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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.

BackgroundThe thyroid hormone receptor-like (THR-like) family is the largest transcription factors family belonging to the nuclear receptor superfamily, which directly binds to DNA and regulates the gene expression and thereby controls various metabolic processes in a ligand-dependent manner. The THR-like family contains receptors THRs, RARs, VDR, PPARs, RORs, Rev-erbs, CAR, PXR, LXRs, and others. THR-like receptors are involved in many aspects of human health, including development, metabolism and homeostasis. Therefore, it is considered an important therapeutic target for various diseases such as osteoporosis, rickets, diabetes, etc.MethodsIn this study, we have performed an extensive sequence and structure analysis of the ligand-binding domain (LBD) of the THR-like family spanning multiple taxa. We have use different computational tools (information-theoretic measures; relative entropy) to predict the key residues responsible for fold and functional specificity in the LBD of the THR-like family. The MSA of THR-like LBDs was further used as input in conservation studies and phylogenetic clustering studies.ResultsPhylogenetic analysis of the LBD domain of THR-like proteins resulted in the clustering of eight subfamilies based on their sequence homology. The conservation analysis by relative entropy (RE) revealed that structurally important residues are conserved throughout the LBDs in the THR-like family. The multi-harmony conservation analysis further predicted specificity in determining residues in LBDs of THR-like subfamilies. Finally, fold and functional specificity determining residues (residues critical for ligand, DBD and coregulators binding) were mapped on the three-dimensional structure of thyroid hormone receptor protein. We then compiled a list of natural mutations in THR-like LBDs and mapped them along with fold and function-specific mutations. Some of the mutations were found to have a link with severe diseases like hypothyroidism, rickets, obesity, lipodystrophy, epilepsy, etc.ConclusionOur study identifies fold and function-specific residues in THR-like LBDs. We believe that this study will be useful in exploring the role of these residues in the binding of different drugs, ligands, and protein-protein interaction among partner proteins. So this study might be helpful in the rational design of either ligands or receptors.

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Electron transfer in POR occurs from NADH to FAD to FMN, and the flexible hinge region in POR is essential for domain movements to bring the FAD and FMN close together for electron transfer. We tested the effect of variations in the hinge region of POR to check if the effects would be similar across all redox partners or there will be differences in activities. Here we are reporting the effects of a POR genetic variant P284T located in the hinge region of POR that is necessary for the domain movements and internal electron transfer between co-factors. Human wild-type and P284T mutant of POR and cytochrome P450 proteins were expressed in bacteria, purified, and reconstituted for enzyme assays. We found that for the P284T variant of POR, the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, indicating intact direct electron transfer from FAD to ferricyanide, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug-metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4, and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR, which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation. The negative impact of P284T mutation in the hinge region of POR seems to be due to disruption of FAD to FMN electron transfer. These results further emphasize the importance of hinge region in POR for protein flexibility and electron transfer within POR as well as the interaction of POR with different redox partners.

Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.

All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN) binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT) and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler syndrome but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations with Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V allele is found in about 30% of all alleles but there are some differences across different population groups.