In silico and in vitro Studies of Human 5α‐reductase Type II Reveal New Loss of Function Variants

Background Androgens are steroid hormones necessary for human sex development. Testosterone (T) and the more potent dihydrotestosterone (DHT) are maybe the best‐known androgens, which exert their effect by binding and activating the androgen receptor. The 5α steroid reductases (SRD5As) catalyze the conversion of T to DHT in the classical androgen production pathway, or from 17‐hydroxyprogesterone to 17OH‐dihydroprogesterone, and androstenedione to androstanedione in alternate pathways leading to DHT. There are two enzymes with differential expression, of which SRD5A2 is expressed in reproductive organs and liver, and catalyzes the reaction of T to DHT more efficiently than the SRD5A1. In humans, SRD5A2 loss‐of‐function mutations are known, and cause severe 46XY undervirilization, while gain‐of‐function variants have been suggested in androgen excess syndromes such as premature adrenarche, the polycystic ovary syndrome or prostate tumors, but these have not been identified so far. Aim We aimed to search for potential gain‐of‐function mutations in the human SRD5A2 gene. Methods We searched sequence databases for genetic variants and performed bioinformatic and functional tests on a set of selected variants. After the amino acid conservation analysis of SRD5A2, a novel 3D protein model was constructed to identify the exact position of amino acids in the tertiary structure and predict their effect on protein function and substrate interaction. We then collected 116 coding SNPs in SRD5A2 from OMIM, dbSNP, Pubmed, Clinvar, HGMD, and UniProt databases. These SNPs were ranked according to their association with phenotypes, physical location in our 3D model, and molecular dynamics simulation studies. Finally, we selected nine coding SNPs for in vitro studies. These SNPs were located within or close to highly conserved areas that form the binding cavities for substrates or the cofactor NADPH. The SRD5A2 variants were expressed in HEK293 cells, and enzyme activity was assessed by conversion of testosterone (T), progesterone (Prog) and androstenedione (Δ4A) to their 5α‐reduced metabolites. Result Variants R50A and P173S showed partial activity with substrates T (34% and 28%) and Δ4A (37% and 22%). With Prog as substrate variants P106L, P106A, L167S, and R168C, showed partial activity (15% to 64%). Functional testing of all other variants showed loss‐of‐function. As predicted in our in silico analysis, all coding SNPs affected enzyme activity. However, none of them showed gain‐of‐function Conclusion We provide a novel protein model for studies of SRD5A2. We have characterized seven human SRD5A2 variants, which although did not show any gain of function, might be of clinical relevance for their loss of enzyme activity. It is possible that individuals carrying these SNPs show a minor, yet unidentified phenotype. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .