Tânia A. S. S. Bachega

Influence of different genotypes on 17‐hydroxyprogesterone levels in patients with nonclassical congenital adrenal hyperplasia due to 21‐hydroxylase deficiency

The diagnosis of the nonclassical form of 21‐hydroxylase (NC‐21OH) deficiency, established before molecular studies, is based on basal 17OH‐progesterone (17OH‐P) values > 15 nmol/l or ACTH‐stimulated 17OH‐P values > 30 nmol/l. This disease is caused by mutations in the structural gene that can be grouped into three categories: A, B and C, according to the predicted level of enzymatic activity. So, the genotype of the nonclassical form is a combination of mutations that cause moderate impairment of enzymatic activity in one allele and mutations which cause total (A), severe (B: 3%) or moderate (C: 20–60%) impairment of enzymatic activity in the other allele.

Low Frequency of CYP21B Deletions in Brazilian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency

The frequency of large mutations was determined in 131 Brazilian patients with different clinical forms of 21-hydroxylase deficiency, belonging to 116 families. DNA samples were examined by Southern blotting hybridization with genomic CYP21 and C4cDNA probes after TaqI and BglII restriction. Large gene conversions were found in 6.6% and CYP21B deletions in 4.4% of the alleles. The breakpoint in these hybrid genes occurred after exon 3 in 92% of the alleles. All rearrangements involving CYP21B gene occurred in the heterozygous form, except in a patient with simple virilizing form who presented homozygous CYP21B deletion. Our data showed that in these Brazilian patients, CYP21B deletions were less frequent than in most of the large series previously reported.

Effects of endocrine disruptors in the development of the female reproductive tract

Environmental agencies have identified a growing number of environmental contaminants that have endocrine disrupting activity, and these can become a major public health problem. It is suggested that endocrine disruptors could account for the higher-than-expected increase in the prevalence of some non-communicable diseases, such as obesity, diabetes, thyroid diseases, and some cancers. Several endocrine Disrupting Chemicals (EDCs), such as pesticides, bisphenol A, phthalates, dioxins, and phytoestrogens, can interact with the female reproductive system and lead to endocrine disruption. Initially, it was assumed that EDCs exert their effects by binding to hormone receptors and transcription factors, but it is currently known that they may also alter the expression of enzymes involved in the synthesis or catabolism of steroids. Biomonitoring studies have identified these compounds in adults, children, pregnant women, and fetuses. Among the diseases of the female reproductive tract associated with EDCs exposure are the following: precocious puberty, polycystic ovary syndrome, and premature ovarian failure. The different populations of the world are exposed to a great number of chemicals through different routes of infection; despite the various available studies, there is still much doubt regarding the additive effect of a mixture of EDCs with similar mechanisms of action.

The degree of external genitalia virilization in girls with 21-hydroxylase deficiency appears to be influenced by the CAG repeats in the androgen receptor gene

Background  Women with 21‐hydroxylase deficiency present much variability in external genitalia virilization, even among those with similar impairments of 21‐hydroxylase (21OH) activity.

Anatomical and functional outcomes of feminizing genitoplasty for ambiguous genitalia in patients with virilizing congenital adrenal hyperplasia

UNLABELLED The ideal surgical technique and appropriate age for performing feminizing genitoplasty are debatable, and few long-term outcome studies have been reported. PURPOSE To report a retrospective study on anatomical and functional outcomes of feminizing genitoplasty in patients with virilizing congenital adrenal hyperplasia. METHODS We selected 34 patients (mean age = 3.4 +/- 2.5 yr) with genital ambiguity classified according to Prader stage. Follow-up ranged from 2 to 16 years. Clitoral length ranged from 1.9 to 5.0 cm; 28 patients had a single perineal orifice, and 6 had a double orifice. The surgical technique included clitorovaginoplasty in a single procedure and was carried out before 2 years of age in 18 patients. Clitoroplasty was performed with glans preservation in all patients. Blood supply was exclusively maintained by the frenular pedicle in 97% of the cases, whereas clitoral dorsal nerves and vessels were preserved in the remaining 3%. The opening of the urogenital sinus was performed using either the Y-V perineal flap procedure (25 patients) or the cut-back incision procedure (8 patients). RESULTS Good morphological and functional results were achieved in 68% of the patients; 21% of the patients had surgical complications, such as incision bleeding (2 cases), glans necrosis (1 girl with Prader V), and vaginal introitus stenosis (4 cases). Three of the latter underwent dilation with acrylic molds in the post-pubertal period with good functional results. CONCLUSIONS We conclude that single-stage feminizing genitoplasty consisting of vulvoplasty, clitoroplasty, and Y-V perineal flap produced good cosmetic and functional results in virilized girls with congenital adrenal hyperplasia, with few complications. In addition, this surgical approach prevented the need for neovaginaplasty even in patients with high vaginal insertion.

Superior discriminating value of ACTH‐stimulated serum 21‐deoxycortisol in identifying heterozygote carriers for 21‐hydroxylase deficiency

Background  Congenital adrenal hyperplasia caused by classic 21‐hydroxylase deficiency (21OHD) is an autosomal recessive disorder with a high prevalence of asymptomatic heterozygote carriers (HTZ) in the general population, making case detection desirable by routine methodology. HTZ for classic and nonclassic (NC) forms have basal and ACTH‐stimulated values of 17‐hydroxyprogesterone (17OHP) that fail to discriminate them from the general population. 21‐Deoxycortisol (21DF), an 11‐hydroxylated derivative of 17OHP, is an alternative approach to identify 21OHD HTZ.

Estudo multicêntrico de pacientes brasileiros com deficiência da 21-hidroxilase: correlação do genótipo com o fenótipo

ABSTRACT Multicentric Study of Brazilian Patients With 21-Hydroxylase Defi-ciency: A Genotype-Phenotype Correlation. We analyzed the clinical and molecular data of 205 patients with thethree different clinical forms of 21-hydroxylase deficiency, in whom theclinical and molecular diagnosis were already defined. The most fre-quent mutations were I2 splice in the salt wasting form, I172N in the sim-ple virilizing and V281L in the nonclassical form, presenting similar fre-quencies as those observed in other populations. We found a lower fre-quency of 21-hydroxylase gene deletion, similar to that previously iden-tified in Argentinean and Mexican populations. Five new mutations weredescribed in our population: G424S, H28+C, Ins 1003^1004 A, R408C andIVS2-2A>G. The genotype was classified in three groups according to theimpairment of enzymatic activity observed in vitro , Group A: 0-2%, GroupB: 3-7% and Group C: >20%. Group A mutations correlated with the saltwasting form, the Group B with simple virilizing form and Group C withthe non classical form. The severity of genotype showed a positive cor-

Molecular analysis of CYP21A2 can optimize the follow-up of positive results in newborn screening for congenital adrenal hyperplasia

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17‐hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3–7%, C > 20%. Twenty‐one children with neonatal symptoms and/or 17OHP levels > 80 ng/ml carried A genotypes, except two virilized girls (17OHP < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP > 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false‐positives and help distinguish between clinical forms of CAH.

Weight-adjusted neonatal 17OH-progesterone cutoff levels improve the efficiency of newborn screening for congenital adrenal hyperplasia

OBJECTIVE To evaluate weight-adjusted strategy for levels of neonatal-17OHP in order to improve newborn screening (NBS) efficiency. SUBJECTS AND METHODS Blood samples collected between 2-7 days of age from 67,640 newborns were evaluated. When N17OHP levels were ≥ 20 ng/mL, and a second sample was requested. We retrospectively analyzed neonatal-17OHP levels measured by Auto DELFIA- B024-112 assay, grouped according to birth-weight: G1: < 1,500 g, G2: 1,501-2,000 g, G3: 2,000-2,500 g and G4: > 2,500 g. 17OHP cutoff values were determined for each group using the 97.5(th), 99(th), 99.5(th) and 99.8(th) percentiles. RESULTS 0.5% of newborns presented false-positive results using the cutoff level ≥ 20 ng/mL for all groups. Neonates of low birthweight made up 69% of this group. Seven full-term newborns presented congenital adrenal hyperplasia (CAH) and, except for one of them, 17OHP levels were > 120 ng/mL. Only the 99.8(th) percentile presented higher predictive positive value (2%), and lower rate of false-positives in all groups. CONCLUSIONS We suggest the use of 99.8(th) percentile obtained by weight-adjusted N17OHP values of healthy newborns to reduce the rate of false-positive results in NBS.

Substitutions in the CYP21A2 promoter explain the simple‐virilizing form of 21‐hydroxylase deficiency in patients harbouring a P30L mutation

The classical and nonclassical phenotypes of 21‐hydroxylase deficiency represent a continuous spectrum of the impairment of 21‐hydroxylase activity due to mutations between the CYP21A2 gene. These mutations occur mainly by microconversion in the homologous nonfunctional CYP21A1P gene. The P30L mutation is associated with the nonclassical form, and it reduces the activity to 30–40% of the normal enzyme. We have described three female patients exhibiting a simple virilizing phenotype and bearing the P30L mutation in compound heterozygosis with a severe mutation. To identify additional mutations causing this phenotype, the promoter region was sequenced and four mutations were identified: −126C → T, −113G → A, −110T → C and −103 A → G. These substitutions are normally present in the promoter region of the pseudogene and in vitro studies demonstrated that they reduced the transcriptional activity fivefold. They might have been converted to the CYP21A2 promoter together with the P30L mutation in these patients. Therefore, these substitutions in synergism with the P30L mutation might decrease the enzyme activity resulting in a more severe phenotype, and a DNA sequence of −167 bases of the CYP21A2 gene should be performed in patients with 21‐hydroxylase deficiency in whom the phenotype is more severe than predicted by the genotype.