The H N mutation in

The H373N mutation in exon 6 has not previously been reported [3], [4], [5], [6]; thus, this mutation appears to be a novel mutation. The functional expression study demonstrated that the H373N mutation results in almost complete loss of enzymatic activity. Thus, it is highly likely that the H373N mutation and the ΔF54 mutation are the causes of combined 17α-hydroxylase/17,20-lyase deficiency in our patient. Of note, another mutation involving codon 373, H373L, has been described in Japanese patients with combined 17α-hydroxylase/17,20-lyase deficiency by us and others [9], [14], [15], [16], [17] and is demonstrated to disturb heme binding and completely abolish both 17α-hydroxylase and 17,20-lyase activities by in vitro expression studies [16]. Computer modeling of human P450c17 suggests that H373, located in a β-sheet, forms a hydrogen bond with the carboxylate of E391 in the adjacent strand of the β-sheet; and thus, the H373L mutation creates a global alteration of P450c17 structure that secondarily prohibits heme binding [18]. Our novel H373N mutation, substituting the highly conserved basic histidine residue to a neutral asparagine residue, reconfirms the importance of the H373 residue for P450c17 structure and function. In patients with combined 17α-hydroxylase/17,20-lyase deficiency, the elucidation of molecular basis generally explains the patient's clinical profiles [4], [5]. The ΔF54 mutant P450c17 is shown to retain 10% to 23% 17α-hydroxylase activity and 5% to 12% 17,20-lyase activity [10], [11]. Given that the H373N mutation almost completely abolishes both 17α-hydroxylase and 17,20-lyase activities, the residual activities of the ΔF54 mutant may, at least in part, account for the presence of clitoromegaly and the absence of hypertension and hypokalemia in our patient. To our knowledge, 6 patients with combined 17α-hydroxylase/17,20-lyase deficiency bearing the ΔF54 mutation at least in 1 allele have been reported in detail; 4 patients (1 genetic male and 3 genetic females) are homozygous for the ΔF54 mutation, and 2 patients (1 genetic male and 1 genetic female) are compound heterozygous for the ΔF54 mutation and the H373L mutation [10], [12], [13], [14], [15]. Blood pressure is described in 5 patients [10], [12], [13], [15]; all of them are hypertensive in contrast with our patient. The external genitalia in the genetic male patient with the homozygous ΔF54 mutation are characterized by hypospadias and cryptorchidism [12]. The genetic male patient with the compound heterozygous ΔF54 and H373L mutation is reported to have female-type external genitalia with no ambiguity [14], whereas our patient has clitoromegaly. In a large-scale study from Brazil, substantial variations in blood pressure and potassium levels as well as in genital differentiation in genetic males can be observed among individuals with the same mutant CYP17A1[19]. Therefore, it is deduced that environmental and other genetic factors than CYP17A1 genotype are also responsible for the clinical presentations of combined 17α-hydroxylase/17,20-lyase deficiency. Recently, it has been demonstrated that the POR gene encoding P450 oxidoreductase (POR), through which all microsomal P450 enzymes receive electrons from reduced nicotinamide Cinobufagin dinucleotide phosphate (NADPH), is very polymorphic and that an A503V variant POR affects the 17α-hydroxylase and 17,20-lyase activities of P450c17 [20]. Thus, it is possible that polymorphisms of the POR gene modify phenotype of combined 17α-hydroxylase/17,20-lyase deficiency; and this possibility can be tested in the large cohort of Brazilian patients with the same mutant CYP17A1[19]. In conclusion, we have demonstrated a novel genetic lesion in the CYP17A1 gene that leads to combined 17α-hydroxylase/17,20-lyase deficiency.
Acknowledgment
Introduction Pyrazoles are an important class of heterocyclic compounds with a broad spectrum of application in the agrochemical industry and medical practice as active substances of pesticides and clinically relevant drugs [1]. The pyrazole scaffold is present in a variety of leading marketed nonsteroidal anti-inflammatory agents [2], and several derivatives have also been found to possess a wide range of bioactivities, such as antihypertensive [3], antibacterial [4], anticonvulsant [5], antidepressant [5], [6], anticancer [7] and others. The incorporation of a pyrazole moiety into the relatively rigid and stereo structurally planar skeleton of sex hormone-derived steroids either condensed with or connected to one of the core-forming rings has also attracted attention from pharmacological aspects. Stanozolol, the best-known steroidal pyrazole with a 2,3-condensed heteroring, is a synthetic anabolic steroid derived from dihydrotestosterone [8], which has been used in both animal and human therapy for a number of conditions. Moreover, 17-exo-azolylandrostanes based on the sterane structure of natural pregnanes (pregnenolone and progesterone) are at the focus of scientific research as a result of their pharmacological importance. Derivatives with an unsubstituted heteroring or with a not too bulky group on the azole moiety are of great relevance as potent inhibitors of 17α-hydroxylase-C17,20-lyase (P450) [9], a key regulatory enzyme of androgen hormone biosynthesis. Although this enzyme is responsible for the catalysis of two subsequent reactions (the 17α-hydroxylation of pregnane precursors and side-chain cleavage between C17 and C20) at a single active site, a direct indicator of the suitability of a compound for the treatment of androgen-dependent diseases is its ability to block the C17,20-lyase activity of this metalloprotein, which is directly involved in the formation of androgens [10]. It has been demonstrated that the inhibitory action of 17-exo-heterocyclic steroids is related to the presence of the attached heteroring, with the nitrogen lone pair coordinating to the heme iron of the enzyme at the binding site. A hydroxy group on C-3 of the sterane core favors the enzyme−substrate interaction by forming a hydrogen-bond with asparagine 202 in the F helix within the protein-binding pocket [11]. The presence of the C16-17 double bond also appears to be of crucial importance, since derivatives lacking this structural element are usually weaker inhibitors than their unsaturated counterparts. With regard to these structural requirements, a large number of derivatives with a triazolyl, tetrazolyl [12], imidazolyl, isoxazolyl [13], oxazolyl [14], thiazolyl [14], [15], oxadiazolyl [16] or pyrazolyl [13], [17] ring on C-17 have been synthesized to date and investigated for their C17,20-lyase inhibitory action. Besides C17,20-lyase inhibition, some 17-exo-heterocyclic steroids have also been reported to exhibit direct antiproliferative effects on cancer cell lines of diverse origins by causing a disturbance in the normal cell cycle and the upregulation of apoptotic pathways [18].