Familial 46,XY disorder of sex development due to a novel CYB5A variant: a rare cause of isolated 17,20-lyase deficiency

Abstract

Primary amenorrhea may be the first clinical presentation of an underlying 46,XY disorder of sex development (DSD). We report 2 siblings assigned female at birth; the index case presented with primary amenorrhea and the younger sibling was identified through family screening. Both had female external genitalia with minimal virilization, absent Mullerian structures, and testes located in the inguinal/labioscrotal region on imaging. Hormonal evaluation showed impaired androgen synthesis with very low testosterone and markedly elevated 17-hydroxyprogesterone:androstenedione ratios, suggestive of isolated 17,20-lyase deficiency. Whole-exome sequencing identified a novel homozygous missense variant in exon 2 of the CYB5A gene (c.140G>T; p.Gly47Val) in both siblings, and segregation analysis confirmed the same variant in both parents as heterozygous carriers. Subsequently, methemoglobin levels were found to be above the normal reference range in both siblings despite the absence of clinical symptoms. Multidisciplinary evaluation confirmed female gender identity in both siblings without any gender incongruence and advised continuation of the assigned female gender role. Gonadectomy and vaginoplasty were planned after 18 years of age. These cases highlight cytochrome b5 deficiency due to a novel variant as a rare cause of 46,XY DSD and the importance of recognizing methemoglobinemia in a DSD.

Introduction

Primary amenorrhea may be the first clinical presentation of an underlying 46,XY disorder of sex development (DSD) in a female-reared individual due to severe undervirilization that goes undiagnosed since birth [1]. 46,XY DSD may present during adolescence as primary amenorrhea, pubertal virilization, or inguinal/labial swellings due to undescended testes. The coexistence of 46,XY DSD with methemoglobinemia due to cytochrome b5 deficiency is exceedingly rare, with fewer than 10 cases reported in the literature [2]. We report 2 siblings with 46,XY DSD due to isolated 17,20-lyase deficiency with asymptomatic methemoglobinemia due to a novel homozygous CYB5A mutation.

Case presentation

Case 1

A 16-year-old female born of a nonconsanguineous marriage presented with complaints of not having attained menarche. However, she had spontaneous and progressive development of both breasts (Fig. 1A) with well-developed pubic and axillary hair. No symptoms of chronic systemic illness, psychosocial stress, excessive physical activity or previous history of chemotherapy and radiotherapy were present. There was no genital ambiguity at birth, neonatal salt-wasting crisis, or hyperpigmentation. Pubertal staging showed Tanner stage B5 breasts, pubic hair stage P3(B5P3) with axillary hair. Anthropometry revealed a height of 174 cm (+1.9 standard deviation score; >97th centile) with a mid-parental height of 160 cm. Examination of the external genitalia revealed normal-appearing female genitalia (Fig. 1B) with no clitoromegaly or labioscrotal fusion, and separate urethral and vaginal openings. No swellings were palpable in the labioscrotal folds or inguinal region. The external masculinization score (EMS) and external genitalia score (EGS) were 0/12.

Figure 1.

Well-developed breasts, Tanner stage 5 (A), with female-appearing external genitalia (B).

Case 2

Her younger sibling, reared as female, was subsequently evaluated at 14 years of age following the genetic diagnosis in case 1. Pubertal staging revealed Tanner stage B3 breast development (Fig. 2A) with absent pubic and axillary hair. Examination of the external genitalia showed asymmetric appearance with a palpable swelling in the left labioscrotal region (Fig. 2B). There was no clitoromegaly, and separate urethral and vaginal openings were present. The EMS and EGS were 1.5/12.

Figure 2.

Tanner stage 3 breast development (A), with asymmetric genitalia and a palpable left gonad (arrow) (B).

Diagnostic assessment

Case 1

The basal hormonal profile showed elevated gonadotropins with high testosterone levels for a phenotypic female, while serum cortisol levels were within the reference range (RR). Pelvic magnetic resonance imaging (MRI) revealed bilateral testes in the inguinal canals (Fig. 3A and 3B) with absent Mullerian structures. Subsequent karyotype analysis confirmed a 46,XY karyotype. Due to the presence of a 46,XY karyotype and bilateral testes on imaging, human chorionic gonadotropin (hCG) stimulation testing was performed, and steroid metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biochemical response showed low androgen precursors, including dehydroepiandrosterone sulfate (DHEAS), androstenedione (A), testosterone (T), and dihydrotestosterone (DHT), with mildly elevated 17-hydroxyprogesterone (17-OHP). This biochemical pattern of elevated 17-OHP (12.1 ng/mL [36.6 nmol/L]; RR, 0.1-2.2 ng/mL [0.3-6.7 nmol/L]), low androstenedione, and a markedly elevated 17-OHP:androstenedione ratio (80.6), in the presence of normal cortisol concentrations, was suggestive of isolated 17,20-lyase deficiency.

Figure 3.

Magnetic resonance imaging showing bilateral testes located in the inguinal canals (arrows) in case 1 (A and B), and the right testis in the inguinal canal and the left testis in the labioscrotal region (arrows) in case 2 (C).

Case 2

The younger sibling also demonstrated a similar hormonal profile with a 46,XY karyotype. Pelvic MRI revealed right testis in the right inguinal canal and left testis in the left labioscrotal region (Fig. 3C) with absent Mullerian structures. Human chorionic gonadotropin stimulation testing showed impaired androgen synthesis with a markedly elevated 17-OHP:androstenedione ratio (90.6), consistent with isolated 17,20-lyase deficiency. The summary of basal and hCG-stimulated hormonal parameters is described in Table 1.

Table 1.

Basal and hCG-stimulated hormonal parameters in both cases

Basal Hormonal parameters	Case 1	Case 2	Reference range
LH	38.44 mIU/mL (38.44 IU/L)	16.83 mIU/mL (16.83 IU/L)	1.7 to 8.6 mIU/mL (1.7-8.6 IU/L)
FSH	20.85 mIU/mL (20.85 IU/L)	9.51 mIU/mL (9.51 IU/L)	1.5 to 12.4 mIU/mL (1.5-12.4 IU/L)
Total testosterone	50.55 ng/dL (1.75 nmol/L)	6.49 ng/dL (0.23 nmol/L)	15 to 38 ng/dL (0.52-1.32 nmol/L)
Estradiol	29.82 pg/mL (109.5 pmol/L)	7.52 pg/mL (27.6 pmol/L)	12.4 to 233 pg/mL (45.5-855.3 pmol/L)
TSH	3.37 µIU/mL (3.37 mIU/L)	2.51 µIU/mL (2.51 mIU/L)	0.3 to 5.5 µIU/mL (0.3-5.5 mIU/L)
Cortisol	15.75 μg/dL (434.5 nmol/L)	12.80 μg/dL (353.2 nmol/L)	6.2 to 18.0 μg/dL (171.1-496.6 nmol/L)
Post hCG stimulation (LC-MS/MS assay)
11-DOC	15.0 ng/mL (45.4 nmol/L)	12.0 ng/mL (36.3 nmol/L)	2 to 15 ng/mL (6.1-45.4 nmol/L)
17-OHP	12.1 ng/mL (36.6 nmol/L)	7.8 ng/mL (23.6 nmol/L)	0.1 to 2.2 ng/mL (0.3-6.7 nmol/L)
DHEA	0.28 μg/L (0.97 nmol/L)	0.06 μg/L (0.21 nmol/L)	1.3 to 6.4 μg/L (4.5-22.2 nmol/L)
DHEAS	252 ng/dL (6.84 nmol/L)	163 ng/dL (4.42 nmol/L)	324 to 3508 ng/dL (8.79-95.20 nmol/L)
Androstenedione (A)	15.0 ng/dL (0.52 nmol/L)	8.6 ng/dL (0.30 nmol/L)	18 to 145 ng/dL (0.63-5.06 nmol/L)
Testosterone (T)	62 ng/dL (2.15 nmol/L)	33 ng/dL (1.14 nmol/L)	2 to 80 ng/dL (0.07-2.77 nmol/L)
Dihydrotestosterone (DHT)a	7.12 ng/dL (0.25 nmol/L)	5.3 ng/dL (0.18 nmol/L)	25 to 99 ng/dL (0.87-3.44 nmol/L)
T/DHT ratio	8.7	6.2	<20
T/A ratio	4.1	3.8	>0.8
17-OHP/androstenedione ratio	80.6	90.6	Not established

Abbreviations: 11-DOC, 11-deoxycorticosterone; 17-OHP, 17-hydroxyprogesterone; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; FSH, follicle-stimulating hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LH, luteinizing hormone; TSH, thyroid-stimulating hormone.

^a DHT was measured by enzyme immunoassay.

Genetic analysis by whole-exome sequencing in the index case identified a homozygous missense variant in exon 2 of the CYB5A gene (c.140G>T; p.Gly47Val), classified as a variant of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) criteria. As variants in CYB5A are known to be associated with congenital methemoglobinemia, methemoglobin levels were measured using spectrophotometric analysis of heparinized whole blood and found to be elevated in the index case (7.4%; RR <1.5%). Segregation analysis was subsequently performed using Sanger sequencing, which demonstrated the same homozygous variant in the sibling (Fig. 4). The younger sibling also had elevated methemoglobin levels (9.8%; RR <1.5%). Parental testing revealed that both parents were heterozygous carriers of the same variant with normal methemoglobin levels (1.1% and 1.2%) in both of them. This segregation pattern, along with biochemical evidence of methemoglobinemia in the affected siblings, provided supportive evidence for the pathogenicity of the identified CYB5A variant.

Figure 4.

Sanger sequencing chromatograms of the index case and the younger sibling.

Treatment

Both cases were discussed in the multidisciplinary DSD committee of Government Rajaji Hospital, which included endocrinologists, pediatricians, pediatric surgeons, and psychiatrists. Gender identity assessment revealed a female gender identity without any evidence of gender incongruence in both siblings. In accordance with the wishes of the adolescents and their parents, continuation in the female gender role was recommended despite the 46,XY karyotype. The presence of spontaneous breast development indicated adequate endogenous estradiol production; therefore, estrogen supplementation was not initiated. According to the existing legal system in India, irreversible cross-gender-affirming surgical procedures are recommended only after 18 years of age; therefore, bilateral gonadectomy was planned after the completion of 18 years in both siblings. In addition to that, both had a blind vaginal pouch and hence vaginoplasty was also considered after 18 years.

Outcome and follow-up

Both adolescents were clinically asymptomatic with respect to methemoglobinemia. They were counseled regarding the avoidance of oxidant drugs, including certain over-the-counter medications and antibiotics known to precipitate methemoglobinemia. Precautions were also advised for future surgical procedures, particularly regarding anesthetic agents that may trigger methemoglobinemia. During follow-up, both patients continued to identify with the female gender identity, with no evidence of gender dysphoria.

Discussion

17,20-lyase catalyzes the conversion of 17-hydroxypregnenolone (17-OH-Preg) to dehydroepiandrosterone (DHEA) and 17-OHP to androstenedione, which are the major substrates for androgen biosynthesis. Isolated 17,20-lyase deficiency can result from mutations in the CYP17A1 (encoding cytochrome P450c17), POR (encoding cytochrome P450 oxidoreductase), or CYB5A (encoding microsomal cytochrome b5) genes. 17,20 lyase reaction depends on the coordinated interaction between these 3 proteins [3]. The first case of isolated 17,20 lyase deficiency with CYB5A mutation was described in 2012 [4]. To date, less than 40 cases of isolated 17,20 lyase deficiency and fewer than 10 with CYB5A mutation have been reported in the published literature [2]. Here, we report a familial case of primary amenorrhea in 46,XY individuals reared as females, with biochemical, imaging, systemic features, and molecular genetics suggestive of cytochrome b5 deficiency.

The microsomal enzyme cytochrome P450c17 (encoded by CYP17A1) is expressed in steroidogenic tissues such as the adrenal cortex, testes, and ovaries. This enzyme catalyzes both 17α-hydroxylase and 17,20-lyase reactions, which are required for glucocorticoid and sex steroid synthesis, respectively. The 17,20-lyase activity depends on the presence of P450 oxidoreductase (POR), an electron-transfer protein for microsomal P450 enzymes. Cytochrome b5 functions as an allosteric cofactor that facilitates the interaction between POR and P450c17, thereby enhancing electron transfer and promoting the 17,20-lyase reaction [3]. In contrast, 17α-hydroxylase activity is unaffected by cytochrome b5, and therefore cortisol synthesis remains unaffected in isolated 17,20-lyase deficiency [5]. Hence, the clinical manifestations differ between combined 17α-hydroxylase/17,20-lyase deficiency and isolated 17,20-lyase deficiency [6].

The differential diagnosis of primary amenorrhea with well-developed breasts in a 46,XY individual reared as female (severely undervirilized and apparently female-appearing external genitalia) includes complete androgen insensitivity syndrome (CAIS) and defects in androgen biosynthesis such as 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) deficiency. However, CAIS was unlikely in the present case (case 1) due to well-developed pubic and axillary hair, which are typically sparse or absent in CAIS. Likewise, HSD17B3 deficiency and partial androgen insensitivity syndrome were considered less likely, as there were no features of pubertal virilization [7] or clitoromegaly in both siblings. In case 1, the presence of pubic hair despite impaired steroidogenesis may be explained by activation of an alternative (backdoor) pathway of androgen biosynthesis. In this pathway, accumulated 17-OHP undergoes sequential conversion to 17-hydroxy-dihydroprogesterone, 17-hydroxy-allopregnanolone, and androsterone, ultimately generating DHT without requiring testosterone as an intermediate [2]. Residual androgen production through this pathway may contribute to pubic and axillary hair development.

The possible mechanisms of well-developed breasts (Tanner stage 5) in case 1, despite only modestly elevated testosterone, may be due to the peripheral aromatase activity that converts the available testosterone into estradiol, which results in relative superiority of estradiol to androgens [8]. In addition, there may be an increase in local tissue/receptor sensitivity to estrogens. In case 1, total testosterone was 50.55 ng/dL (1.75 nmol/L) (RR: 15-38 ng/dL [0.52-1.32 nmol/L]), and estradiol was 29.82 pg/mL (109.5 pmol/L) (RR: 12.4-233 pg/mL [45.5-855.3 pmol/L]).

17-Hydroxyprogesterone elevation is classically seen in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. As a result of isolated 17,20-lyase deficiency, impaired conversion of 17-OH-Preg to DHEA and 17-OHP to androstenedione ultimately leads to low testosterone that manifests as a severe degree of undervirilization in 46,XY individuals. Mild elevations in 17-OHP may be observed in POR deficiency, 17,20-lyase deficiency, and 3β-hydroxysteroid dehydrogenase deficiency, in contrast to the markedly elevated levels in classic 21-hydroxylase deficiency. Although no definite cutoff has been established, the 17-OHP to androstenedione ratio may provide a useful biochemical clue to impaired 17,20-lyase activity (80.6 in case 1 and 90.6 in case 2). With the advent of LC-MS/MS, rare and complex DSDs can be accurately diagnosed using detailed steroid metabolite profiling [2].

Genetic analysis identified a novel homozygous missense variant in exon 2 of the CYB5A gene, c.140G>T (p.Gly47Val), resulting in substitution of glycine by valine at codon 47. This residue is highly conserved across species. The variant is absent from population databases (1000 Genomes, gnomAD), suggesting a novel variant. In silico prediction tools (Sorting Intolerant From Tolerant [SIFT], likelihood ratio test [LRT], MutationTaster) predicted the variant to be deleterious. Initially reported as a VUS, segregation analysis showed the affected sibling to be homozygous, while both parents were heterozygous carriers, consistent with autosomal recessive inheritance. Based on ACMG criteria (PM2, PP3, PP1) and supportive clinical phenotype, the variant was reclassified as likely pathogenic. Despite carrying the same homozygous genetic variant, the 2 cases demonstrated different phenotypic presentations, highlighting that residual enzyme activity may influence the clinical phenotype more than the genotype alone. The second case had asymmetric genitalia with left palpable gonad (Fig. 2B).

Hematological manifestations are uncommon in DSDs; however, when present, they may point toward a rare underlying etiology, such as ATRX mutation in α-thalassemia or CYB5A deficiency in methemoglobinemia. The cytochrome b5 is expressed not only in steroidogenic tissues such as the adrenal glands and gonads but also in erythrocytes, where it facilitates reduction of methemoglobin [9]. Methemoglobinemia results from oxidation of hemoglobin iron from the ferrous (Fe²⁺) to ferric (Fe³⁺) state, impairing oxygen delivery to tissues. Under normal conditions, methemoglobin levels are maintained below 1% to 2% by the reduced nicotinamide adenine dinucleotide (NADH)–cytochrome b5 reductase system. Clinical symptoms usually occur when methemoglobin levels exceed 15%. However, patients with comorbidities such as anemia, cardiovascular disease, lung disease, sepsis, or other abnormal hemoglobin variants (eg, carboxyhemoglobin, sulfhemoglobin, or sickle hemoglobin) may develop moderate to severe symptoms at methemoglobin levels as low as 5% to 8%. Both cases remained asymptomatic without cyanosis, with methemoglobin levels ranging from 7% to 10%. In asymptomatic individuals with mild methemoglobinemia, exposure to oxidant drugs such as local anesthetics (benzocaine, prilocaine, lidocaine), nitrates, and sulfonamides may precipitate life-threatening severe methemoglobinemia. Hence, methemoglobin estimation must be performed preoperatively in all biochemically proven cases of isolated 17,20-lyase deficiency before any surgical procedures to avoid intraoperative and perioperative preventable anesthetic complications [10].

Gender dysphoria has not been evaluated in detail in isolated 17,20-lyase deficiency; however, most patients maintain the gender identity with the gender assigned at birth. Testicular microlithiasis [11] represents intratubular calcifications within seminiferous tubules and has been reported relatively frequently in 46,XY individuals with this condition and therefore warrants periodic surveillance in patients who have not undergone gonadectomy. Malignancy has not been reported to date in isolated 17,20-lyase deficiency. In individuals raised as female, gonadectomy is generally deferred until after completion of puberty, similar to the management approach in CAIS, and estradiol therapy can be initiated after bilateral gonadectomy for psychosocial and bone health.

Learning points

• All individuals with 46,XY DSD warrant genetic evaluation for identification of precise etiology and to facilitate long-term follow-up, malignancy risk, and timing of gonadectomy.

• The 17-OHP:androstenedione ratio is a promising biochemical marker for the diagnosis of isolated 17,20-lyase deficiency.

• Methemoglobinemia is a key distinguishing feature of cytochrome b5 deficiency, helping differentiate it from CYP17A1 and POR defects causing isolated 17,20-lyase deficiency.

Contributors

All authors made individual contributions to authorship. A.K.M., S.S., K.V., A.C., D.T.V., and S.D. were involved in the diagnosis and management of both the patients. A.K.M. and S.S. were involved in writing and editing the manuscript. All authors reviewed and approved the final draft.

Funding

No public or commercial funding.

Disclosures

None declared.

Informed patient consent for publication

Signed informed consent obtained directly from the patient's relatives or guardians.

Data availability

Original data generated and analyzed during this study are included in this published article or in the data repositories listed in References.