Two Cases of 46,XY Differences of Sex Development Due to Gonadal Dysgenesis Associated With Novel NR5A1 Variants

Abstract

Differences of sex development (DSDs) are rare conditions with atypical chromosomal, gonadal, or anatomical sex. We describe 2 cases of 46,XY DSD due to complete gonadal dysgenesis, a 16-year-old female and a 45-year-old female, who both presented with primary amenorrhea and hirsutism. The 16-year-old had tall stature and normal-appearing female external genitalia but absent female secondary sex characteristics. Hormonal assessment revealed hypergonadotropic hypogonadism. The uterus appeared normal on transabdominal ultrasound. Testing revealed 46,XY karyotype and SRY+ using fluorescence in situ hybridization (FISH). 46,XY DSD genetic testing found a novel, heterozygous, likely pathogenic NR5A1 variant (NM_004959.5:c.1166_1180del). Low-dose transdermal estradiol was commenced for pubertal induction, planned for gradual uptitration before progestogen introduction. The 45-year-old female had osteoporosis and had been taking the oral contraceptive pill (OCP) long-term for previously misdiagnosed Turner syndrome. She had normal-appearing female external genitalia, tall stature, and no Turner syndrome features. Hormonal assessment was confounded by previous gonadectomy and OCP use. Testing revealed 46,XY karyotype and SRY+ using FISH. 46,XY DSD genetic testing revealed a novel, heterozygous, likely pathogenic variant of NR5A1 (NM_004959.5:c.489del). Hormone therapy was changed to transdermal estradiol/progestogen. These cases highlight 2 novel NR5A1 variants associated with 46,XY DSD.

Introduction

Normal male sex development can be broadly understood as the consequence of 2 processes which occur in utero: sex determination, in which testes form from the primitive, bipotential gonads as a result of the complex interplay between numerous transcription factors and cells, and sex differentiation, in which male internal and external genitalia differentiate as a result of hormones secreted by the fetal testis [1].

Differences of sex development (DSD) describes a group of rare conditions in which development of chromosomal, gonadal, or anatomical sex is atypical [2]. These conditions can result from a disturbance at any stage of normal male sex development. 46,XY DSDs encompass a spectrum of conditions, relating to the degree of androgenization that occurs in a 46,XY individual. Affected individuals can present with micropenis, atypical, or female external genitalia. Müllerian structures (embryonic precursor of female reproductive organs) can be present or absent [1].

Gonadal dysgenesis, initially termed “Swyer syndrome” after Gerald Swyer who first described the condition in the mid-1950s, is a form of 46,XY DSD [3]. This condition results from an early defect in testis development, leading to complete or partial gonadal dysgenesis. Phenotype ranges from female external and internal genitalia, normal-tall stature, and a lack of female secondary sexual characteristics in the complete form to a spectrum of atypical genitalia with or without Müllerian structures in the partial form [1].

Nuclear receptor subfamily 5 group A member 1 (NR5A1), is a gene located on chromosome 9q33.3 and is commonly known by the protein it encodes: steroidogenic factor-1 (SF-1). SF-1 plays an important role in sex determination via gonadal development, as well as adrenal development and steroidogenesis [4]. Mutations in NR5A1/SF-1 have led to both 46,XY and 46,XX gonadal dysgenesis [4], adrenal insufficiency [4], splenic abnormalities [5], and other organ malformations [6]. NR5A1 variants are a common and increasingly recognized cause of DSD, with a prevalence of 14.7% in a recent Ukrainian DSD register [7]. Here, we present 2 adults from Australia with complete gonadal dysgenesis due to NR5A1 variants.

Case Presentation

Case 1

A 16-year-old Caucasian female presented to an endocrinology outpatient clinic for assessment of primary amenorrhea (PA). She underwent normal childhood development and developed pubic, axillary, and facial hair at age 11. She was the offspring of nonconsanguineous parents. She had 2 female siblings with normal ages of menarche.

Case 2

A 45-year-old female of Indian heritage presented to an Australian endocrinology outpatient clinic for management of PA and osteoporosis. She had a history of PA and hirsutism, troubled by facial hair. She underwent bilateral gonadectomy at age 13 in India. She has 1 child, conceived through surrogacy. She had previously been given a diagnosis of Turner syndrome (45,XO) and was managed with the combined oral contraceptive pill (OCP) (ethinylestradiol 20 mcg/drospirenone 3 mg) daily.

Diagnostic Assessment

Case 1

For case 1, a physical examination revealed a tall stature of 170 cm, which was taller than the midparental height of her father and her 20-year-old brother. Her weight was 108 kg, with a body mass index of 37 kg/m². She was Tanner stage 2 for breast development, Tanner stage 4 for pubic hair, and had normal-appearing female external genitalia. There was mild hirsutism with recently shaved facial hair. There were no cushingoid features.

Laboratory testing revealed hypergonadotropic hypogonadism, mildly elevated testosterone levels, and normal adrenal androgens (Table 1). Electrolytes, thyroid function testing, and prolactin levels were normal.

Table 1.

Serum hormone measurement results for case 1 and case 2 using female reference ranges

Serum hormone measurement	Case 1	Case 2	Reference range
LH	22.0 IU/L	1.9 IU/L	1.5-10.0 IU/L
FSH	50.0 IU/L	4.6 IU/L	2.0-12.0 IU/L
Estradiol	<50.0 pmol/L (<13.6 pg/mL)	<18.0 pmol/L (<4.9 pg/mL)	FP: <332.0 pmol/L (<90.4 pg/mL) LP: 125.0-1300.0 pmol/L (34.0-354.1 pg/mL)
Progesterone	<0.5 nmol/L (<0.2 ng/mL)	<0.2 nmol/L (<0.1 ng/mL)	FP: 0.3-4.0 nmol/L (0.1-1.3 ng/mL) LP: 5.5-90.0 nmol/L (1.7-28.3 ng/mL)
Testosterone	3.1 nmol/L (89.4 ng/dL)	<0.1 nmol/L (<2.9 ng/dL)	0.2-1.8 nmol/L (5.8-51.9 ng/dL)
SHBG	25.0 nmol/L (2.4 µg/mL)	Not available	30.0-110.0 nmol/L (2.9-10.5 µg/mL)
17-hydroxyprogesterone	2.6 nmol/L (85.9 ng/dL)	0.6 nmol/L (19.8 ng/dL)	0.3-5.8 nmol/L (9.9-191.7 ng/dL)
DHEAS	5.9 µmol/L (217.4 µg/dL)	2.1 µmol/L (77.4 µg/dL)	1.0-14.0 µmol/L (36.8-515.8 µg/dL)

Abbreviations: DHEAS, dehydroepiandrosterone-sulfate; FP, follicular phase; LP, luteal phase.

Transabdominal pelvis ultrasound showed a normal-sized uterus measuring 62 × 33 × 31 mm, with a thin 3.8 mm endometrial lining (Fig. 1). Ovarian structures were not confidently identified within the limitations of a transabdominal scan.

Figure 1.

Transabdominal ultrasound images for case 1, demonstrating (A) the presence of a uterus (measuring 62 × 33 × 31 mm) with thin endometrial lining (measuring 3.8 mm); (B) right gonad and (C) left gonad.

The karyotype was 46,XY. The presence of SRY was confirmed by FISH. Peripheral blood for next-generation sequencing 46,XY DSD gene panel (PanelApp Australia version 1.8) found a novel, heterozygous, likely pathogenic variant of NR5A1 (NM_004959.5:c.1166_1180del). This resulted in an in-frame deletion of 5 amino acids (p.Leu389_Ala393del) at the ligand binding domain of the SF-1 protein. Other missense and frameshift variants surrounding this in-frame deletion have been reported in DSD cases, highlighting the importance of this region to protein function.

Case 2

For case 2, a physical examination revealed tall stature for the family with a height of 165 cm. Her weight was 77 kg with a body mass index 28.3 kg/m². There was no evidence of webbed neck, high-arched palate, or wide-spaced nipples. Precordial auscultation revealed dual heart sounds with no murmurs. Examination revealed normal-appearing female external genitalia.

Laboratory testing was completed whilst the patient continued to take the OCP and revealed hypogonadism with low-normal gonadotropins, undetectable testosterone, and normal adrenal androgens (Table 1). Transabdominal pelvis ultrasound showed a small uterus measuring 52 × 23 × 26 mm, with a thin 2.8 mm endometrial lining and absent gonads consistent with a history of resection (Fig. 2).

Figure 2.

Transabdominal ultrasound images for case 2, demonstrating (A) small anteverted uterus (measuring 52 × 23 × 26 mm) with thin endometrial lining (measuring 2.8 mm); (B) absent right gonad and (C) absent left gonad (consistent with history of previous bilateral gonadectomy).

A dual-energy X-ray absorptiometry scan demonstrated osteoporotic bone density at the L1-4 spine (T-score −2.8; Z-score −3.2) and osteopenia at left femoral neck (T-score −1.4; Z-score −1.5) and left total femur (T-score −1.2; Z-score −1.4).

Given the lack of clinical features consistent with Turner syndrome, karyotyping was performed. The karyotype was 46,XY. The presence of SRY was confirmed by FISH. Peripheral blood for a next-generation sequencing 46,XY DSD gene panel found a novel, heterozygous, likely pathogenic variant of NR5A1 (NM_004959.5:c.489del). This resulted in a frameshift leading to the introduction of a premature stop codon 132 residues downstream from the site of change in the hinge region of the SF-1 protein (p.Phe164Leufs*132). The variant mRNA transcript is expected to undergo nonsense-mediated decay [8, 9], resulting in loss of function.

Treatment

Case 1

Case 1 identified as a heterosexual female, desiring breast development and menstruation. Pubertal induction was commenced using transdermal estradiol patch 12.5 mcg/24 hours applied twice weekly. Plans were made to gradually increase the dose over the next 12 months and to introduce progesterone 1 to 2 years into treatment to optimize breast development and for endometrial protection. Clinical psychologist support was arranged throughout diagnosis and treatment.

Case 2

Given the new diagnosis in case 2, hormone therapy was changed from the OCP to transdermal patch estradiol 50 mcg/norethisterone acetate 140 mcg per 24 hours applied twice weekly.

Outcome and Follow-up

Case 1

Follow-up has been difficult in case 1 due to patient relocation. Appropriate psychological review, genetic counseling, and specialist referrals were made. Plans were made for baseline bone mineral density measurement and pelvic magnetic resonance imaging, with consideration of prophylactic gonadectomy in the future to minimize the risk of gonadoblastoma.

Case 2

Bone health will be reassessed in case 2 following a period of treatment with transdermal estradiol.

Discussion

We present 2 unique cases of 46,XY DSD with novel variants in NR5A1 who presented with PA (Table 2). Karyotype is an important investigation for patients with PA associated with hypergonadotropic hypogonadism (abnormal in approximately 70%; mostly Turner syndrome, less commonly 46,XY DSD) or absent Müllerian structures (abnormal in approximately 6%; usually complete androgen insensitivity syndrome) [10, 11]. In our cases, the presence of Müllerian structures and female external genitalia in the presence of 46,XY indicates both anti-Müllerian hormone (AMH)-mediated Müllerian duct regression and androgen-mediated male genitalia differentiation have not occurred (Fig. 3) [1]. This can be explained by defective sex determination leading to an absent testis or complete gonadal dysgenesis. In 46,XY patients with incongruent karyotypes, FISH is performed to detect SRY (known as the testis-determining gene), which is crucial for testis formation [1]. The presence of 46,XY and SRY in the absence of testes should prompt 46,XY DSD genetic testing.

Table 2.

A summary of the clinical, biochemical, and genetic characteristics of case 1 and case 2

	Case 1	Case 2
Age (years)	16	45
Clinical presentation	Primary amenorrhea; failure to progress through female puberty; tall stature; mild hirsutism	Primary amenorrhea and osteoporosis; tall stature; significant hirsutism; previous misdiagnosis of Turner syndrome
External genitalia	Female	Female
Uterus	Present	Present
Hormone assessment	Hypergonadotropic hypogonadism; testosterone 1.5 × female reference range	Confounded by oral contraceptive pill use and previous gonadectomy
Karyotype	46,XY (SRY+)	46,XY (SRY+)
Genetic testing	Heterozygous NR5A1 variant (NM_004959.5:c.1166_1180del)	Heterozygous NR5A1 variant (NM_004959.5:c.489del)

Figure 3.

A simplified representation of the molecular events and role of NR5A1 in normal male development. NR5A1 expression at the urogenital ridge contributes to bipotential gonad, kidney, and adrenal cortex organogenesis. The bipotential gonad also expresses NR5A1, which, in the presence of an SRY-containing Y chromosome, upregulates SRY expression in pre-Sertoli cells. SRY subsequently upregulates SOX9, which leads to a cascade of activation of testis genes, stimulating differentiation of the bipotential gonad to a fetal testis. AMH secreted by testis Sertoli cells causes degradation of Müllerian duct structures. NR5A1-mediated testosterone synthesis by testis Leydig cells causes AR-mediated Wolffian duct development. Testosterone is converted by 5α-reductase type 2 to DHT, which causes AR-mediated prostate, penis, and scrotal development. NR5A1 defects can impair sex determination, with impaired AMH and testosterone secretion from the resultant dysgenetic gonad. In complete gonadal dysgenesis, the testis is absent, and profound deficiency in AMH and testosterone results in female phenotype, with persistence of Müllerian duct structures and lack of male genital development.

Abbreviations: AMH, anti-Müllerian hormone; AR, androgen receptor; DHT, dihydrotestosterone; NR5A1, nuclear receptor subfamily 5 group A member 1.

Notably, case 1 exhibited testosterone levels similar to early-pubertal boys [12], congruent with complete gonadal dysgenesis [1], and case 2 exhibited undetectable testosterone due to previous gonadectomy. Adrenal androgens were not elevated in either case, although testing in case 2 may have been confounded by OCP use. The source of high testosterone in case 1 may be related to her dysgenetic gonads and exacerbated by underlying metabolic syndrome. Tall stature in both cases can be explained by delayed growth plate fusion secondary to reduced estrogen exposure [13], in the presence of 2 copies of the sex-chromosome located short-stature homeobox (SHOX) gene (in contrast to Turner syndrome) [14]. Serum AMH was not measured in case 1 due to cost. The absence of AMH action was inferred by the presence of Müllerian duct structures. If measured, AMH is expected to be undetectable, due to the absence of functional Sertoli cells in complete gonadal dysgenesis [15, 16]. In other 46,XY DSD cases, normal or elevated AMH is consistent with other etiologies, such as androgen insensitivity or androgen synthesis defects [15, 16].

NR5A1 variants are genetically complex, characterized by incomplete penetrance, heterogenous phenotype, and a lack of genotype-phenotype correlation [6, 17]. Neither NR5A1 mutation described in our cases (Fig. 4) has, to our knowledge, been previously reported. In an international study of 197 individuals with NR5A1 gene mutations, 93 of the 113 index cases (82%) had different variants, with the majority located in the ligand-binding domain (41%) (case 1) or the DNA-binding domain (38%) of the SF-1 protein [6]. Hinge-region variants (case 2) accounted for only 11% of variants. Only 25% of relatives with the same variant as an index case had a DSD phenotype. Neither of our cases had a family history of DSD. Furthermore, while NR5A1 knockout in mice results in adrenal and gonadal agenesis, male-to-female sex reversal with present Müllerian structures and spleen hypoplasia [18], NR5A1 variants in human studies are mostly (94%) heterozygous [6]. In the Kouri et al cohort, organ abnormalities were identified in the spleen (15%), central nervous system (5%), and adrenals (4%), although 3 of the 5 patients with adrenal abnormalities were homozygous [6].

Figure 4.

A representation of the NR5A1 gene and location of the variants identified in case 1 and case 2.

Management of patients with DSD is challenging. A multifaceted approach is recommended. Given the heterogeneity of 46,XY DSDs and the complex medical and psychosocial issues, Wisniewski et al recommend multidisciplinary team management incorporating fertility, sexual and genetic counseling, mental health, nursing, endocrinology, and surgical specialty input [19]. For case 1, low-dose transdermal estradiol without progesterone was prescribed to mimic normal female puberty as closely as possible, in which the majority of breast development occurs in the 2 years of unopposed exposure to estrogens prior to menarche [20]. Following appropriate development, the combination of topical estradiol with daily progesterone is preferred due to the favorable effects of transdermal formulations on bone and venous thrombosis risk [21].

In DSDs, the risk of gonadal neoplasm depends on the etiology. The presence of gonadal Y chromosome material (particularly the gonadoblastoma Y locus), intraabdominal gonadal location, and earlier stage of disruption of Sertoli cell differentiation are associated with higher tumor risk [1, 22]. Gonadal neoplasm risk in 46,XY DSD due to gonadal dysgenesis is estimated to be 15% to 50% in the complete form and 16% to 30% in the partial form, compared to <5% in complete androgen insensitivity syndrome and ovotesticular DSD [22]. Most neoplasms are gonadoblastomas [23] and occur after puberty [22], although prepubertal cases have been reported [24]. Optimal timing of gonadectomy remains controversial; some recommend surgery at puberty given low prepubertal risk, rather than in infancy [22]. Management should therefore be individualized, and consideration should be given to phenotype, location of gonads, and assigned sex [25]. In complete gonadal dysgenesis, gonadectomy at diagnosis is generally recommended [22, 25]. The indication for gonadectomy in case 2 is unclear; however, it can be theorized that case 2 may have been misdiagnosed as Turner syndrome with Y chromosome mosaicism and prophylactic gonadectomy performed due to the risk of gonadal tumor. Access to prior medical records was not available.

In summary, we describe 2 cases of 46,XY DSD due to 2 novel NR5A1 gene mutations, highlighting the clinical complexity associated with these conditions.

Learning Points

• Karyotype is useful for the assessment of PA associated with hypergonadotropic hypogonadism or absent uterus and may diagnose a 46,XY DSD.

• Genetic testing should be considered in SRY+ 46,XY cases with absent testes.

• 46,XY DSDs due to NR5A1 mutations can result in complete gonadal dysgenesis, female phenotype, and hypogonadism.

• A patient-centered multidisciplinary team can help guide complex management decisions surrounding pubertal induction, bone health, gonadal malignancy, and psychosocial issues in these patients.

Contributors

All authors made individual contributions to authorship. J.V.G. is the primary author of this manuscript, with principal contribution to case write-up and literature review, with M.W. and A.W. serving as supervisors. M.W. was involved in the diagnosis and management of case 1. A.W. was involved in the diagnosis and management of case 2. P.L.C. and S.Z. carried out the molecular genetic testing for both cases and were involved in the interpretation of the genetic test results. All authors reviewed and approved the final draft.

Funding

No public or commercial funding.

Disclosures

None declared.

Informed Patient Consent for Publication

In case 1, signed informed consent could not be obtained from the patient or a proxy but has been approved by the treating institution. Informed patient consent was obtained directly from the patient in case 2.

Data Availability Statement

Original data generated and analyzed during this study are included in this published article.