Clinical Characteristics and Molecular Aetiology of Cytochrome P450 Oxidoreductase Deficiency Diagnosed in 46 XX Patients

Patients and clinical data

Patients with PORD were included if the peripheral blood karyotype was 46 XX and if clinical and biochemical manifestations confirmed PORD, with medical histories being completely documented. The study was approved by the Ethics Committee of Peking Union Medical College Hospital (PUMCH) (IRB Number: JS-2510). Written consent was obtained from all adults or legal guardians of minors under 18 years old.

The clinical presentations of patients with PORD with 46 XX included irregular menses, primary or secondary amenorrhea, underdeveloped breast, sparse axillary and pubic hair, virilizing genitalia, and skeletal malformation.

The laboratory results of PORD were increased serum adrenocorticotropic hormone (ACTH) with increased 17α-hydroxy progesterone (17OHP) and 11-deoxycorticosterone (DOC). Sex hormones usually presented as hypergonadotropic hypogonadism—the oestradiol (E2) level remained at the early follicular stage level with a luteal-phase progesterone (P) level [6].

Medical data, including age, social sex, chief complaint, clinical investigation, management, menstrual history and family history from 2004 to 2022, were retrospectively collected from medical records. Sequencing of the POR gene is crucial for final validation, but clinical diagnosis depended primarily on the patients’ manifestations and laboratory results.

Physical examination was recorded at their very first visits. Abnormal skeletal morphology was recorded. Breast, axillary and pubic hair development were assessed using the Tanner rating scale, while the external genitalia were allocated to the proper Prader stage. Laboratory investigations included (1) gonadal function: anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2, P and T and pelvic sonography for the internal reproductive organs; (2) adrenal function: ACTH, 17OHP, cortisol (F), aldosterone (ALDO), androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DS), 11-deoxycorticosterone (DOC), serum sodium (Na+), and serum potassium (K+). Peptide hormone tests were performed via an automated Elecsys Immunoanalyzer (Beckman Coulter UniCel DXI800, Beckman Coulter; Brea, CA, USA), and the mass spectrometric method (LC‒MS/MS, Quest, USA) was used for the comprehensive analysis of steroid metabolism. The follow-up time ranged from two months to more than 11 years.

Mutation analysis of the POR gene by Sanger sequencing

Blood samples were collected for sequencing of the POR gene (NM_000941.3). Genomic DNA from peripheral blood was extracted using a Lab-Aid 820 Automatic DNA Extraction Kit (Xiamen Zhishan Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Exons and flank areas of POR were tested through Sanger sequencing.

General clinical characteristics

Seven patients were eligible for analysis (Table 1). They all presented as females with a 46 XX karyotype. Their first visits were between 11 and 19 years old. The majority camewere due to abnormal menses (5/7, 71.4%), and the second most common complaints were ovarian cysts (3/7, 42.9%). All patients experienced spontaneous but less-developed breast development (Tanner stage II-V), while the axillary and pubic hair were almost in line with their ages (Tanner stage II-IV). Five patients’ external genitalia exhibited virilizing patterns, staged as Prader I-IV, and three (Cases 2, 3 & 6) had received perineoplasty to correct the clitomegaly and/or labial fusion. No patient complained of retardation of height growth or hypertensive disorder.

Gonadal axis function

Their serum E2 levels followed an early-follicular-stage pattern. Specifically, serum P levels were abnormally elevated (6.60-14.78 ng/mL), serum T levels ranged from normal to slightly higher, and gonadotropins (FSH and LH) were moderately to severely elevated (Table 2). All seven patients had bilateral enlarged multilocular ovaries. Cases 2, 6 & 7 had received ovarian cystectomy, and their pathologies confirmed the diagnosis of luteinized cysts. All patients were prescribed combined oral contraceptives (COC) after diagnosis of PORD, and no relapse of ovarian cysts occurred thereafter. They were negative for ovarian tumour markers, including CA12-5, CA19-9, HE4, CEA, and AFP.

Adrenal axis function

For impaired glucocorticoid metabolism, four patients (Cases 2, 3, 6 & 7) had significantly high ACTH, but only Cases 2 & 7 presented reduced serum F levels (Table 2). The 17OHP level was exclusively above the upper limit, and those of Cases 2, 5, 6 & 7 were even higher than 10 ng/mL. The mineralocorticoid axis was also involved. The serum DOC level extended beyond the reference range, compared to patients’ shortage of ALDO (except for the unavailable data of Case 1). However, all patients’ blood pressure was normal, and the serum interference with Na⁺ and K⁺ levels was not observed. No case reported an abnormal DS or androstenedione level in circulation.

Detection of genetic mutations

The detected POR mutations are summarized in Table 3. Cases 4 & 7 were originally diagnosed as isolated 17,20-lyase deficiency (a rare form of 17OHD) according to their clinical manifestations. However, Sanger sequencing revealed no mutation in P450c17A1. Therefore, a further analysis of the POR gene was arranged. Cases 1 to 6 carried accordant mutations, which were uniformly c.1370G > A (p. Arg457His) for two alleles, while case 7 was a carrier of two heterozygous allele mutations, including this prevalent mutation and a novel mutation (c.1684dupG, p. Glu562Gly fs*13). This newly discovered variance was situated within exon 14, encoding a nonsense mutation in the NADPH binding area. The most frequent mutation, c.1370G > A, was located on exon 11 and belonged to the missense category. This variation appeared in all seven patients, with an allele frequency of 92.9% (13/14).

Genotype-Phenotype Analysis

Although we describe cases particularly in 46 XX females, PORD involves both chromosomal sexes at a roughly equal rate, and the majority are of European or Japanese descent[7]. In line with the autosomal recessive pattern of inheritance, biallelic mutations in the POR gene were detected in all seven cases. The POR gene contains 16 exons. Exons 1 to 15 encode the POR enzyme containing 680 amino acids, while an extra exon does not encode any protein[8]. Spread is the flavin mononucleotide domain of POR directly transferring electrons for P450 enzymes, while most mutations are clustered in the NADPH-binding domain serving as the electron donor. Nevertheless, data from three large PORD cohorts have reached only a poor genotype–phenotype correlation[9–11]. Among all seven patients, exon 11 of POR harbours at least one mutation (c.1370G > A), which leads to a conversion of arginine at amino acid position 457 to histidine. This POR mutation, p.Arg457His, is most widely reported in Japanese people [4, 7], and it is also the most prevalent in our cohort, present in 13 out of 14 alleles. However, in patients of Caucasian origin, the p.Ala287Pro is the most frequently reported[10]. Previously, an in vitro study found that p.Ala287Pro does not impair aromatase activity but that p.Arg457His abolishes it; both mutations lead to 46 XX virilization if present in the homozygous state[12]. Therefore, the p.Arg457His is reported to be associated with virilization in 46,XX patients but likely results in normal male genital development[6]. In addition, because cholesterol production in skeletal tissues occurs in a simple one-way manner without alternative reactions, the skeletal phenotype is supposed to depend obviously on the p.Arg457His dosage, reflecting the residual activity of POR[6]. As a result, the threshold for the development of severe skeletal phenotypes resides between a single copy and two copies of the p.Arg457His residual activity. In Case 7, we reported a newly discovered null mutation c.1684dupG (p. Glu562Gly fs*13). Since the frameshift variance contributes to a premature termination of peptide chain, leaving almost 100 amino acids unattached, the mutated enzyme is presumably to be loss-of-function. No PORD-affected live-born individual has been found to carry this kind of null mutation on both alleles thus far. This suggests that such a genotype is incompatible and lethal to postnatal life, which is in accordance with the observation of early intrauterine foetal death in the Por deletion murine model[13].

Endocrine characteristics

Most newly discovered PORD cases are infants or even foetuses, while adolescents and adults account for only 24%[7]. The most prevalent clinical manifestations were an aberrant hormonal profile at birth or developmental delay since puberty (89.2%), ABS-like skeletal deformity (82.7%), disorders of sex development or hermaphroditism (75%), nonclassical CAH-related symptoms (74.6%), ovarian multilocular morphology in females (46.7%) and maternal virilization during pregnancy (40.8%)[7]. These figures refer to PORD as a typical endocrinological issue. The low serum E2 levels (21.7–40.7 pg/mL) of our seven cases are attributed to decreased ovarian androgen production (except Case 2) and its subsequent aromatization[14]. The hypogonadism is partially responsible for the negative feedback of high FSH. This could be mixed with ovarian insufficiency, particularly when patients have menstrual problems and low AMH (Cases 5, 6 & 7). Low-to-normal AMH can exist in these patients, and this scenario is commonly seen in other CAH subtypes, such as 17 OHD[15]. Since patients with severe POR mutations suffer from severe P450 enzymatic defects, their follicular reservoir can undergo premature failure due to impaired granular cell steroid metabolism; mild forms of mutation that leave partial secretive function of follicles contribute to the residual ovarian follicles’ response to the persistent stimulation of raised FSH. Interestingly, regardless of the level of AMH or FSH, all patients are found to have ovarian multilocular cysts, and these cysts are frequently seen in compromised function of POR[16]. The underlying cause is that the relatively young age of these patients is distinguished from the postmenopausal exhaustion of the ovarian reserve. Even a limited number of remaining follicles will respond to FSH recruitment and grow into a multilocular ovarian morphology. In addition to the accumulation of P as a steroid precursor, excessive LH will mediate luteinization of these cysts and further promote P production in these PORD-CAH patients[17]. All affected individuals from our cohort exhibited a simultaneous increase in serum 17OHP and P, which misleadingly suggested nonclassical (NC) 21OHD. Moreover, impaired catalytic activity of 17,20-lyase is responsible for decreased circulating androgen levels, which mimics the isolated 17,20-lyase (ILD) form of 17OHD, but differs from 21OHD and PCOS[16]. Surprisingly, 75% of PORD patients with 46 XX are commonly accompanied by ambiguous genitalia[7]. Four of our cohorts (4/7, 57.1%, Cases 2, 3, 5 & 6) underwent virilization of external genitalia. This is an apparent dichotomy, given that DHEA and androstenedione production in the foetus should be disturbed due to impaired 17,20-lyase activity. Even the modest amount of androgens produced could be accumulated due to the deficiency of placental aromatase affected by PORD[18]. In addition, elevated 17OHP, which cannot be efficiently metabolized via P450c21 or P450c17 activities, seeks an alternative pathway via 5α-reduction and is ultimately converted to DHT[19].

Skeletal deformities are associated with lanosterol 14α demethylase (CYP51A1), which is involved in cholesterol biosynthesis[20] and retinoic acid metabolism by microsomal CYP26 enzymes[21]. Compound heterozygous and homozygous mutations in the POR gene are associated with more serious skeletal deformities, resulting from the effect of various mutations on the activity of related enzymes[10]. Cases 1 to 6 harbour the same homozygous mutation but have various skeletal deformities, illustrating the heterogeneity of this symptom in PORD.

Ovarian cysts and fertility outcomes

Ovarian cysts can trigger significant morbidity ranging from mild abdominal tenderness to acute ovarian cyst rupture or torsion. All patients from our cohort had fluid-filling luteinized cysts of at least one ovary. Cases 2, 6 & 7 have even previously undergone ovarian cystectomy, and their pathologies confirmed the diagnosis of luteinized cysts. All patients are prescribed COC after the operation, and this provides effective conservative management for this type of recurrent cyst. Large cysts shrink soon after COC administration in most patients by suppressing gonadotropin stimulation from the hypophysis; however, ovarian cysts may reappear after the cessation of COC treatment, and it is still effective to use it again to repress the cyst[22]. Their ovarian tumour markers were negative at the beginning. However, if ovarian cysts persist after COC intervention, the possibility of nonfunctional tumours should be excluded. In addition, recurrent functional ovarian cysts in 46XX PORD must be differentiated from other endocrine-related diseases, such as 46 XX partial 17OHD, Van Wyk–Grumbach (VWG) syndrome, and functional gonadotroph adenomas (FGAs) [23].

Fertility preservation is a challenging issue for 46 XX patients with PORD. Natural conception has not been reported in these females, and almost all patients of reproductive age encounter primary infertility[16]. PORD will implicate CYP51A1, an enzyme responsible for the conversion of lanosterol to meiosis-activating sterols, which in turn cause defective oocyte maturation[24]. For patients with severe mutations of POR leading to the complete loss of enzymatic function, the premature depleted ovarian reservoir offers them a rare chance to conceive[16]. For patients still equipped with remaining primordial follicles and diagnosed with a partial loss of POR activity, endocrinological elements lead to a barrier to natural fertility. The main contributing factor is excessive P levels without cyclic change, which in turn exert an anti-gonadotropic effect on the hypothalamus and pituitary gland. Without the physical periodic fluctuation of gonadotropin, even in those cases with oligomenorrhea, ovulation seldom occurs spontaneously. This is presumably due to the blockage of LH positive feedback on rising E2 by high P levels. Persistently high circulating P levels reverse the endometrial receptivity synchronized with embryo development[25] and alter the cervical mucus. Moreover, anovulation could also be blamed for defective follicular maturation due to steroidogenesis failure, particularly androgen deficiency[26]. Thanks to the advancement of assisted reproductive technology (ART), pregnancy can be achieved by controlled ovarian stimulation (COS) and in vitro fertilization[22] while suppressing P with potent corticosteroids and creating an artificial menstrual cycle with hormone replacement for frozen-thawed embryo transfer[27]. Bosch et al revealed that serum P levels < 1.5 ng/ml over COS are associated with higher live pregnancy rates[28]. Consequently, pregnancy can be achieved in females through ART if PORD is correctly diagnosed as early as possible and if the ovaries are well protected by avoiding unnecessary operation due to the spontaneous disappearance of cysts following the commencement of COC[22]. This is essential for dealing with multilocular cysts and protecting the diminished ovarian reserve in 46 XX PORD patients, maximizing their chance for future ART.

Limitations

Several limitations exist in our study. Primarily, since PORD is a rare type of CAH, the sample size is very limited, but we are working on the collection and updating of new cases. Second, some laboratory studies can be modified without consideration of cost and accessibility in our hospital. The pathogenicity of the newly identified mutation (c.1684dupG) was not evaluated by an in vitro functional study. The skeletal evaluation was based on physical examination and plain radiographs in some cases; peripheral quantitative computed tomography for the study of bone lesions might be a better option. In addition, the assessment of adrenal function by serum steroid variation with ACTH stimulation is more precise and solid[29]. Given that PORD affects multiple systems, its management is challenging and should encompass transdisciplinary cooperation, including paediatric endocrinologists and orthopaedists. Herein, we provide management experience dominantly from the perspectives of gynaecological endocrinology and reproduction and hope to promote patients’ sexual-developmental and fertility outcomes.