Variant predictions in congenital adrenal hyperplasia caused by mutations in CYP21A2.

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.

Enhanced UGT1A1 Gene and Protein Expression in Endometriotic Lesions.

The cellular function in endometriosis lesions depends on a highly estrogenic milieu. Lately, it is becoming evident that, besides the circulating levels of estrogens, the balance of synthesis versus inactivation (metabolism) of estrogens by intralesion steroid-metabolizing enzymes also determines the local net estrogen availability. In order to extend the knowledge of the role of estrogen-metabolizing enzymes in endometriosis, we investigated the gene and protein expression of a key uridine diphospho-glucuronosyltransferase (UGT) for estrogen glucuronidation, UGT1A1, in eutopic endometrial samples obtained from nonaffected and endometriosis-affected women and also from endometriotic lesions. Although UGT1A1 messenger RNA (mRNA) expression was detected at similar frequencies in endometriotic lesions and in eutopic endometrial samples, the levels of mRNA expression were greater in deep-infiltrating endometriotic lesions and in non-deep-infiltrating lesions when compared with either control endometrium or eutopic endometrium from women with endometriosis. Overall, we observed that protein expression of UGT1A1 was significantly more frequent in samples from endometriotic lesions in comparison with endometria. In addition, expression of UGT1A1 protein was greater in deep-infiltrating than in non-deep-infiltrating endometriotic lesions. We suggest that the finding of increased expression of UGT1A1 in lesions versus endometria might be related to impairment of regulatory mechanisms, in response to a highly estrogenic milieu, and that this enzyme may be a new target for therapy.

Steroid metabolism in vitro during final oocyte maturation in white croaker Micropogonias furnieri (Pisces: Scianidae)

Final oocyte maturation (FOM) is a process involving a complex set of genetical, biochemical, and morphological mechanisms. FOM involves the shift of a post-vitellogenic follicle to a pre-ovulated oocyte, which is necessary for fertilization by spermatozoan to occur. This process is regulated by a maturation-inducing steroid (MIS) at the follicular level. In other species of scienids fish the MIS, a hydroxilated derivatives of progestagen 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S), was identified. Although Micropogonias furnieri is the second fishery resource of Uruguay, basic knowledge about its endocrine process is very scarce. The aim of this work was to investigate what steroids are synthesized in vitro by the oocyte follicle of M. furnieri during the maturation process. Fragments of ovary (1 g) in three stages: post-vitellogenic (PV), maturing (Mtg), and mature (M) were incubated with 1 mug.g-1 of tritiated progesterone (P) at 30, 60, and 180 min. After extraction with ethanol and dichloromethane, steroid metabolites were purified by TLC and rpHPLC. Two progesterone derivatives with identical chromatographic properties of 20beta-S and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were purified. In other Teleost fish these steroids are biologically activ as MIS. The 17,20beta-P was clearly detected in Mtg and M stages and confirmed by enzymatic oxidation with enzyme 20beta-HSD. The 20beta-S was strongly detected in all Mtg oocytes. The results do not corroborate 20beta-S as a major hormone synthesized in the ovary in FOM as occurs in other scienid fish. A differential steroid synthesis in the advanced oocyte stages suggests that the 20beta-S is acting as a MIS in M. furnieri.

A maturação ovocitária final (FOM) é um processo complexo que envolve mecanismos genéticos, bioquímicos e morfológicos que conduzem à transformação de um ovócito pós-vitelogênico em um ovócito apto a ser fertilizado. Esse processo está regulado pelo hormônio esteróide indutor da maturação ovocitária final (MIS), o qual é sintetizado no folículo. Em outras espécies de Sciaenidae, o MIS foi identificado como um derivado hidroxilado da progesterona 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S). Micropogonias furnieri é um recurso superexplorado na costa uruguaia, contudo, seus processos endócrinos são pouco conhecidos. O objetivo deste trabalho foi pesquisar quais esteróides são sintetizados in vitro pelos folículos em maturação de M. furnieri. Fragmentos de ovários (1 g) foram incubados em três estágios diferentes: pós-vitelogênese (PV), em maturação (Mtg) e maduros (M) com 1 mig/g de progesterona tritriada (P) durante 30, 60 e 180 min. Depois da extração dos esteróides com etanol e diclorometano, esses foram purificados e identificados utilizando-se TLC, rpHPLC e oxidação enzimática. Foram identificados dois derivados de progesterona com idênticas propriedades cromatográficas ao 20beta-S e 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), os quais, em outras espécies de peixes, apresentam atividade biológica como o MIS. A 17,20beta-P foi observada claramente nos estágios Mtg e M e confirmada pela oxidação com a enzima 20beta-HSD. A 20beta-S foi claramente observada nos ovários em maturação (Mtg). Os resultados não permitiram confirmar que 20beta-S é o hormônio mais sintetizado nos estágios estudados, como ocorre em outras espécies de ciaenídeos, mas a presença de uma síntese diferencial no estágio de maturação sugere que 20beta-S esteja atuando como o MIS em M. furnieri.