The CYP11A1 gene located in the long arm of chromosome 15 (15q23–q24) encodes the cholesterol side‐chain cleavage enzyme which converts cholesterol to pregnenolone, the first step for corticoid, mineralocorticoid, and sexual hormone biosynthesis (Miller and Auchus 2011). CYP11A1 deficiency results in complete or partial adrenal insufficiency with a wide range of clinical manifestations. Severe or classical CYP11A1 deficiency manifests with feminization of external genitalia in 46XY males and severe early onset adrenal failure in the first few hours or days of postnatal life. Mild or nonclassical CYP11A1 deficiency is associated with adrenal insufficiency that can develop later in childhood and is accompanied by a spectrum of sexual development disorders including precocious or delayed puberty (Katsumata et al. 2002; Hiort et al. 2005; Slominski et al. 2015).

The patient is a 12‐year‐old male, who is the second child of nonconsanguineous parents. He was delivered via vaginal route after induction of labor on the 34th week of pregnancy due to HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count) syndrome and toxemia and was in the neonatal intensive care unit for 3 weeks for recovery and treatment of neonatal jaundice. His birth weight and length were 2.24 kg and 47 cm, respectively. Upon examination of the external genitalia, hypospadias were detected and later surgically repaired; cryptorchidism was not a feature.

At 3 years of age, he began having severe diurnal episodes of lethargy and hypoglycemia. He was also noted to have skin hyperpigmentation. Testing of hormone levels revealed high adrenocorticotropic hormone (ACTH) and low cortisol levels, suggestive of a primary adrenal insufficiency. An ultrasound and CT scan of the adrenal glands at 3 years of age did not show abnormal findings. Genetic testing for the NR0B1 (DAX1), which was involved in X‐linked congenital adrenal hypoplasia (MIM# 300200), was negative. At 8 years of age, his bloodwork showed an ACTH of 6 pg/mL (normal range 10–60 pg/mL), 17‐hydroxyprogesterone of ˂10 ng/dL (Prepubertal males: <110 ng/dL), and testosterone levels below 2.5 ng/dL (normal rage for males 6 months to 9 years: <7–20 ng/dL). By 9 years of age, his ACTH was abnormally high at 261 pg/mL. Then at 10 years of age, his ACTH level increased to 321.9 pg/mL and repeat testing a year later revealed levels of 405 pg/mL. During his last medical visit, no signs of altered puberty were noticed, and initiation of corticosteroid treatment lead to the normalization of ACTH levels (32.4 pg/mL post treatment). This also improved his hyperpigmentation. No investigations were performed for precocious puberty as his growth and development were age appropriate. He did have a bone age, however, at age 8 9/12 which showed a bone age of 8. The patient was also diagnosed with attention‐deficit/hyperactivity disorder (ADHD) and migraine. Currently, he is under treatment with hydrocortisone (10 mg/m²/day in the mornings and 5 mg/m²/day at night), fludrocortisone (50 μg/day), amphetamine‐dextroamphetamine (15 mg/day), and vitamin D (1000 units/day).

The patient presented to the Department of Clinical Genomics (Mayo Clinic, Jacksonville, FL) with the finding of primary adrenal insufficiency of unknown genetic cause. Informed consent was obtained and blood samples were collected from the patient and his parents for WES. There was no family history of adrenal insufficiency or urogenital anomalies (Fig. 1). Previous genetic testing included mitochondrial DNA (mtDNA) sequencing which revealed no relevant pathogenic variants.

WES revealed compound heterozygous variants in the proband, including a novel paternally inherited CYP11A1 splice site variant (Chr15(GRCh37): g.74640240C>T, NM_000781.2: c.425+1G>A) and a previously reported CYP11A1 missense variant (Chr15(GRCh37): g.74635368C>T, NM_000781.2: c.940G>A) that was maternally inherited (Table 1). The c.425+1G>A variant is considered pathogenic and the c.940G>A (p.E314K) variant likely pathogenic based on HGVS guidelines. Variants were deposited in ClinVar under accession numbers RCV000403766.1 and RCV000413593.1, respectively.

Adrenal insufficiency (AI) is characterized by defects in cortisol, aldosterone, and sexual hormone production (Miller 2017). Common manifestations of the disease include hyperpigmentation, hypoglycemia, lethargy, fatigue, muscle weakness, weight loss, dizziness and hypotension (Ucar et al. 2016). Biochemical criteria for AI diagnosis include very low serum cortisol levels (<80 nmol/L), and elevated ACTH plasma levels (>200 pmol/L) (Neary and Nieman 2010; Ucar et al. 2016).

AI can be produced either by congenital or acquired pathologies. Congenital causes include autosomal recessive congenital lipoid adrenal hyperplasia (CAH, MIM# 201710) (caused by pathogenic variants in STAR); congenital adrenal insufficiency with partial or complete 46XY sex reversal (AICSR, MIM# 613743) (caused by variants in CYP11A1), congenital adrenal hyperplasia due to 3β‐hydroxysteroid dehydrogenase 2 deficiency (caused by variants in HSD3B2); congenital adrenal hyperplasia due to 21‐hydroxylase deficiency (MIM# 201910) (caused by variants in CYP21A2); 11β‐hydroxylase deficiency (caused by variants in CYP11B1) which can result in autosomal recessive congenital adrenal hyperplasia (MIM# 202010) or autosomal dominant glucocorticoid‐remediable aldosteronism (MIM# 103900); 17‐α‐hydroxylase/17,20‐lyase deficiency (MIM# 202110) (caused by variants in CYP17A1); P450 oxidoreductase deficiency (MIM# 201750) (caused by variants in POR) (Katsumata et al. 2002; Hiort et al. 2005; Slominski et al. 2015); familial glucocorticoid deficiency (FGD) (caused by defects in STAR, MC2R, MRAP, MCM4, NNT, TXNRD2, GPX1 and PRDX3) (Miller 2017); and autoimmune disorders such as polyendocrinopathy syndrome type I, with or without reversible metaphyseal dysplasia (MIM# 240300, caused by defects in AIRE) (Fig. 2) (Miller and Auchus 2011; Turcu and Auchus 2015; Manna et al. 2016). Acquired causes, include infectious diseases (Waterhouse‐Frederichsen syndrome), vascular diseases (hemorrhage and coagulopathy), surgery (bilateral adrenalectomy), and exposure to certain medications (rifampicin, phenytoin, and ketoconazole) (Zaloga and Marik 2001).

Congenital AI is an autosomal recessive disorder characterized by elevated ACTH and plasma renin levels with low or absent adrenal steroids. AI has two different expression forms: classical and nonclassical (Pomahacova et al. 2016). The classical phenotypic spectrum ranges from miscarriage and prematurity during pregnancy, to complete under‐androgenization and severe early or late onset adrenal failure at postnatal stages. In the classic presentation of the disease, clitoromegaly is a common finding for feminization of external genitalia in 46XY patients (Neary and Nieman 2010). In the nonclassic form, patients can present with adrenal function impairment with minimal effects on primary sexual characteristics and male patients may present with only cryptorchidism or hypospadias (Hauffa and Hiort 2011; Tee et al. 2013).

Adrenal insufficiency can be caused by pathogenic variants in CYP11A1. CYP11A1 is located at chromosome 15q23–24, and encodes the cytochrome P450 side‐chain cleavage enzyme (P450scc), which initiates steroidogenesis (Fig. 3). P450scc catalyzes the conversion of cholesterol to pregnenolone and isocaproic aldehyde in the inner mitochondrial membrane. These reactions represent the first step for steroid‐hormone biosynthesis. Biallelic pathogenic variants in CYP11A1 are a rare cause of adrenal insufficiency in patients (Tajima et al. 2001; Hauffa and Hiort 2011; Parajes et al. 2011, 2012; Tee et al. 2013). Compared with CAH, AI patients can present mild to severe adrenal insufficiency symptoms without adrenal hyperplasia, these symptoms can manifest at any time from infancy to early childhood, depending on the degree of P450scc dysfunction (Hauffa and Hiort 2011; Tee et al. 2013).

In this report, we found a c.425+1G>A pathogenic variant in CYP11A1 in our 12‐year‐old male patient. This splice site variant disrupts the canonical splice donor site in intron 2, and is predicted to cause abnormal gene splicing. The c.425+1G>A variant is a rare variant and has not been seen in NHLBI Exome Sequencing Project or the Genome Aggregation Database (gnomAD) (insert database references) (Table 1). To the best of our knowledge, the c.425+1G>A pathogenic variant in CYP11A1 has not been reported previously. Regarding ACTH levels, records indicate no cosyntropin stimulation test was performed, and perhaps would have led to earlier diagnosis of adrenal insufficiency and initiation of steroids.’

In addition to the c.425+1G>A variant, we identified a missense variant (c.940G>A p.(E314K)) in exon 5. The p.(E314K) variant is a nonconservative amino acid substitution, which likely impacts secondary protein structure as these residues differ in polarity, charge, and size. However, this substitution occurs at a position that is not conserved, and in silico analysis is inconsistent in its predictions as to whether or not the variant is predicted to be damaging. The p.(E314K) variant is a strong candidate for a pathogenic variant. In two unrelated individuals with primary adrenal insufficiency, the p.(E314K) variant was previously reported; one of this cases was accompanied by another CYP11A1 variant in trans with another variant whereas the other reported case had no additional molecular findings (Chan et al. 2015). According with the NHLBI ESP Exome Sequencing Project, p.(E314K) variant is present in 29/8592 cases from individuals of European American ancestry and in 710/277190 individuals in gnomAD, including four homozygotes, (Table 1) suggesting that this variant may retain partial function and could result in disease when in trans cwith another inactivating mutation. Molecular modeling of the p.E314K variant was performed and Monte Carlo simulations suggest that the orientation of the E and K residues flips and the mutant may disrupt cholesterol binding and lead to minor changes in helicity at the end of the helix when K314 is introduced (Fig. 3).

In conclusion, the compound heterozygous presence of the c.425+1G>A and p.(E314K) variants in CYP11A1 found in this patient, explain the reported primary adrenal insufficiency diagnosis. The c.425+1G>A splice variant in CYP11A1 is novel and may be a pathogenic variant for AI, the p.(E314K) variant has been cited in the literature as pathogenic when present as a compound heterozygous variant (Chan et al. 2015). In addition to the novel variant description, this case remarks the application of WES as a powerful diagnostic tool to provide accurate diagnosis, medical management and family counseling to improve patient care.

The authors declare no conflicts of interest.

M.L.V., A.P.P., P.R.B., and P.S.A. designed the study and wrote the manuscript. T.R.C. performed the in silico analyses. P.R.B. and J.M.G. assisted with data collection and provided critical review of the manuscript. P.S.A. collected the clinical data. All authors approved the final version of the manuscript for publication.