Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder associated with inborn errors of steroid metabolism. 21-hydroxylase enzyme deficiency occurs in 90 to 95% of all cases of CAH, with accumulation of 17 hydroxyprogesterone (17-OHP). Early diagnosis of CAH based on newborn screening is possible before the development of symptoms and allows proper treatment, correct sex assignment, and reduced mortality rates. This study describes the results obtained in the first year of a public CAH screening program in the state of Rio Grande do Sul, Brazil. We reviewed the screening database in search of babies with suspected CAH, that is, altered birth-weight adjusted 17-OHP values at screening. The following data were analyzed for this population: screening 17-OHP values, retest 17-OHP values, serum 17-OHP values for those with confirmed CAH on retest, maternal and newborn data, and family history of CAH. For the screening program, 17-OHP levels are determined on dried blood spots obtained in filter paper with GSP solid phase time-resolved immunofluorescence. Of 108,409 newborns screened, eight were diagnosed with CAH (four males, four females). The incidence of CAH in the state was 1:13,551. Six cases were identified as classic salt-wasting CAH and two were cases of virilizing CAH. The positive predictive value (PPV) of the initial screening (before diagnostic confirmation) was 1.6%. The overall rate of false positive results was 0.47%. The number of false positive results was higher among newborns with birth weight < 2000 g. The present results support the need for CAH screening by the public health care system in the state, and show that the strategy adopted is adequate. PPV and false positive results were similar to those reported for other states of Brazil with similar ethnic backgrounds.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia (CAH) cases, a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provide critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Here, we analyzed the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of CYP21A2. SNVs of CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. All SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy of between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews’ correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). From our analysis, we identified four predictors of CYP21A2 variant pathogenicity with good performance, CADD, ConSurf, DANN, and PolyPhen2. These results can be used for future analysis to infer the impact of uncharacterized SNVs in CYP21A2.

Objective: To describe the results obtained in a neonatal screening program after its implementation and to assess the clinical and molecular profiles of confirmed and suspicious congenital adrenal hyperplasia cases. Methods: A cross-sectional study was conducted. Newborns with suspected disease due to high 17-hydroxyprogesterone levels and adjusted for birth weight were selected. Classical congenital adrenal hyperplasia (salt-wasting and simple virilizing forms) was diagnosed by an increase in 17-hydroxyprogesterone levels as confirmed in the retest, clinical evaluation, and genotype determined by SNaPshot and multiplex ligation-dependent probe amplification. Results: After 24 months, 15 classic congenital adrenal hyperplasia cases were diagnosed in a total of 217, 965 newborns, with an estimated incidence of 1:14, 531. From 132 patients, seven non-classical and 14 heterozygous patients were screened for CYP21A2 mutations, and 96 patients presented false positives with wild type CYP21A2. On retest, increased 17-hydroxyprogesterone levels were found in classical congenital adrenal hyperplasia patients and showed significant correlation with genotype-related classical genital adrenal hyperplasia. The most frequent mutations were IV52-13A/C >G followed by gene deletion or rearrangement events in the classical form. In non-classical and heterozygous diseases, p.Val282Leu was the most common mutation. Conclusions: The results underscore the effectiveness of congenital adrenal hyperplasia neonatal screening in the public health system and indicate that the adopted strategy was appropriate. The second sample collection along with genotyping of suspected cases helped to properly diagnose both severe and milder cases and delineate them from false positive patients. (C) 2018 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Pediatria.

Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of 21OH deficiency.