Molecular modeling and clinical characterization of a CYP17A1 variant (p.Asp298Asn) causing 17α-hydroxylase/17,20-lyase deficiency in two siblings

Highlights

● Two siblings with 17α-hydroxylase/17,20-lyase deficiency carry a homozygous CYP17A1 variant (c.892G>A; p.Asp298Asn) that was not previously registered in public databases at initial submission and was previously uncharacterized at the case level.

● Early diagnosis and multidisciplinary management are critical to prevent major complications in 46,XY individuals (e.g., uncontrolled hypertension, osteoporosis, psychosocial impact, and increased gonadal malignancy risk).

Introduction

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases resulting from defects in enzymes involved in steroidogenesis. One of the rare causes of CAH, 17α-hydroxylase/17,20-lyase deficiency, occurs due to pathogenic variants in the CYP17A1 gene. The CYP17A1 gene encodes the enzymes 17α-hydroxylase and 17,20-lyase which play critical roles in steroidogenesis and androgenesis. (1) This case report presents a previously uncharacterized homozygous variant, c.892G>A(p.Asp298Asn), in the CYP17A1 gene (NM_000102.4), classified as “likely pathogenic” according to ACMG criteria, detected in two siblings diagnosed with 17α-hydroxylase/17,20-lyase deficiency.

Case Report

The first patient was referred to pediatric endocrinology at the age of 3.5 yr after intra-abdominal testicular tissue was incidentally detected during an appendectomy. The patient had phenotypically female external genitalia. Physical examination revealed no palpable gonads, dysmorphic features, hyperpigmentation, or hypertension. The parents were first cousins, and the patient had two healthy sisters. Pelvic magnetic resonance imaging confirmed the absence of uterus and ovaries, and the presence of intra-abdominal testes. In the standard-dose intravenous ACTH stimulation test, the peak cortisol response was 1.18 μg/dL, indicating significantly low adrenal reserve (Table 1). Bone age was consistent with chronological age. Treatment with hydrocortisone (13 mg/m²/d) was initiated. Karyotype analysis revealed 46,XY. Considering the low adrenal reserve combined with 46,XY differences of sex development (DSD), 17α-hydroxylase/17,20-lyase deficiency was suspected. Supporting this, 11-deoxycorticosterone levels were markedly elevated at 7.27 pmol/mL (reference range: 0.12–0.60). Genetic analysis identified a homozygous CYP17A1 variant: c.892G>A (p.Asp298Asn). The patient was reared as female from birth and, during repeated follow-up visits in early childhood, demonstrated consistent female social identification and female-typical gender role behavior reported by caregivers and documented by the multidisciplinary DSD team (including child psychiatry), acknowledging the limitations of formal gender identity assessment at preschool age; bilateral orchiectomy was performed at 4.5 yr. Estradiol replacement was initiated at age 14 for pubertal induction. The patient is currently monitored by child psychiatry for psychosocial support and pediatric surgery for vaginal stenosis. Low-dose amlodipine was added due to borderline hypertension during follow-up.

Table 1. Hormonal and biochemical characteristics of the cases.

The second patient was evaluated at 6 d of life due to the sibling’s diagnosis. Born at term (3400 g), the patient had completely feminized external genitalia with bilateral palpable gonads in the labia majora. No dysmorphism, hypertension, or hyperpigmentation were observed. The ACTH stimulation test showed a peak cortisol response of 1.43 μg/dL. The family history, female external genitalia with palpable gonads, and hormone profile (Table 1) supported the diagnosis. Hydrocortisone (10 mg/m²/d) was initiated. Karyotype analysis revealed 46,XY. Genetic analysis confirmed homozygosity for the same CYP17A1 c.892G>A (p.Asp298Asn) variant. Gonadectomy decisions were made by the multidisciplinary DSD board, adhering to current international recommendations. Written informed consent was obtained from the patients and their family for this case report.

Mutation Analysis

The detected variant had not been previously reported in public databases (ClinVar, gnomAD) (accessed March 2026). In silico analyses predicted a deleterious effect: SIFT 0.01 (detrimental), PolyPhen-2 0.99 (probably detrimental), CADD 27.7 (among the most detrimental 1% variants), and REVEL 0.638 (likely detrimental) (2). Multiple sequence alignment showed that the Asp298 residue is highly conserved (VarSome PhyloP score: 7.4). Pathogenicity evaluation followed the 2015 ACMG-AMP guidelines. The following criteria were met: PM1, located in a functionally important region lining the catalytic cavity (I-helix/active-site helix vicinity) of cytochrome P450 enzymes (3), as illustrated in Fig. 1; PM2, the variant was absent or detected at extremely low frequency in control populations (gnomAD v4.0); PP3, multiple in silico tools predicted a deleterious effect for the variant; and PP4, the clinical phenotype of patients showed a high degree of concordance with the CYP17A1-related disease spectrum. Consequently, the homozygous variant c.892G>A (p.Asp298Asn) was evaluated as “likely pathogenic” according to the ACMG–AMP classification criteria (4).

Fig. 1.

Three-dimensional modeling was performed in Discovery Studio using the CYP17A1 crystal structure (PDB ID: 6WR1), which contains the heme prosthetic group and the bound inhibitor abiraterone. All interactions were identified using the same software criteria/settings in panels A and B. (A) In the wild-type model, Asp298 participates in a local network of non-covalent interactions within the heme-containing catalytic cavity. Green dashed lines indicate predicted hydrogen-bond contacts (e.g., backbone amide contacts from Ile112 and Ala113 to Asp298 OD2, and contacts involving Thr294/Thr295 and Ala302), and the red dashed line indicates a predicted charge-based interaction between Arg239 (NH1) and Asp298 (OD1). In addition, a hydrophobic π–alkyl packing interaction is predicted between Ile112 and the heme porphyrin ring. (B) In the p.Asp298Asn model, replacement of a negatively charged aspartate by an uncharged asparagine is associated with reorganization and reduction of predicted local contacts around residue 298 under the same interaction criteria. (C) Spatial view highlighting the proximity of the residue-298 interaction network to the heme-containing active-site cavity.

Discussion

The CYP17A1 gene encodes the activity of both 17α-hydroxylase and 17,20-lyase enzymes, which play critical roles in steroidogenesis. 17,20-lyase activity is essential for androgen and estrogen synthesis. Therefore, in the absence of this enzyme activity, fetal testosterone and dihydrotestosterone production is insufficient in both 46,XX and 46,XY individuals, and both karyotypes are born phenotypically with female external genitalia. However, in 46,XY individuals, Müllerian structures regress due to the continued production of testicular anti-Müllerian hormone (AMH). On the other hand, 17α-hydroxylase activity is also necessary for glucocorticoid synthesis. In the absence of this enzyme, cortisol production is impaired, but corticosterone accumulation provides glucocorticoid activity, preventing the development of clinically significant adrenal insufficiency in most patients. In clinical practice, 46,XY individuals are frequently diagnosed incidentally during inguinal hernia surgery due to the detection of testicular tissue or the identification of internal genital structures inconsistent with the phenotypic female genital appearance. In the presented cases, both patients had a 46,XY karyotype and were phenotypically female. The first case was diagnosed at 3.5 yr of age upon the detection of intra-abdominal testicular tissue during appendectomy. The second case was evaluated immediately after birth due to family history, thus allowing for earlier diagnosis and follow-up. From a treatment approach perspective, physiological glucocorticoid deficiency is not clinically apparent in most patients due to the glucocorticoid activity provided by corticosterone. However, reducing deoxycorticosterone (DOC) levels through ACTH suppression may be beneficial in preventing or controlling the development of hypertension. In cases where hypertension persists despite glucocorticoid treatment, mineralocorticoid antagonists constitute an effective treatment option. During puberty, estrogen replacement therapy is necessary for the development of secondary sexual characteristics (1).

While the c.892G>A variant was recently listed in a large regional cohort study (5), its clinical features and functional impact remained uncharacterized. Here, we provide the detailed clinical presentation and a structural rationale consistent with pathogenicity. The structural consequences of p.Asp298Asn provide a plausible molecular rationale for impaired CYP17A1 function. In the CYP17A1 crystal structure (PDB: 6WR1), residue 298 lies in the active-site–lining helix region (I-helix vicinity) within the heme-containing catalytic cavity. Using a model that includes the bound steroidal inhibitor abiraterone as a structural proxy ligand, Asp298 is predicted to contribute to the local polarity and geometry of the active-site pocket through a network of polar and charge-based contacts. In the wild-type model, Asp298 engages in backbone amide hydrogen-bond contacts (including OD2-mediated contacts with Ile112/Ala113) and a charge-based interaction with Arg239, and the local packing environment includes a predicted hydrophobic π–alkyl interaction between Ile112 and the heme porphyrin ring (Fig. 1). Substitution with the uncharged Asn298 removes the negatively charged side chain and is associated with reorganization and reduction of predicted contacts around residue 298 under the same interaction criteria, consistent with perturbation of the heme-proximal active-site microenvironment rather than direct heme coordination. Together with the highly specific biochemical phenotype, these structural considerations support the likely pathogenicity of p.Asp298Asn. The clinical relevance of this segment is supported by nearby missense variants p.Ile296Thr and p.Thr306Asn, both classified as likely pathogenic in ClinVar (accessed March 2026), supporting the plausibility that substitutions in the 290–310 region lining the catalytic cavity may perturb the local active-site microenvironment.

In conclusion, we report two siblings with 17α-hydroxylase/17,20-lyase deficiency carrying the likely pathogenic CYP17A1 p.Asp298Asn variant and provide case-level clinical characterization with supportive structural interpretation. Early diagnosis through family-based evaluation and multidisciplinary management are critical to reduce complications and guide individualized care.

Conflict of interests

The authors declare no conflict of interest.