Abstract

Background: Classical congenital adrenal hyperplasia is a genetic disorder characterized by defects in the steroidogenesis cascade, mainly due to 21-hydroxylase enzyme deficiency. The phenotype can vary from the most severe salt-wasting syndrome to the less severe simple-virilizing form. The genotype-phenotype correlation is complex, and it is expected that the higher the number of mutations detected, the worse the phenotype would be. Moreover, when more than one mutation occurs in the same patient, the phenotype should be the result of the most severe mutation detected.

Case Presentation: This is the case of a 49-year-old Caucasian man with simple virilizing congenital adrenal hyperplasia, diagnosed at two years of age due to neonatal presentation of ambiguous genitalia. The karyotype was 46, XX, the phenotype was male, and hormonal evaluations highlighted cortisol deficiency, which required cortisone acetate replacement therapy. Gonads were removed during infancy, surgical interventions were performed to align physical attributes with his male gender identity, and the patient underwent testosterone replacement therapy. During adulthood, while compensation for cortisone acetate was reached, managing testosterone therapy proved to be challenging, achieved after the introduction of a low dose of dexamethasone (0.125 mg daily) and testosterone gel 2% 30 mg daily. Genetic analysis unraveled five different heterozygous pathogenic variants on the CYP21A2 gene. One mutation was detected on the maternal allele, while the remaining four were found on the second allele. Three of these mutations were proven to be pathognomonic for the salt-wasting form.

Conclusion: This case underscored the intricate and heterogeneous correlation between genotype and phenotype in simple-virilizing congenital adrenal hyperplasia, illustrated by a patient with a rare occurrence of five distinct pathogenic mutations on the CYP21A2 gene. Although three of the five mutations detected are related to the salt-wasting congenital adrenal hyperplasia, only a simple-virilizing form was detected here. Moreover, although it is supposed that the higher the number of mutations detected, the worse the phenotype should be, here we described a simple-virilizing form in a patient with five mutations on the CYP21A2 gene.

Introduction

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder characterized by a defect in the enzymatic steroidogenic cascade, which impairs the physiological production of both cortisol and aldosterone. The most common enzyme altered in the pathogenesis of CAH, accounting for approximately 90-95% of all cases, is microsomal P450c21. This enzyme catalyzes the 21-hydroxylation of progesterone into deoxycorticosterone and of 17-hydroxyprogesterone (17-OHP) into 11-deoxycortisol, serving as precursors for mineralocorticoids and glucocorticoids, respectively1, 2. When this enzyme is deficient or ineffective, precursors such as 17-OHP accumulate, along with lesser amounts of progesterone, 17-hydroxypregnenolone, and androstenedione, leading to a varying degree of deficiency in the final compounds, cortisol, and aldosterone1, 3.

The overall prevalence of CAH is estimated to range from 1 in 10,000 to 1 in 20,000 live births worldwide, although geographical and ethnic variations exist. Based on the severity of enzyme dysfunctions, two recognized forms of CAH exist: classic and non-classic. Classic CAH is estimated to occur in approximately 1 in 15,000 to 1 in 16,000 live births worldwide4, 5 and can be further categorized into two clinical presentations: salt-wasting (SW) and simple virilizing (SV) forms4. SW CAH, representing 75% of classic cases, is associated with the most severe clinical manifestations due to the complete absence of 21-hydroxylase activity, resulting in total deficiency of cortisol and aldosterone production and thus a lack of hormones with mineralocorticoid activity. This phenotype is compounded by hyperandrogenemia, leading to the accumulation of precursors with androgenic activity, potentially causing sexual ambiguity in affected female newborns. In contrast, the SV form of CAH is characterized by reduced enzymatic activity, estimated to be less than 2%, resulting in varying degrees of hyperandrogenemia. This can lead to external genital virilization in female newborns and precocious puberty in males, without overt cortisol/aldosterone deficiency1, 6. Classic CAH may interfere with genital system development and is classified as a disorder of sexual differentiation (DSD)7. Alongside classic forms, non-classic forms are prevalent, affecting approximately 0.1 – 0.2% of the general Caucasian population (1). Non-classic CAH is less severe, as genetic mutations do not completely impair 21-hydroxylase activity, which remains preserved at 20-60% compared to physiological levels8. Symptoms of non-classic CAH are primarily associated with hyperandrogenism and are typically diagnosed during adolescence or young adulthood in females, who may experience oligomenorrhea, infertility, and hirsutism, while diagnosis in males can be challenging4, 9, 10. The therapeutic approach to CAH involves hormone replacement therapy, management of symptoms, and addressing potential complications11. The primary goals are to normalize hormone levels, manage electrolyte imbalances, and promote normal growth and development. Considering the high variability in the CAH phenotype, this treatment must be highly individualized and could involve hormone replacement therapy (with glucocorticoids and mineralocorticoids), androgen suppression, surgical interventions, and psychosocial support. This latter should be accurately evaluated since CAH can impact the quality of life substantially and in multifaceted ways12. Indeed, the diagnosis and management of CAH is well-known to affect physical health, psychological well-being, social interactions, and overall life satisfaction. Obviously, the extent of this impact varies depending on the severity of the condition, effectiveness of treatment, and individual coping mechanisms, but requires accurate evaluation from the clinician.

The 21-hydroxylase gene, CYP21A2, consists of 10 exons13 and is located on chromosome 6p21.34, 14. In the literature, several gene variations associated with CAH are described, including large deletions, gene conversions, insertions, and single-nucleotide variants9, 10, 15. The continual discovery of new CYP21A2 mutations has led to the identification of over 300 mutations to date13, 16. Although a strong correlation between genotype and phenotype is suggested, it is not universally confirmed13. The clinical presentation is typically heterogeneous and complicated by the occasional detection of more than one mutation on the same allele, occurring in 5-6% of cases17. Numerous mutations have been described in patients with CAH, and it is even possible for multiple mutations to coexist in the same patient, potentially leading to a more severe phenotype4. While case reports of two to three concomitant mutations are relatively common in the literature, finding more than three mutations in the same gene is rare4, 16.

Here, we present the case of a patient with five different heterozygous pathogenic variants in the CYP21A2 gene, resulting in an SV form of CAH.

Case Presentation The Diagnosis and Childhood

A 49-year-old man presented at the Unit of Endocrinology in Modena, Italy, in 2022 with a previously established diagnosis of CAH. Inquiring about his medical history revealed that he was the only child of non-consanguineous healthy parents. During infancy, he attended medical consultations at the Pediatrics department in Modena due to ambiguous genitalia. At the age of two, a suspicion of SV CAH syndrome arose, as indicated by a 46, XX karyotype and the absence of signs or symptoms of the SW form during the perinatal period. Biochemical examinations revealed reduced cortisol serum levels, prompting the initiation of corticosteroid replacement therapy. The therapy was adjusted to maintain cortisol serum levels within reference ranges and prevent androgen excess. Throughout childhood, the patient was raised as male and underwent several surgical procedures for sex reassignment. Specifically, at the age of nine, he underwent partial removal of his vagina and complete removal of his uterus and ovaries at the University Hospital of Liege in Belgium. Subsequently, hypospadias correction surgery was performed, and testicular prostheses were implanted. Starting at the age of ten, replacement androgen therapy was added to the corticosteroid treatment to induce male pubertal development. During adolescence, the patient also underwent lower limb elongation surgery to address short stature. The patient's subjective gender identity has consistently been male.

Table 1.

Hormonal examinations performed during endocrinological evaluations, according to both steroid and androgen replacement therapies

Date Steroid therapy Androgen therapy ACTH (pg/mL) 17-OHP (nmol/L) Total Testosterone (nmol/L) Reference ranges 4.3-52 1.21-7.23 7.29 – 23.6 July 2018 Cortisone Acetate 25 mg daily Testosterone gel 2%, 60 mg daily 69 5.5 >55.2 November 2018 Cortisone Acetate 25 mg daily Testosterone gel 2%, 30 mg daily 56 n.a. 1.7 April 2019 Cortisone Acetate 25 mg daily Testosterone gel 2%, 30 mg daily 75 11.4 5.2 November 2019 Cortisone Acetate 18.75 mg daily Testosterone gel 2%, 30 mg daily 151 62.0 53.0 June 2020 Cortisone Acetate 25 mg daily Testosterone gel 2%, 20 mg daily 171 52.0 12.1 January 2021 Cortisone Acetate 25 mg daily Testosterone gel 2%, 20 mg daily 187 76.3 33.9 September 2021 Cortisone Acetate 31.25 mg daily Testosterone gel 2%, 20 mg daily 157 152.6 >55.2 November 2021 Cortisone Acetate 31.25 mg daily Androgen withdrawal 189 101.6