The activity of Cytochrome P450 side-chain cleavage enzyme (CYP11A1, EC 1.14.15.6) is the prerequisite for all mineralocorticoid, glucocorticoid, and sex steroid biosynthesis as it catalyzes its first step, the conversion of cholesterol to pregnenolone ⁽¹⁾. It is encoded by the CYP11A1 gene, localized on the long arm of chromosome 15 (15q23–q24). CYP11A1 synthesizes pregnenolone from cholesterol in three consecutive catalytic steps: 20α-hydroxylation, 22R-hydroxylation, and cleavage of the C20–C22 carbon side chain of cholesterol ⁽¹⁾.

Only 11 patients with CYP11A1 deficiency (OMIM #118485) have been reported so far. The clinical phenotype is similar to congenital lipoid adrenal hyperplasia (CLAH; OMIM #201710) that is caused by deficient mitochondrial cholesterol import due to mutations in the steroidogenic acute regulatory protein (STAR) ⁽²⁾. Severe CYP11A1 deficiency manifests with female external genitalia irrespective of chromosomal sex and with early onset adrenal insufficiency, usually manifesting within the first hours or days of life ^{(3, 4)}. A milder form of CYP11A1 deficiency has also been described, associated with delayed onset of adrenal insufficiency and variable degrees of 46,XY disorder of sex development (DSD) ^{(5, 6, 7, 8, 9)} or also normal male genital development ⁽¹⁰⁾. Adrenal glands are normal size or absent in CYP11A1 deficiency ^{(10, 11)}, in contrast to CLAH ^{(2, 12, 13, 14)}. In vitro expression studies assessing the residual activity of mutant CYP11A1 suggest that, overall, the genotype correlates well with the degree and onset of adrenal insufficiency ⁽¹⁰⁾.

Herein, we report a case of delayed diagnosis of adrenal insufficiency in a patient manifesting at birth with severe penoscrotal hypospadias and inadequate adrenal steroidogenesis caused by two novel CYP11A1 mutations. Our data highlights the importance of considering adrenal insufficiency in the differential diagnosis of 46,XY DSD patients. Adrenal function should be assessed in cases with suspicion of adrenal insufficiency to prevent life-threatening adrenal crises.

As a first step, based on the incidence of mutations in SF1 or STAR associated with AI and 46,XY DSD, the mutation analysis of the SF1 and STAR genes was performed. No disease-causing mutations were found in either of these candidate genes. The CYP11A1 mutation analysis was performed as the next diagnostic procedure. Two novel CYP11A1 mutations were found: a cytosine to thymine transition at nucleotide position 27 921 (c.1078C>T, p.R360W) on the maternal allele and a cytosine to thymine transition at nucleotide position 28 327 (c.1213C>T, p.R405X) on the paternal allele.

The p.R360W mutation retained 38 and 30% of wild-type CYP11A1 in vitro activity for the conversion of cholesterol and 22R-hydroxycholesterol to pregnenolone respectively (Fig. 1A). Western blot analyses consistently showed lower protein levels for the p.R360W mutation than for the wild-type, suggesting that p.R360W may impair protein stability, also indicated by consistent evidence of protein degradation in the mutant protein blots (Fig. 1B).

The arginine residue at position 360 of the CYP11A1 protein is highly conserved across species, but not across mitochondrial cytochrome P450 (CYP) type I enzymes (Fig. 2). This residue is localized at the amino-terminus of the J-helix. Substitution of a positive-charged arginine by an aliphatic nonpolar tryptophan results in a polarity change on the protein surface, but otherwise no major structural changes were apparent (Fig. 1C, D and E).

The arginine at codon 405 is not well conserved (Fig. 2) and localizes to the C-terminal part of the loop between the β2-1 and β2-2 sheets (Fig. 1C). A premature truncation of the protein at this position eliminates protein domains crucial for CYP11A1 activity ⁽¹⁵⁾. Therefore, the novel nonsense p.R405X mutation is predicted to result in a complete loss of CYP11A1 function.

Herein, we present a case of delayed diagnosis of adrenal insufficiency in a patient with penoscrotal hypospadias caused by two novel CYP11A1 mutations, p.R360W and p.R405X. In vitro and in silico analyses were consistent with the clinical findings and confirmed the diagnosis of CYP11A1 deficiency in the patient. This report demonstrates that diagnosis of CYP11A1 deficiency can be challenging and highlights the importance of adrenal function assessment to provide adequate clinical diagnosis and to prevent life-threatening adrenal crises.

Partially CYP11A1 inactivating mutations are usually associated with onset of adrenal insufficiency between the age of 2 and 9 years ^{(7, 8, 9, 10)}. Earlier onset of adrenal symptoms has been reported in a patient compound heterozygote for the mild p.L141W and the severe p.V415E mutations ⁽⁴⁾. Signs and symptoms suggest that adrenal steroidogenesis was already partially impaired in our patient soon after birth, with generalized hyperpigmentation, low cortisol levels, and insufficient cortisol response during a UTI sepsis episode. Furthermore, it is tempting to speculate that the episode of premature labor at 12 weeks of gestation may be due to insufficient placental CYP11A1 activity ⁽¹⁶⁾.

Our current data together with previous findings ⁽⁴⁾ suggest that compound heterozygosity for a mild and a severe mutation may advance onset of signs and symptoms of impaired adrenal function in patients with mild CYP11A1 deficiency. The diagnosis of adrenal insufficiency can be missed by normal baseline cortisol concentrations and lack of adequate adrenal function assessment. Similarly, to our previous report of two siblings with partial CYP11A1 deficiency ⁽¹⁰⁾, our patient had normal plasma renin activity at the age of 2 years when not stressed. Mild CYP11A1 deficiency can be thereby misdiagnosed as familial glucocorticoid deficiency in 46,XX patients and 46,XY patients with no or very mild DSD and apparently normal mineralocorticoid activity.

Prenatally, partial impairment of pregnenolone synthesis is associated with a broad phenotypic spectrum with regard to genital presentation ⁽¹¹⁾ and postnatally with gonadal insufficiency ^{(7, 4, 10)}. Similarly to other inborn errors of steroidogenesis such as CLAH ⁽¹³⁾ and HSD3B2 deficiency ⁽¹⁷⁾, the CYP11A1 genotype correlates poorly with the phenotype of the external genitalia. CYP11A1 mutations with residual enzyme activities similar to p.R360W were associated with either normal female external genitalia in 46,XY patients (p.L141W and p.A359V) ^{(4, 7)} or normal male genital development (p.R451W) ⁽¹⁰⁾. The moderate degree of 46,XY DSD in our patient indicates that gonadal steroidogenesis was only partly impaired during early development. However, increased gonadotropin levels at age 2 years suggest progressive loss of Leydig cell function. The reassessment of gonadal function at early puberty and clinical follow-up will be essential to assess the requirements for sex hormone replacement during puberty and the potential for fertility in our patient.

We describe two novel CYP11A1 mutations, p.R360W and p.R405X, associated with early onset and mild adrenal insufficiency and moderate 46,XY DSD. Our data demonstrate the partially inactivating nature of the novel p.R360W mutation. Importantly, this report exemplifies the importance to properly assess adrenal function at the time of first clinical presentation to ensure timely diagnosis of patients with mild inborn and possibly progressively developing adrenal insufficiency. Adrenal insufficiency must be considered in the differential diagnosis of patients presenting with 46,XY DSD including severe hypospadias to establish an early correct diagnosis and to prevent potential life-threatening adrenal crises.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

This study was supported by a Marie Curie Intra-European fellowship (grant no. IEF-GA-2009-255424 to S Parajes), the Medical Research Council UK (program grant no. 0900567 to W Arlt), and the Wellcome Trust (Clinician Scientist Fellowship GR079865MA to N Krone).