Clinical and molecular characterization of 10 Chinese children with congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases. The most common form of CAH originates from steroid 21-hydroxylase deficiency (21OHD), which accounts for about 90%–95% of all cases, followed by 11beta-hydroxylase deficiency (11βOHD) (OMIM 202010), which accounts for about 5%–8% of cases [1]. The incidence of 11β-OHD in the total population is approximately one in 100,000–200,000 [2, 3], and it is common in the Israeli population of Jewish origin in the Middle East and North Africa, affecting 1:5000–7000 live births [4]. 11β-OHD is caused by mutations in the cytochrome P450 family 11 subfamily B member 1 (CYP11B1) gene. CYP11B1 mutation leads to a reduction in the conversion of 11 deoxycorticosterone (DOC) to corticosterone and 11 deoxycorticosterol to cortisol, which leads to an increase in the secretion of adrenocorticotropic hormone (ACTH) and the accumulation of steroid precursors. Thus, 11β-OHD is characterized by increased serum concentrations of 11β-deoxycorticosterone, 17-hydroxyprogesterone (17-OHP) and androgens, resulting in hypertension with low renin levels, hypokalemia and genital ambiguity in affected individuals with a 46,XX karyotype. The milder nonclassical form is very similar to the nonclassical form of 21-OHD and is characterized by mild virilization, irregular menstruation, and male precocious puberty; hypertension does not occur frequently. According to the Human Gene Mutation Database (HGMD, www.​hgmd.​cf.​ac.​uk), a total of 155 CYP11B1 gene mutations were included, of which missense and nonsense mutations are among the most common types, and their effects on enzyme activity have been studied mainly through three-dimensional protein structure simulation and in vitro protein expression experiments.

Here, we report the clinical and molecular characterization of 10 Chinese patients with CAH due to 11β-OHD. Novel mutations and three previously reported but not functionally studied mutations were explored in vitro to monitor the residual enzyme activity and to analyze the correlation between the genotype and phenotype of the new mutations, providing help for clinical diagnosis and analysis of 11β-OHD. Three-dimensional protein simulations may provide additional support for the physiopathological mechanism of genetic mutations.

In this study, we describe the clinical and molecular characterization of 11β-hydroxylase deficiency and the genotype–phenotype correlation of 11β-OHD in 10 Chinese patients from 10 unrelated families. Five female patients were affected by excessive fetal adrenal androgens in the uterus, resulting in ambiguous external genitalia and varying degrees of masculinization at birth [5]. Therefore, the female patients were treated at an early age, at an average age of 2.08 ± 1.66 years old. The age of the 46,XY patients at the time of diagnosis ranged from 4.25 to 12 years, with the difference between bone age and chronological age being 3.69 ± 2.82 years. The age of diagnosis and treatment of female patients was significantly earlier than that of male patients. All 10 patients displayed different degrees of skin pigmentation. Four patients were initially misdiagnosed with 21-OHD. Due to the increase in DOC, some patients develop hypertension with low aldosterone and low renin with hypokalemia. Therefore, the levels of DOC and deoxycortisol are important diagnostic criteria for 11β-OHD. Unfortunately, they are not routinely detected in  China. In our clinical practice, hypertension is the main clue to differentiate 11β-OHD from 21-OHD. However, most patients do not have manifestations of hypertension and hypokalemia at birth, and some patients do not have hypertension [6, 7]. Therefore, it is generally detected later in childhood or even in adolescence. In our study, three male patients presented hypertension and hypokalemia due to delayed diagnosis of 11β-OHD and delayed treatment. The female patients were diagnosed at an early age and were treated in a timely manner without hypertension or hypokalemia. Complications such as cardiomyopathy, retinal vein occlusion, and even blindness from long-lasting, uncontrolled hypertension have been reported in patients with 11β-OHD [8]. Early diagnosis and treatment are key points to prevent the complications of hypertension. The lack of negative feedback of glucocorticoids leads to increased secretion of ACTH, such that adrenal crisis rarely occurs, but patients generally have hyperpigmentation. Therefore, molecular analysis of the CYP11B1 gene is particularly important to confirm a suspected clinical diagnosis of 11β-OHD.

11β-OHD is caused by mutations in CYP11B1 located on chromosome 8q21-22. The CYP11B1 gene consists of nine exons and encodes a 503 amino acid protein. To date, the HGMD has reported 98 missense and nonsense mutations. These mutations are concentrated in exons 2 and 6–8, with a small number in exons 1 and 9. In this study, we detected 13 mutations, including three point mutations and one nonsense mutation that have been described in other reports [9], of which nine mutations are novel (Y286X, S315R, D317V, Q121del, L464del, exon 3–4 deletion, R384Wfs × 45, E459fs, c.396-1G > A). The mutation sites are mainly concentrated in exons 2, 5, 7, and 8. Consistent with previous reports, four patients (P2, P5, P9, P10) carried the R454C pathogenic variation, which is found only in the Chinese population [10]. The nine novel mutations included two missense mutations (S315R and D317V), one nonsense mutation (Y286X), five deletion frameshifts (Q121del, L464del, exon 3–4 deletions, R384Wfs × 45 and E459fs) and one splice mutation (c.396-1G > A). Nonsense mutations, deletion frameshift mutations and splicing mutations all indicate loss of enzymatic activity. Therefore, newly discovered and previously reported missense mutations that have not been tested for enzymatic activity were selected for enzymatic activity function detection, and the encoded enzymatic protein after gene mutation was analyzed. For the D317V mutation detected in patient 4, enzyme activity was 9.4% (< 10%); the patient showed clinically accelerated growth with peripheral precocious puberty, which was misdiagnosed as 21-OHD in other hospitals, and developed hypertension and hypokalemia several years later. Therefore, the disease was considered to be classic 11β-OHD. For patient 8 (46, XX), N152K mutant enzyme activity was 12.3%, this patient had obvious masculinization of external genitalia at birth with renin in infancy of 0.1 ng/mL/h, and serum potassium of 3.67 mmol/L. Therefore, the case was also considered classical 11β-OHD. In 2010, Parajes et al. [11] detected M88I and P159L mutations in patients with the nonclassical form; these mutations decreased the enzyme activity to 40% and 25% of the wild-type level, respectively. While studies have shown that CYP11B1 mutations that cause classic 11β-OHD usually reduce enzyme activity to less than 5% or result in complete loss of activity, for each specific mutation, there is no significant correlation with the severity of the clinical manifestations for each specific mutation. Indeed, patients with the same gene pathogenic variant may have mild or severe hypertension, and the manifestations of androgen excess vary in severity. Thus, the correlation between genotype and phenotype needs to be further studied.

Through the three-dimensional protein structure model, we attempted to infer the influence of the mutation site on the three-dimensional structure of the protein conformation. The helix structure is the heme-binding region and is highly conserved. The cysteine sulfhydryl group at position 450 binds with a heme iron atom to form one of the enzyme active sites. Therefore, the amino acid changes around C450 can affect binding of heme and cause the loss of enzyme activity. For example, Y423X, Q426X, P427H, V441G, G444D, G446V, G446S, R448C, R448H, R448P, R453Q, R454C, R461P and other mutations can cause a loss of enzyme activity [10, 12], of which R454C has been reported only in the Chinese population. The R454 residue is highly conserved; protein model predictions show increased hydrogen bonding with L451 for the p.R454C mutant, which may enhance local interaction strength and reduce domain flexibility.

As part of the substrate binding pocket, the I-helix contains many hydrophobic amino acids and potential enzyme active sites and is involved in the recognition of and binding to substrates. When its conformation changes, the enzyme activity of CYP11B1 may be impaired. For example, L299P, A368D, P94L, A331V, E371G and other mutations can reduce or even eliminate the activity of the enzyme [13‐15]. The G-helix, the I-helix and the B-C loop between them are important channels for the substrate to enter the active site the G-helix is necessary for the initial recognition of the substrate, and the change in the hydrophobic environment around it can hinder the binding of the substrate to the enzyme. For example, W116C, W116G, V129M, V148G, A259D, T318M, T318R, T318P, R384Q, R384G and other mutations can reduce or even cause loss of enzyme activity [15]. The c.945C > A mutation was detected in patient 1, resulting in the amino acid change S315R (Ser315Arg), which is a new missense mutation. The amino acid conservation analysis of CYP11B1 in seven species was carried out through the UniProt database, and S315 is not highly conserved; PolyPhen-2 (score 1.000) software predicts the p. S315R variant to be deleterious, but with SIFT (score 0.073) and CADD (score 17.80) software, the deleterious threshold was not reached. The protein model prediction showed that the p.S315R mutant does not form hydrogen bonds with T312 but shows increased the hydrogen bonding with S200. The c.456C > G mutation was detected, leading to the amino acid N152K (Asn315Lys), was detected in patient 8. This missense mutation has been reported, but no functional experiments have been performed.Amino acid conservation analysis of CYP11B1 in seven species was performed through the UniProt database. N152 is a variable amino acid; PolyPhen-2 (score 0.000), SIFT (score 1.000), and CADD (score 0.024) predict that the p. N152K variant to be benign. The protein model prediction showed that the p. N152K mutant folds in the same manner as the wild-type protein, and in both cases, the residue at this position forms hydrogen bonds only with R156. This patient is 46,XX, with clinical manifestations of clitoral hypertrophy, visible vaginal opening, urethral opening, Prader stage II, and Tanner stage B1PH1, but no manifestations of hypertension. Therefore, this current missense mutation of CYP11B1 can lead to changes in the three-dimensional conformation of the CYP11B1 enzyme, leading to different degrees of changes in enzyme activity.