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, , 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 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.

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	1.4
January 2022	Cortisone Acetate 31.25 mg + dexamethasone 0.125 mg daily	Testosterone gel 2%, 10 mg daily	189	36.4	>55.2
May 2022	Cortisone Acetate 25 mg + dexamethasone 0.187 daily	Testosterone gel 2%, 5 mg daily	n.a.	8.0	>55.2
June 2022	Cortisone Acetate 25 mg + dexamethasone 0.187 daily	Testosterone gel 2%, 5 mg daily	36.4	8.3	0.3
September 2022	Cortisone Acetate 25 mg dexamethasone 0.187 daily	Testosterone gel 20 mg/g, 23 mg daily	11.4	1.4	44.0
December 2022	Cortisone Acetate 25 mg + dexamethasone 0.187 mg daily	Testosterone gel 20 mg/g, 23 mg every other day	n.a.	n.a.	1.0
March 2023	Cortisone Acetate 25 mg + dexamethasone 0.125 mg daily	Testosterone gel 20 mg/g, 11.5 mg daily	20.2	3.5	23.2

Mutation	Protein change	Site	Molecular Effect
c.293-13A/C>G	-	Intron 2	Abnormal splicing of the CYP21A2 pre-mRNA, leading to: 1. Skipping of exon 3 during mRNA splicin 2. Retention of intron 2 within the mRNA transcript 3. Cryptic splice sites within intron 2 or exon 3, resulting in a transcript with altered sequence or premature termination codons
c.844G>T	p.Val282Leu	Exon 7	Change in the DNA sequence of the CYP21A2 gene, affecting the structure and function of the enzyme
c.923dup	p.Leu308PhefsTer6	Exon 7	Lead to truncated protein is usually unstable and rapidly degraded within the cell
c.955C>T	p.Gln319Ter	Exon 8	Affect the structure and function of enzyme, altering the enzyme's catalytic activity, substrate binding affinity, or stability
c.1069C>T	p.Arg357Trp	Exon 8	Affect the structure and function of the enzyme, altering the enzyme's catalytic activity, substrate binding affinity, or stability. The specific consequences depend on the location of the amino acid change within the protein structure and its role in enzyme function.

Discussion

This case serves as a seminal illustration of the heterogeneous nature of CAH syndrome resulting from 21-hydroxylase deficiency. The extensive variability of the locus is well-documented, with several genetic variants now recognized as pathogenic19. The phenotype is determined by the type of mutation detected and the number of mutations. Specifically, some mutations are linked to the most severe phenotype, such as SW-CAH, while others are associated with milder forms. Although encountering multiple mutations within a single gene is common, cases with more than three mutations are rarely reported. It might be expected that a greater number of mutations would correlate with increased severity of the clinical presentation. However, the phenotype was moderate, characterized by an SV form, despite the identification of five different heterozygous mutations in the same individual, three of which are known to be associated with SW-CAH. Thus, in our case, the severity of the phenotype does not align with the detected genotype. The literature largely demonstrates that understanding genotype-phenotype relationships in CAH is crucial for accurate diagnosis, predicting disease severity, and tailoring treatment strategies to improve patient outcomes20, 21. Identifying multiple mutations responsible for CAH in a single patient allows for precise diagnosis, personalized treatment plans, and improved patient outcomes2, 22. This genetic information helps tailor glucocorticoid and mineralocorticoid dosing, predict enzyme activity, and manage adrenal crisis risks more effectively, optimizing hormone levels, reducing complications, and enhancing quality of life, including better psychological well-being and social integration. Additionally, genetic counseling for families becomes more accurate, aiding in carrier detection and informed family planning. Overall, detailed genetic insights contribute to the advancement of research and the development of targeted therapies.

The genotype of our patient is notably peculiar. Specifically, the first mutation detected in the maternal-origin allele, the CYP21A2 c.293-13A/C>G (rs6467), is a point mutation within intron 2, already described as causative for both SW and SV-CAH forms23. The correlation between this variant and the clinical phenotype remains incompletely understood. One possible mechanism is the synthesis of a small amount of correctly spliced product, hypothesized to activate a cryptic upstream three-splice acceptor site, causing aberrant splicing in an inconsistent manner and allowing for some correct splicing of mRNA capable of mitigating the severity of the disease10. The other four mutations detected in our patient are located on the second allele. The CYP21A2 c.844G>T p.Val282Leu mutation, resulting in an amino acid substitution in the I-helix region of the final protein chain, leads to steric collisions, reducing enzyme activity24, 25. This variant, supported by functional studies, is considered pathogenic for CAH 26. The CYP21A2 c.923dup p.Leu308PhefsTer6 mutation involves the insertion of a nucleotide, resulting in a premature termination codon and complete loss of 21-hydroxylase enzyme activity27. The CYP21A2 c.955C>T p.Gln319Ter mutation causes a nonsense mutation, resulting in an inactive protein3, 4. The CYP21A2 c.1069C>T p.Arg357Trp mutation describes a pathogenic variant associated with the SW phenotype28, 29, 30. Despite three of the five mutations being pathognomonic for SW-CAH, only an SV form was identified, indicating that the clinical manifestation is primarily influenced by residual enzymatic activity. Hence, given CAH resulting from 21-hydroxylase deficiency is a recessive disorder, the phenotype of each patient reflects the allele with the lesser degree of impairment1.

A total of 1,248 genetic variants causative for CAH are cataloged, with 460 affecting the translated region, and 52 mutations associated with classic CAH, primarily the SW form13. In our patient, three of these mutations were detected, although the phenotype did not manifest as severely as expected. It is probable that the combined effect of these mutations resulted in a different residual enzymatic activity, moderating the clinical presentation. Twenty-seven out of 35 associated mutations described in the literature are linked to SW-CAH, underscoring the potential additive effect of mutations on the clinical phenotype, which was not observed in our patient. Thus, our case report confirms that different phenotypes can be presented in patients with identical compound mutations24, 31, 32.

As a further complication, our patient experiences significant challenges in achieving testosterone serum levels within reference ranges during androgen replacement therapy. The absence of androgens renders testosterone serum levels nearly undetectable, as expected in the absence of testicles. Conversely, when low to intermediate doses of testosterone replacement therapy are administered, testosterone serum levels exceed the upper limit of reference ranges. This pattern persists across different testosterone formulations, suggesting that challenges in achieving adequate testosterone serum levels are not attributable to differences in transdermal absorption related to excipients. While the patient underwent a psychiatric evaluation in the past, which did not reveal any pathological features, further investigation into therapeutic compliance was not pursued. Therefore, we cannot definitively exclude non-compliance with androgen therapy, currently the most probable hypothesis.

The most intriguing aspect of the described case is the unique phenotype seemingly disconnected from the genotype. To the best of our knowledge, we are unable to justify this genotype/phenotype misalignment. Nonetheless, we could hypothesize that the presence of three copies of the gene, instead of the typical two, could play a significant role in determining the phenotype.

Conclusion

This case presentation should serve as the starting point for future research aimed at advancing our understanding of genotype-phenotype relationships in CAH. Enhancing this understanding will facilitate more precise genetic counseling for CAH patients and improve the personalization of therapy. These efforts are essential for developing novel targeted therapies aimed at enhancing patient management and quality of life.

Abbreviations

ACTH - Adrenocorticotropic Hormone, CAH - Congenital Adrenal Hyperplasia, CT - Computed Tomography, CYP21A2 - Cytochrome P450 Family 21 Subfamily A Member 2 (a gene associated with CAH), DSD - Disorder of Sexual Differentiation, LC-MS/MS - Liquid Chromatography with Tandem Mass Spectrometry, MLPA - Multiplex Ligation-dependent Probe Amplification, MRI - Magnetic Resonance Imaging, PCR - Polymerase Chain Reaction, SV - Simple Virilizing, SW - Salt-Wasting, 17-OHP - 17-Hydroxyprogesterone

Acknowledgments

None.

Author’s contributions

GA, GS, SV, EM and DS performed the clinical evaluation of the patient. GA, GS and DS wrote the manuscript. SM performed genetic analyses. AF, ARM and MS revised the manuscript. All authors read and approved the final manuscript.

Funding

None.

Availability of data and materials

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Competing interests

The authors declare that they have no competing interests.