ABSTRACT Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is an autosomal recessive disorder caused by CYP21A2 gene mutations, and its molecular diagnosis is widely used in clinical practice to confirm the hormonal diagnosis. Hence, considering the miscegenation of the Brazilian population, it is important to determine a mutations panel to optimise the molecular diagnosis. The objective was to review the CYP21A2 mutations’ distribution among Brazilian regions.Two reviewers screened Brazilian papers up to February 2020 in five databases. The pair-wise comparison test and Holm method were used in the statistical analysis. Nine studies were selected, comprising 769 patients from all regions. Low proportion of males and salt-wasters was identified in the North and Northeast regions, although without significant difference. Large gene rearrangements also had a low frequency, except in the Center-West and South regions (p < 0.05). The most frequent mutations were p.I172N, IVS2-13A/C>G, p.V281L and p.Q318X, and significant differences in their distributions were found: p.V281L was more frequent in the Southeast and p.Q318X in the Center-West and Northeast regions (p < 0.05). Thirteen new mutations were identified in 3.8%-15.2% of alleles, being more prevalent in the North region, and six mutations presented a founder effect gene. Genotype-phenotype correlation varied from 75.9%-97.3% among regions. The low prevalence of the salt-wasting form, affected males and severe mutations in some regions indicated pitfalls in the clinical diagnosis. The good genotype-phenotype correlation confirms the usefulness of molecular diagnosis; however, the Brazilian population also presents significant prevalence of novel mutations, which should be considered for a molecular panel.

As diversas agências de controle ambiental têm identificado um crescente número de contaminantes ambientais que apresentam atividade de desregulador endócrino e estes poderão se tornar um dos maiores problemas de saúde pública. Sugere-se que os Desreguladores Endócrinos (EDCs) poderiam justificar o aumento na prevalência de algumas doenças não transmissíveis acima do esperado, como, por exemplo, obesidade, diabetes, doenças tireoidianas e alguns tipos de cânceres. Vários EDCs, como pesticidas, bisfenol A, ftalatos, dioxinas e fitoestrógenos, podem interagir com o sistema reprodutivo feminino e levar à desregulação endócrina. Inicialmente, supunha-se que os EDCs exercessem seus efeitos através da ligação com receptores hormonais e fatores de transcrição, mas, atualmente, sabe-se que também podem alterar a expressão de enzimas envolvidas na síntese ou no catabolismo dos esteroides. Estudos de biomonitoramento têm identificado esses compostos em adultos, crianças, gestantes e em fetos. Entre as patologias do trato reprodutor feminino associadas à exposição aos EDCs, destacam-se: puberdade precoce, síndrome dos ovários policísticos e falência ovariana prematura. As diversas populações estão expostas a um grande número de substâncias químicas, através de diferentes vias de contaminação. Apesar dos diferentes estudos disponíveis, ainda permanece uma grande dúvida sobre o efeito aditivo de uma mistura de EDCs com similar mecanismo de ação.

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.

OBJETIVO: Avaliamos retrospectivamente os valores da 17OHP ajustados para o peso ao nascimento para melhorar a eficiência da triagem neonatal. SUJEITOS E MÉTODOS: 67.640 recém-nascidos com amostras coletadas entre 2-7 dias de vida. Uma segunda amostra foi solicitada na presença de testes com valores da 17OHP > 20 ng/mL. 17OHP dosada pelo método DELFIA- B024-112 e correlacionada com o peso ao nascimento: G1 < 1.500 g, G2 1.501-2.000 g, G3 2.000-2.500 g e G4 > 2.500 g. Pontos de corte da 17OHP foram determinados para cada grupo usando os percentis 97,5th, 99th, 99,5th e 99,8th. RESULTADOS: Falso-positivos ocorreram em 5% dos resultados com o ponto de corte > 20 ng/mL, dos quais 69% eram prematuros. Sete recém-nascidos apresentaram deficiência da 21-hidroxilase e, exceto em um, os valores da 17OHP foram > 120 ng/mL. Somente o valor da 17OHP do 99,8th apresentou maior valor preditivo positivo (2%) e menor índice de falso-positivos. CONCLUSÕES: Sugerimos o valor da 17OHP do 99,8th ajustado para o peso ao nascimento para se reduzir a taxa de resultados falso-positivos da triagem neonatal.

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 97.5th, 99th, 99.5th and 99.8th 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 99.8th percentile presented higher predictive positive value (2%), and lower rate of false-positives in all groups. CONCLUSIONS: We suggest the use of 99.8th percentile obtained by weight-adjusted N17OHP values of healthy newborns to reduce the rate of false-positive results in NBS.

INTRODUCTION: 21-hydroxylase deficiency is an autosomal recessive disorder that causes glucocorticoid deficiency and increased androgen production. Treatment is based on glucocorticoid replacement; however, interindividual variability in the glucocorticoid dose required to achieve adequate hormonal control has been observed. OBJECTIVE: The present study aimed to evaluate the association between polymorphic variants involved inglucocorticoid action and/or metabolism and the mean daily glucocorticoid dose in 21-hydroxylase deficiency patients. METHODS: We evaluated 53 patients with classical forms of 21-hydroxylase deficiency who were receiving cortisone acetate. All patients were between four and six years of age and had normal androgen levels. RESULTS: The P450 oxidoreductase A503V, HSD11B1 rs12086634, and CYP3A7*1C variants were found in 19%, 11.3% and 3.8% of the patients, respectively. The mean ± SD glucocorticoid dose in patients with the CYP3A7*1C and wild-type alleles was 13.9 ± 0.8 and 19.5 ± 3.2 mg/m²/d, respectively. We did not identify an association between the P450 oxidoreductase or HSD11B1 allelic variants and the mean glucocorticoid dose. CONCLUSION: Patients carrying the CYP3A7*1C variant required a significantly lower mean glucocorticoid dose. Indeed, the CYP3A7*1C allele accounted for 20% of the variability in the cortisone acetate dose. The analysis of genes involved in glucocorticoid metabolism may be useful in the optimization of treatment of 21-hydroxylase deficiency.

INTRODUCTION: PXR polymorphisms have been implicated in modulating CYP3A4 and PXR expression, potentially accounting for interindividual differences in drug metabolism. The prevalence of PXR polymorphisms varies among ethnic groups and data on the allelic distribution in the highly mixed Brazilian population is lacking. The aim of this study was to analyze genetic variations in the PXR gene in Brazilians and to compare the results to other ethnic groups. METHODS: DNA samples from 117 healthy Brazilians underwent PCR amplification and sequencing. RESULTS: Eleven polymorphisms were identified, 3 of which are highly associated with differences in CYP3A4 expression. We also identified 1 new synonymous variant in 1.3% of the alleles. Among the functional polymorphisms, -25913 C>T and -6994T>C occurred at a higher frequency comparedtothe Africanalleles (p < 0.05) but at a lower frequency compared to Caucasian alleles. The 8055 C>T allele was found at a similar frequency to those described in Caucasians and Africans (p > 0.05). CONCLUSION: We observed that functional variants of the PXR were frequent in our sample of the Brazilian population. Our results suggest that PXR gene variants may be of interest in pharmacogenetic studies involving Brazilians.

OBJETIVO: Padronizar a técnica de Southern blotting usando hibridização com material não radioativo para detectar grandes rearranjos no gene CYP21A2 em uma amostra da população brasileira com hiperplasia adrenal congênita. MÉTODO: Foram estudados 42 pacientes, 2 dos quais aparentados, totalizando 80 alelos não relacionados. As amostras de DNA foram obtidas de sangue periférico, digeridas com enzima de restrição Taq I, realizado Southern blotting e hibridizadas com sonda marcada com biotina. RESULTADOS: O método se mostrou eficaz, com resultados similares aos encontrados ao utilizar a metodologia com material radioativo. Foram encontradas 2,5% de deleção do CYP21A2, 8,8% de grandes conversões, 3,8% de deleção do CYP21A1P e 6,3% de duplicação do CYP21A1P. Estas freqüências foram similares às encontradas em nosso estudo prévio, onde um número significante de casos foi estudado. Um bom padrão de hibridização foi alcançado utilizando menor quantidade de DNA (5mg) e a emissão de sinais foi observada entre 5 minutos e 1 hora de exposição. CONCLUSÕES: Padronizamos uma técnica de Southern blotting/ hibridização com material não radioativo (biotina) para a pesquisa de grandes rearranjos no gene CYP21A2 com bons resultados. Apesar de ser mais trabalhoso, este método é mais rápido, utiliza menores quantidades de DNA e, principalmente, evita problemas com o uso de radioatividade.

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.