Novel phenotypes and genotypes in Antley-Bixler syndrome caused by cytochrome P450 oxidoreductase deficiency: based on the first cohort of Chinese children.
ABSTRACT: BACKGROUND:Antley-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. METHODS:We 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. RESULTS:Eight 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. CONCLUSIONS:We 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.
Project description:P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available.The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort.The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries.We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17?-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles.We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.
Project description:Antley-Bixler syndrome (ABS) is rare form of craniosynostosis of both autosomal dominant and autosomal recessive inheritance. We are reporting a female term appropriate for gestational age newborn with clinical features of frontal bossing, brachycephaly, proptosis, synostosis of radioulnar joints, hemangioma over nose and philtrum, hydrocephalus suggestive of ABS.
Project description:P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.
Project description:Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.
Project description:Cytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. It can be difficult to differentiate with other diseases such as 21-hydroxylase deficiency (21-OHD), polycystic ovary syndrome (PCOS) and Antley-Bixler syndrome (ABS). Nearly 100 cases of PORD have been reported worldwide. However, the genetic characters and clinical management are still unclear, especially in China.In this study, we report a 27-year-old female Chinese patient who first presented with amenorrhea and recurrence of large ovary cyst. She was misdiagnosed with PCOS and non-classical 21-OHD due to ovary cysts and elevated 17-hydroxy-progesterone. The patient's complaining of a mild difficulty of bending the metacarpophalangeal joints reminded us to consider PORD, which usually presents with skeletal deformities and sexual dysfunction. The diagnosis of PORD was confirmed by genetic analyses, which showed the patient harboring a homozygous missense mutation in the POR gene (R457H) and her parents carrying the heterozygous mutation. The patient was treated with low-dose corticosteroids and estrogen/progesterone sequential therapy, and her ovarian cyst gradually reduced with regular menstruation in the follow-up. Moreover, the clinical and genetic characteristics of 104 previously reported PORD cases were also summarized and analyzed.PORD is a very rare disease which can be easily misdiagnosed in mild cases. Clinicians should keep in mind of this disease in patients with sexual dysfunction, especially combined with special skeletal deformities. Our data could provide a consciously understanding of this disease for clinic practicers. Low-dose corticosteroids combined with estrogen/progesterone sequential therapy will be effective in PORD patients with recurrence of large ovary cyst. The fact that the reported PORD patients in China carrying an identical variant R457H in POR gene also give us a viewpoint that R457H mutation in POR gene maybe important in causing PORD in Chinese as same as in Japanese.
Project description:Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR(null), POR(wt), POR(YH), and POR(VE), for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR(null), not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR(YH) and POR(VE) models than in POR(wt), indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.
Project description:Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders. It comprises a group of autosomal recessive disorders caused by the deficiency of one of four steroidogenic enzymes involved in cortisol biosynthesis or in the electron donor enzyme P450 oxidoreductase (POR) that serves as electron donor to steroidogenic cytochrome P450 (CYP) type II enzymes. The biochemical and clinical phenotype depends on the specific enzymatic defect and the impairment of specific enzyme activity. Defects of steroid 21-hydroxylase (CYP21A2) and 11beta-hydroxylase (CYP11B1) only affect adrenal steroidogenesis, whereas 17alpha-hydroxylase (CYP17A1) and 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) deficiency also impact on gonadal steroid biosynthesis. Inactivating POR gene mutations are the cause of CAH manifesting with apparent combined CYP17A1-CYP21A2 deficiency. P450 oxidoreductase deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant: skeletal malformations and severe genital ambiguity in both sexes.
Project description:NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por (lox/lox) and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.
Project description:Importance:Cytochrome 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. Objective:To perform an exome analysis and identify the pathogenic cause in order to assist clinicians to obtain a proper evaluation on the genetic condition. Methods:The 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. Results:A 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. Interpretation:We 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.
Project description:Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.