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 21-hydroxylase deficiency (21OHD), accounting for 90–95% of all CAH cases (2). In contrast, CAH due to the deficiency of 11β-hydroxylase (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 11β-hydroxylase 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.

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 (14–16), 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. 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, this larger deletion variant (Exons 1–6del) is predicted to result in completely impaired enzymatic activity.

Patient 2 with compound heterozygous variant c.[1398+5G>C];c.[1360C>T] was diagnosed at 2 years of age with severe virilization (Prader IV) and significantly advanced bone age. Missense variant c.1360C>T [p.(Arg454Cys)] is a hotspot mutation for Han Chinese (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).

Patient 3 carried compound heterozygous nonsense variants p.(Arg141^*); p.(Gln73^*). Vitro expression data showed that p.(Arg141^*) mutation retained 4.9% of residual 11β-hydroxylase enzymatic activity and almost completely abolished enzymatic activity (22). Thus, the novel variant p.(Gln73^*) is predicted to result in completely impaired enzymatic activity in the truncated protein, which corresponded to the severe phenotype in patient 3 with hypertension and secondary central precocious puberty at the age of 4.7 years.

Patient 4 harboring compound heterozygous missense variant p.(Gly267Ser); p.(Gly134Arg) presented relatively mild clinical phenotype. Missense variant c.799G>A [p.(Gly267Ser)] activated an alternative splice site within exon 4, leading to a 45-bp deletion in the transcript and a truncated protein (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.

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.

The datasets generated for this study can be found in the LOVD database at the following links: https://databases.lovd.nl/shared/individuals/00306720, https://databases.lovd.nl/shared/individuals/00306721, https://databases.lovd.nl/shared/individuals/00306716, https://databases.lovd.nl/shared/individuals/00306718.

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.

QZ, DW, and CW collected samples, performed the experimentation, and analyzed the data. WG and ZJ conceived and designed this study. ZZ and QL collected the clinical samples and clinical data. CW and BZ performed NGS analysis. WG, QZ, and DW wrote the manuscript. All authors have read and approved the final manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.