Letteratura scientifica selezionata sul tema "3-Oxo-5-alpha-Steroid 4-Dehydrogenase"

BACKGROUND 5α-Reductase 2 deficiency (5ARD) is a known cause of 46,XY disorders of sex development (DSD). Traditionally, the diagnosis relies on dihydrotestosterone (DHT) measurement, but the results are often equivocal, potentially leading to misdiagnosis. We reviewed alternative approaches for diagnosis of 5ARD. METHODS We conducted a retrospective review of the results of urinary steroid profiling (USP) by GC-MS and mutational analysis of SRD5A2 [steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2)] by PCR and direct DNA sequencing of all 46,XY DSD patients referred to our laboratory with biochemical and/or genetic findings compatible with 5ARD. We also performed a literature review on the laboratory findings of all 5ARD cases reported in the past 10 years. RESULTS Of 16 patients diagnosed with 5ARD between January 2003 and July 2012, 15 underwent USP, and all showed characteristically low 5α- to 5β-reduced steroid metabolite ratios. Four patients had DHT measured, but 2 did not reach the diagnostic cutoff. In all 12 patients who underwent genetic analysis, 2 mutations of the SRD5A2 gene were detected to confirm the diagnosis. Twenty-four publications involving 149 patients with 5ARD were published in the review period. Fewer than half of these patients had DHT tested. Nearly 95% of them had the diagnosis confirmed genetically. CONCLUSIONS 5ARD can be confidently diagnosed by USP at 3 months postnatally and confirmed by mutational analysis of SRD5A2. Interpretation of DHT results may be problematic and is not essential in the diagnosis of 5ARD. We propose new diagnostic algorithms for 46,XY DSD.

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

Abstract Introduction Invasive aspergillosis (IA) represents a critical complication that frequently emerges during induction chemotherapy for acute leukemia or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality rates. During the last years, routine administration of antimold prophylaxis based on triazole regimens provided a major advance in controlling fungal infections in patients at high risk for IA. However, several studies recently reported azole resistant fungal strains, which are detectable in patients at risk with rapidly increasing frequency. While mutations in the 14α-sterol demethylase (cyp51A) gene have been implicated in mediating azole resistance, in approximately 50% of resistant patient isolates the underlying genetic cause is unknown (van der Linden, Emerging Infectious Diseases, 2015). In view of a rising prevalence of azole resistance in the high risk clinical setting we performed whole genome sequencing (WGS) of azole-resistant cyp51A-wildtype Aspergillus fumigatus (Afu) specimen, the main effector of IA, with the aim to identify novel genetic alterations leading to azole resistance. Methods Afu clinical strains were isolated from six different patients with IA undergoing intensive chemotherapy for acute leukemia or after allo-HSCT. Phenotypic resistance screening confirmed azole resistance in all samples. Moreover, Sanger sequencing revealed characteristic resistance associated cyp51A mutations ("TR46/Y121F/T289A") in four of the six specimen, while two samples harbored cyp51A wildtype sequences. High molecular weight genomic DNA (gDNA) was isolated using a modified DNeasy Plant Mini Kit protocol (Qiagen). WGS sequencing libraries were prepared utilizing the transposome fragmentation based Nextera XT DNA Library Prep Kit (Illumina) and sequenced on a MiSeq next generation sequencing (NGS) system (Illumina) with V2 sequencing chemistry (2x250bp). Raw paired reads were mapped to the NCBI Af293 (ASM265v1) reference genome using BWA followed by calibration with GATK. Subsequently, genomic variants were identified by GATK Haplotypecaller module and ANNOVAR software was used for functional variant annotation. Results WGS of four cyp51A mutated and two cyp51A wildtype samples resulted in an average genomic coverage of 26x (range 21.2x - 30.1x) allowing robust detection of genomic sequence variants. Of note, the presence or absence of cyp51A "TR46/Y121F/T289A" mutational genotype was reliably validated by WGS data using both our variant calling pipeline and manual screening of mapped reads with the IGV browser. In an attempt to screen for novel, previously undocumented candidate genes that are mechanistically involved in azole resistance we first identified all mutations that are exclusively present in the cyp51A wildtype samples and undetectable in the cyp51A mutated cohort. Next, we excluded all variants that have previously been reported as being polymorphic in WGS data from 22 different Afu strains sequenced by the J. Craig Venter Institute. Using this strategy we identified a core candidate cluster of 112 nucleotide variants in 62 genes that were exclusively found in the cyp51A wildtype group and predicted to have an impact on the amino acid sequence of the corresponding translational products. In particular, exemplary identified mutations affected genes coding for glycosyl hydrolase family 31 proteinimplicated in carbohydrate metabolic processes and 3-oxo-5-alpha-steroid 4-dehydrogenase family protein, which is, similar to cyp51A, involved in steroid metabolism. Conclusion The rapid increase in detection of triazole resistant fungal strains in clinical contexts represents a critical problem, particularly for immunocompromised patients at high risk for IA, and highlights the need to explore the underlying molecular causes. Pending further validation in a larger cohort, our NGS-based approach for sequencing the genomes of azole resistant Afu strains identified several candidate genomic alterations that might pinpoint alternative pathways to acquisition of resistance, independent of the previously described cyp51A mutations. Ultimately, our approach for discovery of previously undescribed resistance pathways in fungal strains might aid in exploration of novel therapeutic targets and identify suitable biomarkers for effective clinical and epidemiological surveillance. Disclosures Buchheidt: Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Other: travel grant, Research Funding, Speakers Bureau; Astellas: Other: travel grant, Speakers Bureau; Merck Sharp & Dohme: Other: travel grant, Speakers Bureau.

Une exposition excessive aux glucocorticoïdes (GC), comme observée chez les patients recevant une corticothérapie, peut entraîner un dysfonctionnement des cellules β et un diabète chez jusqu'à 40% des patients. Dans l'obésité, une surexposition locale au cortisol secondaire à une altération du métabolisme du cortisol contribue à l'apparition du diabète. Des doses élevées de GC comme la dexaméthasone (DEX) inhibent la sécrétion d'insuline stimulée par le glucose (SISG), mais les effets de doses plus faibles et des autres GC, tels que l'hydrocortisone (HC) et la prednisone (PRED), restent peu étudiés. L'enzyme 5α-réductase de type 1 (SRD5A1) est une enzyme cruciale pour la dégradation des GC, modulant ainsi leur biodisponibilité. L'inhibition de SRD5A1 est associée à une altération de la sensibilité à l'insuline et à un risque accru de diabète. La première partie de ma thèse étudie l'impact de doses "thérapeutiques faibles" de PRED (équivalentes à 5 à 10 mg par voie orale) et d'autres GC sur la SISG étudiée par périfusion dans des îlots isolés de pancréas humains. Tous les GCs diminuent significativement la SISG, la DEX ayant un impact plus important que la PRED et l'HC. L'IMC, l'âge ou le sexe n'influencent pas significativement l'impact de la PRED sur la sécrétion d'insuline. La deuxième partie du travail caractérise le métabolisme des GC dans les îlots humains. SRD5A1 est la seule réductase A-ring dans les îlots, et son expression, ainsi que celle de HSD11B1, est localisée dans les cellules β des îlots. Nous avons démontré l'existence d'un métabolisme intracrine du cortisol dans des cultures primaires d’îlots humains. L’expression de HSD11B1 et SRD5A1 est significativement diminuée dans les îlots des donneurs atteints de DT2 par rapport aux donneurs normoglycémiques. La dernière partie visait à prouver que la diminution de la biodisponibilité du cortisol via la surexpression de SRD5A1 dans les îlots humains atténue l'effet inhibiteur des GC sur la SISG. La surexpression de SR5DA1 a permis d’atténuer l'impact de l'HC sur la première phase de la SISG, mais pas de la PRED. En conclusion, même à faibles doses, les GC altèrent la SISG. La diminution de l'expression de SRD5A1 dans les îlots peut contribuer au développement du diabète dans un contexte métabolique. La surexpression de SRD5A1 protège contre l'impact délétère du cortisol sur la SISG. Ces résultats supportent le rôle de SRD5A1 dans la surexposition locale au cortisol et le développement du diabète. Cependant, l'augmentation de l'activité de SRD5A1 ne semble pas efficace pour protéger contre les complications métaboliques induites par la corticothérapie. D'autres aspects de la fonction des cellules β, en particulier la viabilité cellulaire, vont être étudiés. Par ailleurs, le bénéfice potentiel de SRD5A1 dans la modulation de la résistance à l'insuline et de la stéatose hépatique doivent être étudiés. Ces études complémentaires permettront de mieux comprendre le potentiel du gène SRD5A1 dans la modulation de la résistance à l'insuline et de la maladie du foie gras
Excessive glucocorticoid (GC) exposure, as seen in patients receiving GC therapy, can lead to β-cell dysfunction and diabetes in up to 40% of the cases. In obesity, increased local cortisol exposure due to altered metabolism contributes to diabetes onset. High doses of GCs like dexamethasone (DEX) are known to inhibit glucose-stimulated insulin secretion (GSIS), but the effects of lower doses and other GCs, such as hydrocortisone (HC) and prednisone (PRED), remain underexplored. The enzyme 5α-reductase type 1 (SRD5A1) is a crucial enzyme for GC degradation, modulating their bioavailability. Inhibition or knockout of SRD5A1 is associated with impaired insulin sensitivity and increased diabetes risk. This first part of my thesis investigates the impact of “low therapeutic” doses of PRED (equivalent to 5 to 10 mg administrated orally) and other GCs on glucose stimulated insulin secretion (GSIS). We showed that PRED significantly decreases GSIS, with DEX having a worse effect compared to PRED and HC. BMI, age, or sex do not significantly influence the direct impact of PRED on insulin secretion. The second part of the work aimed to characterize GC metabolism in human islets. SRD5A1 is the only A-ring reductase expressed in islets, and its expression, along with HSD11B1, is localized within the β-cells of human islets. We demonstrated evidence of intracrine metabolism of cortisol in intact primary human islets cultured under dynamic experimental settings. Expression data reveals significantly diminished expression of both HSD11B1 and SRD5A1 in T2D donors compared to normoglycemic donors. The last part aimed to provide proof of concept that decreased cortisol bioavailability via the overexpression of SRD5A1 in human islets mitigates the inhibitory effect of GCs on GSIS. SR5DA1 overexpression attenuated the impact of HC on the first phase of insulin secretion, but not the PRED impact. To conclude, even at low doses, GCs impair GSIS. The decrease in SRD5A1 expression in islets may contribute to the development of diabetes in metabolic context. SRD5A1 overexpression protects against the deleterious impact of cortisol on GSIS, providing additional evidence to support the enzyme's role in local cortisol overexposure and the development of diabetes. However, increasing SRD5A1 activity may not be an effective approach to protect against metabolic complications induced by GC therapy. Other aspects of β-cell function, especially cell viability, need to be studied. Moreover, the potential benefits of SRD5A1 in modulating insulin resistance and fatty liver disease should be investigated. These further studies will provide more insight into the potential of SRD5A1 as a therapeutic target