a flavoprotein that donates electrons for all microsomal P450s, including P450c21, an essential step for P450s activity. The second hypothesis was that other enzymes that not P450c21 could perform extra-adrenal 21-hydroxylation of progesterone and 17OHprogesterone (17OHP), modulating salt balance and virilization. Hepatic drugmetabolizing P450 enzymes CYP2C19 and CYP3A4 can 21-hydroxylate progesterone; however, this activity was never compared to 21-hydroxylation performed by P450c21, in order to determine the importance of this extra-adrenal activity in vivo. The present cohort consisted of 11 patients with nonclassic form and incomplete genotype and 6 patients with genotype/phenotype discrepancies...

Functional Expression of Human Cytochrome P450 Enzymes in Escherichia coli / 中国生物工程杂志

Functional expression of human cytochrome P450(P450,CYP)in E.coli is important for new drug R&D,clinical drug therapy and the study of early ADME/T properties.Escherichia coli is the most extensively utilized host in the production of recombinant human P450 enzymes.However,it is challenging to obtain sufficient P450s with catalytic activity.Depict current developments in the heterologous expression of human P450 enzymes,strategies for the expression of human P450s in E.coli,factors affecting high-level expression,and coexpression.The recent works of authors in expression optimization are also reviewed.

Genetic polymorphisms of cytochrome P450 oxidoreductase and its effect on drug metabolism / 中国药理学通报

Cytochrome P450 oxidoreductase (POR) is the only electron donor for all microsome Cytochrome P450 monooxygenases which are phase I metabolizing enzymes responsible for the metabolism of more than 80% drugs used in clinic.Also,POR metabolizes some anti-tumor prodrugs directly.Therefore,the alteration in POR activity caused by the polymorphisms of POR gene will be of great clinical significance.This review summarizes the newest advancement on the effects of POR polymorphisms on drug metabolism.