Tania A. S. S. Bachega

Extraadrenal 21-Hydroxylation by CYP2C19 and CYP3A4: Effect on 21-Hydroxylase Deficiency

CONTEXT 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known. OBJECTIVE We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD. METHODS Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia. RESULTS Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype. CONCLUSIONS CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.

The Common P450 Oxidoreductase Variant A503V Is Not a Modifier Gene for 21-Hydroxylase Deficiency

CONTEXT 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene. OBJECTIVES We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity. PATIENTS AND METHODS We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [(14)C]progesterone to deoxycorticosterone and [(3)H]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging. RESULTS The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR. CONCLUSION The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

Disorders of sex development: effect of molecular diagnostics

Disorders of sex development (DSDs) are a diverse group of conditions that can be challenging to diagnose accurately using standard phenotypic and biochemical approaches. Obtaining a specific diagnosis can be important for identifying potentially life-threatening associated disorders, as well as providing information to guide parents in deciding on the most appropriate management for their child. Within the past 5 years, advances in molecular methodologies have helped to identify several novel causes of DSDs; molecular tests to aid diagnosis and genetic counselling have now been adopted into clinical practice. Occasionally, genetic profiling of embryos prior to implantation as an adjunct to assisted reproduction, prenatal diagnosis of at-risk pregnancies and confirmatory testing of positive results found during newborn biochemical screening are performed. Of the available genetic tests, the candidate gene approach is the most popular. New high-throughput DNA analysis could enable a genetic diagnosis to be made when the aetiology is unknown or many differential diagnoses are possible. Nonetheless, concerns exist about the use of genetic tests. For instance, a diagnosis is not always possible even using new molecular approaches (which can be worrying for the parents) and incidental information obtained during the test might cause anxiety. Careful selection of the genetic test indicated for each condition remains important for good clinical practice. The purpose of this Review is to describe advances in molecular biological techniques for diagnosing DSDs.

Quality of life of patients with 46,XX and 46,XY disorders of sex development

Disorders of sex development (DSD) result from abnormalities in the complex process of sex determination and differentiation. An important consideration to guide the assignment of social sex in newborns with ambiguous genitalia is the quality of life (QoL) of these patients in adulthood. The rarity of most DSD conditions makes it difficult to conduct a long‐term follow‐up of affected patients through adulthood. This review of papers on the QoL of DSD patients evaluated in developing and developed countries by qualitative and quantitative instruments revealed a large spectrum of QoL, ranging from very poor to similar to, or even better than, the normal population. A more adequate QoL was found in patients from tertiary centres, indicating that the medical care of DSD patients should be multidisciplinary and carried out by specialized teams.

Quality of life in a large cohort of adult Brazilian patients with 46,XX and 46,XY disorders of sex development from a single tertiary centre

Few studies have focused on the quality of life (QoL) of patients with disorders of sex development (DSD). Our aim was to evaluate QoL in DSD patients with defined diagnoses followed until adulthood in a single tertiary centre.

Molecular CYP21A2 Diagnosis in 480 Brazilian Patients with Congenital Adrenal Hyperplasia Before Newborn Screening Introduction

BACKGROUND Most congenital adrenal hyperplasia (CAH) patients carry CYP21A2 mutations derived from conversion events involving the pseudogene, and the remaining carry new mutations. OBJECTIVE To review causal mutations and genotype-phenotype correlation in 480 Brazilian patients. METHODS DNA was extracted from 158 salt-wasters (SWs), 116 simple virilizing (SV), and 206 nonclassical (NC) patients. Fourteen point mutations were screened by allele-specific PCR, large rearrangements by Southern blotting/MLPA, and sequencing was performed in those with incomplete genotype. The gene founder effect was analyzed by microsatellite studies. Patients were divided into six genotypes (Null; A: <2%; B: 3-7%; C: >20% of residual enzymatic activity (EA); D: unknown EA; E: incomplete genotype). RESULTS Targeted methodologies defined genotype in 87.6% of classical and in 80% of NC patients and the addition of sequencing in 100 and 83.5%, respectively. The most frequent mutations were p.V281L (26.6% of alleles), IVS2-13A/C>G (21.1%), and p.I172N (7.5%); seven rare mutations and one novel mutation (p.E351V) were identified. Gene founder effect was observed in all but one (p.W19X) mutation. Null, A, B, and C genotypes correlated with SW (88%), SW (70%), SV (98%), and NC forms (100%), respectively. In group D, the p.E351V mutation correlated with classical form and group E comprised exclusively NC-patients. ACTH-stimulated 17OHP level of 44.3ng/mL was the best cutoff to identify NC-patients carrying severe mutations. CONCLUSIONS We identified a good genotype-phenotype correlation in CAH, providing useful data regarding prediction of diseases severity; moreover, we suggest that ACTH-stimulated 17OHP levels could predict carrier status for severe mutations. Sequencing is essential to optimize molecular diagnosis in Brazilian CAH patients.

Increased expression of ACTH (MC2R) and androgen (AR) receptors in giant bilateral myelolipomas from patients with congenital adrenal hyperplasia

BackgroundAlthough chronic adrenocorticotropic hormone (ACTH) and androgen hyperstimulation are assumed to be involved in the pathogenesis of adrenal myelolipomas associated with poor-compliance patients with congenital adrenal hyperplasia (CAH), the expression of their receptors has not yet been demonstrated in these tumors so far.MethodsWe analyzed Melanocortin 2 receptor (MC2R), Androgen Receptor (AR), Leptin (LEP), and Steroidogenic factor 1 (SF1) expression using real-time qRT-PCR in two giant bilateral adrenal myelolipomas from two untreated simple virilizing CAH cases and in two sporadic adrenal myelolipomas. In addition, the X- chromosome inactivation pattern and CAG repeat numbers in AR exon 1 gene were evaluated in the 4 cases.ResultsThe MC2R gene was overexpressed in myelolipomas from 3 out of 4 patients. AR overexpression was detected in 2 tumors: a giant bilateral myelolipoma in a CAH patient and a sporadic case. Simultaneous overexpression of AR and MC2R genes was found in two of the cases. Interestingly, the bilateral giant myelolipoma associated with CAH that had high androgen and ACTH levels but lacked MC2R and AR overexpression presented a significantly shorter AR allele compared with other tumors. In addition, X-chromosome inactivation pattern analysis showed a polyclonal origin in all tumors, suggesting a stimulatory effect as the trigger for tumor development.ConclusionThese findings are the first evidence for MC2R or AR overexpression in giant bilateral myelolipomas from poor-compliance CAH patients.

Mineralocorticoid replacement during infancy for salt wasting congenital adrenal hyperplasia due to 21-hydroxylase deficiency

OBJECTIVE: The protocols for glucocorticoid replacement in children with salt wasting 21-hydroxylase deficiency are well established; however, the current recommendation for mineralocorticoid replacement is general and suggests individualized dose adjustments. This study aims to retrospectively review the 9-∝-fludrocortisone dose regimen in salt wasting 21-hydroxylase deficient children who have been adequately treated during infancy. METHODS: Twenty-three salt wasting 21-hydroxylase deficient patients with good anthropometric and hormonal control were followed in our center since diagnosis. The assessments of cortisone acetate and 9-∝-fludrocortisone doses, anthropometric parameters, and biochemical and hormonal levels were rigorously evaluated in pre-determined intervals from diagnosis to two years of age. RESULTS: The 9-∝-fludrocortisone doses decreased over time during the first and second years of life; the median fludrocortisone doses were 200 μg at 0-6 months, 150 μg at 7-18 months and 125 μg at 19-24 months. The cortisone acetate dose per square meter was stable during follow-up (median = 16.8 mg/m2/day). The serum sodium, potassium and plasma rennin activity levels during treatment were normal, except in the first month of life, when periodic 9-∝-fludrocortisone dose adjustments were made. CONCLUSIONS: The mineralocorticoid needs of salt wasting 21-hydroxylase deficient patients are greater during early infancy and progressively decrease during the first two years of life, which confirms that a partial aldosterone resistance exists during this time. Our study proposes a safety regiment for mineralocorticoid replacement during this critical developmental period.

Utilidad del estudio molecular de CYP21A2 en el manejo prenatal de hiperplasia suprarrenal congénita: detección de dos nuevas mutaciones en Chile

Prenatal treatment of pregnancies at risk of congenital adrenal hyperplasia (CAH) may prevent ambiguous genitalia in female fetuses. We present the prenatal treatment performed in an extended family with two mutations. The proband, a boy with CAH-salt losing form, and his relatives were studied. The probands paternal uncles/aunts were married to the maternal aunts/uncles, respectively. The relatives had normal basal and stimulated 17OHProgesterone levels, which did not clarify their carrier status. The CYP21A2 gene was sequenced. The proband and the paternal relatives harbored a Q318X, R483W mutation in one allele. The maternal relatives and the proband exhibited an R483 frameshift mutation. Early dexamethasone treatment was given during two pregnancies and stopped when male gender was confirmed by early ultrasonography Both newborns were healthy and had normal 17OHProgesterone levels. This family had three mutations which abolish the 21-hydroxylase activity. Two mutations were detected in codon 483 of CYP21A2 gene, exon 10, which have not been reported previously in Latin-America. The molecular study performed in this family allowed us to give an appropriate genetic counseling and prenatal treatment.

Long-term follow-up of a female with congenital adrenal hyperplasia due to P450-oxidoreductase deficiency

P450 oxidoreductase deficiency (PORD) is a variant of congenital adrenal hyperplasia that is caused by POR gene mutations. The POR gene encodes a flavor protein that transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 type II (including 21-hydroxylase, 17α-hydroxylase 17,20 lyase and aromatase), which is fundamental for their enzymatic activity. POR mutations cause variable impairments in steroidogenic enzyme activities that result in wide phenotypic variability ranging from 46,XX or 46,XY disorders of sexual differentiation, glucocorticoid deficiency, with or without skeletal malformations similar to Antley-Bixler syndrome to asymptomatic newborns diagnosed during neonatal screening test. Little is known about the PORD long-term evolution. We described a 46,XX patient with mild atypical genitalia associated with severe bone malformation, who was diagnosed after 13 years due to sexual infantilism. She developed large ovarian cysts and late onset adrenal insufficiency during follow-up, both of each regressed after hormone replacement therapies. We also described a late surgical approach for the correction of facial hypoplasia in a POR patient.