Clinical and Molecular Analysis of Four Patients With 11β-Hydroxylase Deficiency

Objective: 11β-hydroxylase deficiency (11βOHD) is a rare autosomal recessive disorder caused by mutations in the CYP11B1 gene. It is characterized by virilization, hypertension, and significant final height impairment. In this study, we aim to investigate the clinical and molecular characteristics of four unrelated Chinese patients with 11βOHD disorder. Methods: The clinical information of four 11βOHD patients were carefully reviewed. Genetic analysis was performed using next-generation sequencing (NGS) based panel analysis. NGS coverage depth was analyzed to detect exonic copy-number variants (CNVs) on patient 1. Quantitative PCR (qPCR) was subsequently performed to confirm the CNVs detected from the NGS coverage depth analysis. Results: The mean age of the patients at diagnosis was 4.7 years (range, 2.0–9.3 years). Two genetically female patients (patients 1 and 2) with 11βOHD presented severe virilization of external genitalia and were raised as males. Two genetically male patients (patients 3 and 4) presented precocious puberty. Additionally, patients 1, 3, and 4 presented with hypertension. In patient 4, unilateral adrenal mass was detected and removed at the age of 9 years. Interestingly, the height of patient 4 (174.4 cm, +6.7 SD) wasn't impaired and reached his mid-parental height (173 cm). Three novel variants in the CYP11B1 gene (c.1150_1153del, c.217C>T, and c.400G>C) were identified by NGS. Various bioinformatics tools revealed potential pathogenic effects for the novel variants, and evolutionary-conservation revealed that the novel missense variant affected an amino acid that is highly conserved among species. Furthermore, NGS coverage depth analysis and qPCR identified a novel heterozygous deletion of exons 1–6 in patient 1. Conclusion: Our study expands the spectrum of mutations of the CYP11B1 gene in Chinese population. In addition, We reported the first case of a patient with classical 11βOHD disorder, whose final height wasn't compromised.


INTRODUCTION
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by defects in various enzymes that are involved in the synthesis of cortisol from cholesterol by the adrenal cortex (1). The most common form of CAH is 21hydroxylase deficiency (21OHD), accounting for 90-95% of all CAH cases (2). In contrast, CAH due to the deficiency of 11βhydroxylase (11βOHD) accounts for ∼5-8% of all cases and has an annual incidence of 1/100,000-200,000 live births (3). Female newborns can be identified with ambiguous genitalia as in 21OHD. However, boys are usually not diagnosed by newborn screening of 17-hydroxyprogesterone (17OHP), which is not as high in 11βOHD as it is in 21OHD (4,5).
11βOHD is caused by mutations in the CYP11B1 gene which is located on chromosome 8q21, ∼40 kb from the highly identical CYP11B2 gene (6). The CYP11B1 gene includes 9 exons and encodes a 503 amino acid protein which belongs to the cytochrome P450 system. Variants that lead to deactivated CYP11B1 can abolish or reduce the activity of the 11β-hydroxylase which converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone (DOC) to corticosterone in the mitochondria of the adrenal cortex. Reduced activity of 11βhydroxylase leads to decreased cortisol secretion, accumulation of glucocorticoid and mineralocorticoid precursors, and excessive adrenal androgen biosynthesis. The classical and more severe form of 11βOHD is most likely caused by severe impairment of 11β-hydroxylase activity (residual activity of <10%) (7). Hypertension due to accumulation of the potent mineralocorticoid DOC, occurs in approximately two-third of the cases, often early in life (8,9). In affected females, the excess androgens result in varying degrees of enlargement of the clitoris, fusion of the labioscrotal folds, and the vaginal and urogenital confluence. Both girls and boys with classical 11βOHD undergo rapid postnatal growth with sexual precocity and skeletal maturation, leading to short stature in adulthood (10,11). In this study, we performed a genetic and phenotypic analysis of four Chinese patients with 11βOHD, including two 46,XX females misassigned to the male gender at birth and reared as males, and two 46,XY males.

Subjects
Four unrelated Chinese patients were investigated in this study. Patients 1 and 2 were genetically females but raised as males due to severe virilization before the diagnosis. The clinical diagnosis of the patients was based on evaluation of clinical symptoms and laboratory findings.

Bioinformatics Analysis
Three software: PolyPhen-2 (http://genetics.bwh.harvard.edu/ pph2/), Provean (http://provean.jcvi.org/), and SIFT (http://sift. jcvi.org/) were performed to predict the damaging effect of the identified mutations on CYP11B1 function. The amino acid sequences of human CYP11B1 protein were aligned with those in the homologous proteins from different species. The putative effect of the novel missense mutation was analyzed using the X-ray structure of the human CYP11B1 as a template (PDB accession number: 6M7X).

Clinical Characteristics and Serum Hormone Levels
Clinical features and laboratory evaluations of the patients are listed in Tables 1, 2. All patients were born to nonconsanguineous parents at full-term gestation. Patients 1 and 2 were found to have a (46,XX) karyotype while patients 3 and 4 had a karyotype of (46,XY). Patients 1 and 2 were raised as boys based on the appearance of the male external genitalia after birth. They were admitted to the local hospital for cryptorchidism at about the age of two. No gonads were palpable in the scrotum or bilateral inguinal canals. Uterus and ovaries were revealed by pelvic ultrasound. Regarding patient 1, laparoscopic exploration of the gonad was performed and tissue biopsy revealed the ovaries at the local hospital. Additionally, physical examination showed high blood pressure (120/80 mmHg, >99th), and external genitalia showed Prader score of 5 (penis 4 cm in length and complete fusion of the labioscrotal folds without hypospadias) while in patient 2 the Prader score was 4 (penis 3.5 cm in length and complete fusion of the labioscrotal folds with hypospadias). Abdominal ultrasound revealed enlargement of bilateral adrenal glands in  both patients and hydrocortisone treatment was subsequently started. For patient 1, spironolactone was combined for 3 months, then stopped due to normal blood pressure. In both patients, surgery of feminizing genitoplasty was performed after affirmative diagnosis. Patient 3 was suspected the diagnosis of CAH with the chief complaint of skin pigmentation and began therapy with glucocorticoid therapy from 3.5 year-old in the local hospital. However, patient compliance with therapy was very poor. He was thereafter admitted to our department for enlargement and erection of penis at the age of 4.7 years. His height was 128.5 cm (+4.7 SD) with advanced bone age (13 years). The following observations were revealed upon physical examination: high blood pressure (121/80 mmHg, 95-99th), skin hyperpigmentation, penile enlargement, and enlarged testicular volume, no pubic hair. Further Gonadotropin-releasing hormone (GnRH) stimulation test showed luteinizing hormone (LH)peak of 16.9 mIU/mL and follicle-stimulating hormone (FSH)peak of 6.22 mIU/mL, suggesting that hypothalamic-pituitarygonadal axis (HPGA) was activated. Abdominal ultrasound revealed enlargement of bilateral adrenal glands. Based on the clinical features and biochemical results, patient 3 was diagnosed with CAH due to 11βOHD, and central precocious puberty was considered. The treatment started with hydrocortisone, spironolactone and GnRH agonist (triptorelin acetate). Patient 4 was admitted to the local hospital for hypokalemic paralysis (Serum K + : 2.95 mmol/L, normal range: 3.5-5.5 nmol/L) and hypertension (140/95 mmHg, >99th) at the age of 9 years. Medical imaging showed a mass on the left adrenal gland (5 cm × 2.2 cm) and bilateral adrenal hyperplasia. The patient underwent left adrenal tumor resection. Pathology findings showed adrenocortical oncocytic neoplasm and adrenal cortical hyperplasia. Postoperative clinical examination showed high blood pressure (150/96 mmHg, >99th) that was difficult to control with antihypertensive therapy (nifedipine, valsartan and hydrochlorothiazide). Patient 4 was thereafter admitted to our hospital 3 months after the surgery. Notably, patient 4 showed a tall stature of 174.4 cm (+ 6.7 SD), while his mid-parental height was 173 cm, calculated following the standard procedure from the mean height of the parents (average of the father's and mother's height + 6.5 cm) (13). His puberty advanced to Tanner stage V for pubic hair and testicular volume increased to 20 mL (Tanner IV) with advanced bone age of 17 years. Dexamethasone combined with valsartan and spironolactone treatment was subsequently started, and his blood pressure levels were maintained at the upper limit of normal. The evolutionary-conservation alignment showed that the 134 residue G is a highly conserved residue (Figure 1B). The three-dimensional molecular model of CYP11B1 showed that Gly134 lies in a loop between B3-and C-helix ( Figure 1C).

DISCUSSION
We identified eight variants of the CYP11B1 gene in four Chinese patients with 11βOHD, four of which are novel variants. The clinical examination findings of these patients are generally consistent with classical 11βOHD. Notably, serum aldosterone levels in patients 1 and 4 were elevated and normal, respectively, which were expected to be low. We measured aldosterone by chemiluminometric immunoassay (Autobio, China) with a cross reactivity level of <0.1% to other steroid compounds like DOC, 11-deoxycortisol, testosterone and cortisol. Similar to our study, previous reports have shown that elevated aldosterone levels in CAH may be associated with cross-reactivity of high levels of adrenal steroid precursors (14)(15)(16), which could explain the aldosterone levels in our patients.
Since salt-wasting is not commonly seen in 11βOHD, the diagnosis of this disorder in these patients was made relatively late. In our study, the diagnosis in all four cases was delayed as well. In addition, about 70% of 46,XX patients with 11βOHD had Prader score 4 or 5 at presentation, so females with 11βOHD are more commonly mis-assigned to the male gender at birth than those with 21OHD (6). Meanwhile, most of males were diagnosed with precocious puberty, hypertension, hyperpigmentation or hypokalemia. Final height was severely impaired in children with 11βOHD, irrespective of age at diagnosis and quality of therapeutic control. The mean adult height was (156.8 ± 7.2) cm in male patients and (153.1 ± 2.9) cm in female patients (10). Bone age was significantly advanced in our cases, yielding a severely compromised predicted final adult height. Structural visualization of the identified CYP11B1 missense alteration using X-ray structure of the human CYP11B1 as a template (PDB accession number: 6M7X). The residues present at our mutation sites are shown as sticks.
Unexpectedly, the height of patient 4 wasn't impaired, and he reached his genetical final height, which was not compatible with classic 11βOHD. The underlying mechanism is not clear, though. The three older patients: patient 1, patient 3 and patient 4 presented varying severity of hypertension, which was not the case in the youngest patient (patient 2). This suggests that there is a significant correlation between older age at diagnosis and higher blood pressure. Glucocorticoids can effectively improve blood control, while it might be required to add anti-hypertensive medication in patients with delayed diagnosis of 11βOHD (3). Moreover, some reports have described the role of adrenalectomy in the management of refractory hypertension in 11βOHD (17,18). Hypokalemia as a result of accumulation of DOC and other steroid precursors is found in a minority of patients with 11βOHD and is not correlated to blood pressure. Similar to the reported patients, hypokalemia in patient 4 resolved with glucocorticoid replacement (19). Adrenal tumor in patient 4 may be caused by chronic ACTH elevation. Long-term stimulation from elevated ACTH levels play a role in the development of adrenocortical tumors in CAH patients. Glucocorticoid treatment can cause the reduction in size of the adrenal masses (20). We support previous recommendation that patients with adrenal masses should be checked for CAH.
The functional severity of CYP11B1 genotypes may be correlated with age at onset, degree of virilization in female, and age at hypertension. Patient 1, who presented with severe virilization (Prader V) and hypertension at diagnosis before 3 years of age, carried compound heterozygous variants c.
[1150_1153del]; [c.Exons1-6del]. The novel variant c.1150_1153del [p.(Arg384Trpfs * 45)] will probably generate a premature stop codon at the amino acid 429, which will result in the loss of 73 amino acids with severely impaired 11β-hydroxylase enzymatic activity. In close proximity to this variant, c.1179_1180dup [p.(Asn394Argfs * 37)] was described in a female newborn with severe virilization and a male presenting with hypertension before 2 years of age, respectively (21). The large deletion variant (Exons 3-5del) was reported in a female with complete virilization and raised as male at birth (5). Thus,  (22,23). A Chinese patient was homozygous for this variant, who was raised as male at birth with severe virilization (Prader IV) and diagnosed with hypertension at the age of 16 years (24). The residue Arg454 is localized in close vicinity to Arg453. Therefore, p.(Arg454Cys) may alter heme binding site and result in complete loss of enzyme activity as p.(Arg453Gln) (25). The c.1398+5G>C homozygous mutation was previously described in a 46,XX female with severe virilization of external genitalia at birth (5).  (26). The residues His133 and Gly134 are highly conserved and located on a loop between B'-and C-helix. p.Asn133His previously reported in non-classical form of 11βOHD, resulted in ∼30-50% residual 11β-hydroxylase activity (27,28). It seems likely that p.(Gly134Arg) may result in partially disrupted enzyme activity, which is consistent with relatively mild clinical phenotype in patient 4. Taken together, our results support that truncated mutations or mutations that altered the heme binding site in CYP11B1 gene may account for a more severe phenotype of patients.

CONCLUSION
In conclusion, we have found four novel mutations in the CYP11B1 gene. These results may expand the spectrum of mutations of the CYP11B1 gene in the Chinese population. Significant heterogeneity of clinical phenotype and low prevalence are largely responsible for the late diagnosis of 11βOHD. Early diagnosis and glucocorticoid treatment are important to prevent complications and improve long-term outcomes.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Children's Hospital of Nanjing Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.