Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene

doi:10.1016/j.jsbmb.2015.10.011

The Journal of Steroid Biochemistry and Molecular Biology

Volume 155, Part A, January 2016, Pages 126-134

https://doi.org/10.1016/j.jsbmb.2015.10.011 Get rights and content

Highlights

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The urinary steroid metabolome of 6 CAH patients was analyzed by GC–MS.
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Three novel mutations in the CYP11B1 gene were detected.
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These mutations were analyzed in COS-1 cells.
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These mutations explained the phenotype of the patients.

Abstract

Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis. Steroid 11β-hydroxylase deficiency (11β-OHD) due to mutations in the CYP11B1 gene is the second most common form of CAH.

In this study, 6 patients suffering from CAH were diagnosed with 11β-OHD using urinary GC–MS steroid metabolomics analysis. The molecular basis of the disorder was investigated by molecular genetic analysis of the CYP11B1 gene, functional characterization of splicing and missense mutations, and analysis of the missense mutations in a computer model of CYP11B1.

All patients presented with abnormal clinical signs of hyperandrogenism. Their urinary steroid metabolomes were characterized by excessive excretion rates of metabolites of 11-deoxycortisol as well as metabolites of 11-deoxycorticosterone, and allowed definite diagnosis. Patient 1 carries compound heterozygous mutations consisting of a novel nonsense mutation p.Q102X (c.304C > T) in exon 2 and the known missense mutation p.T318R (c.953C > G) in exon 5. Two siblings (patient 2 and 3) were compound heterozygous carriers of a known splicing mutation c.1200 + 1G > A in intron 7 and a known missense mutation p.R448H (c.1343G > A) in exon 8. Minigene experiments demonstrated that the c.1200 + 1G > A mutation caused abnormal pre-mRNA splicing (intron retention). Two further siblings (patient 4 and 5) were compound heterozygous carriers of a novel missense mutation p.R332G (c.994C > G) in exon 6 and the known missense mutation p.R448H (c.1343G > A) in exon 8. A CYP11B1 activity study in COS-1 cells showed that only 11% of the enzyme activity remained in the variant p.R332G. Patient 6 carried a so far not described homozygous deletion g.2470_5320del of 2850 bp corresponding to a loss of the CYP11B1 exons 3–8. The breakpoints of the deletion are embedded into two typical 6 base pair repeats (GCTTCT) upstream and downstream of the gene.

Experiments analyzing the influence of mutations on splicing and on enzyme function were applied as complementary procedures to genotyping and provided a rational basis for understanding the clinical phenotype of CAH.

Introduction

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by the loss of one of the enzymes involved in cortisol synthesis. More than 90% of all cases are due to steroid 21-hydroxylase deficiency and about 3–8% are cases caused by 11β-hydroxylase deficiency (11β-OHD) [1], [2], [3]. This is equivalent to an incidence of 1 in 100,000–200,000 live births.

Human CYP11B1 is a member of the cytochrome P450 superfamily and catalyzes as a monooxygenase the reductive cleavage of molecular oxygen (O₂) and the subsequent incorporation of one oxygen atom into the substrate and the other one into water. CYP11B1 is located in the inner mitochondrial membrane where it receives electrons for the reaction from NADPH via the electron transfer proteins adrenodoxin and adrenodoxin reductase. The major function of human CYP11B1 (11β-hydroxylase) is the 11β-hydroxylation of corticosteroids and thereby it is primarily responsible for the formation of cortisol from 11-deoxycortisol [4].

The 11β-OHD causes decreased synthesis of cortisol and corticosterone in the zona fasciculata of the adrenal gland, resulting in accumulation of the steroid precursors 11-deoxycortisol and 11-deoxycorticosterone, which, redirected into the androgen synthesis, cause the clinical symptoms of hyperandrogenism. Phenotypical expression of classic 11β-OHD leads to virilization of external genitalia in newborn females and precocious pseudopuberty, accelerated somatic growth, and advanced bone maturation due to reactive androgen overproduction in both sexes. A non-classic 11β-OHD form can manifest later and reveals milder virilization than the classic form [5].

CYP11B1 gene is located on chromosome 8q22 approximately 40 kb apart from the paralog CYP11B2 gene, which encodes the aldosterone synthase [6], [7]. The CYP11B1 consists of nine exons and encodes a protein of 503 amino acids. At present, over 85 different mutations causing congenital adrenal hyperplasia have been identified and distributed over the entire coding region, whereas mutation hot spots are reported to be around exons 2, 6, 7, and 8 [8], [9], [10], [11], [12]. Mutations have been identified in classic and non-classic 11β-OHD form.

In this study, we performed a molecular genetic analysis of the CYP11B1 gene from six patients suffering from CAH. Sequence analysis of the CYP11B1 gene led to the identification of the deficiency causing mutations. Among them, 3 novel mutations were identified including a missense mutation, a nonsense mutation, and a deletion of 6 exons of the CYP11B1 gene. The influence of the splice-site mutation and the missense mutation were further analyzed in COS-1 cells and showed intron retention and reduced enzyme activity proving understanding of the rational basis of the influence of the mutation on the enzyme deficiency.

Section snippets

Patients

Clinical data of the patients at their first presentation are summarized in Table 1. All patients with karyotype 46,XX had severely virilized external genitalia at birth. Premature adrenarche and growth acceleration were leading symptoms in 46,XY patients. The 11β-hydroxylase deficiency was diagnosed by urinary GC–MS steroid metabolome analysis in untreated patients.

Gas chromatography-mass spectrometry (GC–MS) urinary steroid profiling

Steroid profiles in urine samples were analyzed using quantitative data that were generated by GC–MS analysis as described

Urinary steroid metabolome in patients with 11β-OHD

Fig. 1 reveals a typical chromatogram obtained by GC–MS in a patient with 11β-OHD. In contrast to healthy individuals, the chromatogram is dominated by metabolites of 11-deoxycortisol (substance S;4-Pregnene-17α,21-diol-3,20-dione) which accumulates before the enzymatic block. Stemming from the zona fasciculata, the tetrahydrated metabolites of substance S, 5β- (THS,5β-Pregnane-3α,17α,21-triol-20-one, Table 1) and 5α-tetrahydro substance S (a-THS,5α-Pregnane-3α,17α,21-triol-20-one) are secreted

Discussion

Beside 21-hydroxylase and 3β-hydroxysteroiddehydrogenase deficiencies, 11β-hydroxylase deficiency is one of the virilizing forms of CAH [21]. As can be seen in Table 1, all patients with karyotype 46,XX presented with severely virilized external genitalia. In 46,XY individuals, premature adrenarche and growth acceleration indicated clinical hyperandrogenism. Severe bone age acceleration was present in all patients over 2 years of age. In younger CAH patients, bone age acceleration does not

Acknowledgement

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-YS.02-2013.20 to H.H.N.

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Humans have two isozymes with 11β-hydroxylase activity that are respectively required for cortisol and aldosterone synthesis. CYP11B1 (11β-hydroxylase) converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, is expressed at high levels, and is regulated by ACTH. CYP11B2 (aldosterone synthase) is normally expressed at low levels and is regulated mainly by angiotensin II and potassium levels. The latter enzyme also has 18-hydroxylase and 18-oxidase activities and thus can synthesize aldosterone from deoxycorticosterone. Mutations in the CYP11B1 gene cause steroid 11β-hydroxylase deficiency, a form of congenital adrenal hyperplasia. Mutations in CYP11B2 result in aldosterone synthase deficiency, which can cause hyponatremia, hyperkalemia, and hypovolemia in infancy. These are both recessive disorders. Unequal crossing over between the CYP11B genes can generate a duplicated chimeric gene, causing glucocorticoid-suppressible hyperaldosteronism, an autosomal dominant form of hypertension. Frequent polymorphisms in these genes can affect aldosterone secretion and risk of hypertension.
Compound heterozygosity of a novel Q73X mutation and a known R141X mutation in CYP11B1 resulting in 11β-hydroxylase deficiency in a Chinese boy with congenital adrenal hyperplasia
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To date, more than 100 pathogenic CYP11B1 mutations—including missense/nonsense, splice-site, small/ gross deletions, insertions, and complex rearrangements with CYP11B2—have been identified in the Human Gene Mutation Database (HGMD) [18]. Among the coding regions, the mutation sites are generally clustered in exons 2, 6, 7, and 8 [19–21]. Here, we report a case of CAH with classic 11β-hydroxylase deficiency in a Chinese boy who was compound heterozygous for a known nonsense mutation p.R141X (c.421C > T) in exon 3 and also possessed an undescribed nonsense mutation p.Q73X (c.217C > T) in exon 1 of CYP11B1.
Steroid 11β-hydroxylase deficiency (11β−OHD), which is caused by mutations of the CYP11B1 gene, is the second leading cause of congenital adrenal hyperplasia (CAH), an autosomal recessive inherited disorder. Here, we report a case of classic 11β−OHD in a Chinese boy characterized by hypertension, penile enlargement, skin pigmentation, and acne. Molecular analysis of CYP11B1 revealed that the patient was compound heterozygous for a c.217C > T (p.Q73X) mutation in exon 1 and a c.421C > T (p.R141X) mutation in exon 3. His parents carried the novel c.217C > T (p.Q73X) mutation and the prevalent c.421C > T (p.R141X) mutation. Furthermore, we identified a novel 217-bp substitution mutation (Q73X) in CYP11B1 that generates a truncated protein without biological activity, which is likely to be pathogenic. Pursuant to the phenotype of the proband and his family, the Q73X mutation is inferred to exacerbate the disease burden of the R141X mutation, a known pathogenic variant. To further explore this possibility, selecting the x-ray structure of human CYP11B2 as a template, we built three-dimensional homologous models of the normal and mutant proteins. In the mutant model, a change from a helix to terminal structure in amino acids 73 and 141 occurred that affected the binding capacity of CYP11B1 with heme and impaired 11β-hydroxylase activity. Taken together, our findings expand the spectrum of known mutations leading to 11β−OHD and provide evidence to study genotype-phenotype concordance, confirm early diagnosis and treatment of 11β−OHD, and prevent most complications.
Challenges in defining the role of intron retention in normal biology and disease
2018, Seminars in Cell and Developmental Biology
Citation Excerpt :
Several individual protein-coding intron-retaining isoforms have been associated with the development of human disease. IR results in the production of a smaller CYP11B1 protein isoform associated with the development of the steroidogenesis disorder congenital adrenal hyperplasia [90]. IR can also have a protective effect, with the retention of introns 12 and 13 in the calcineurin gene producing an isoform which improves cardiac function and reduces scar formation after myocardial infarction [91].
RNA sequencing has revealed a striking diversity in transcriptomic complexity, to which alternative splicing is a major contributor. Intron retention (IR) is a conserved form of alternative splicing that was originally overlooked in normal mammalian physiology and development, due mostly to difficulties in its detection. IR has recently been revealed as an independent mechanism of controlling and enhancing the complexity of gene expression. IR facilitates rapid responses to biological stimuli, is involved in disease pathogenesis, and can generate novel protein isoforms. Many challenges, however, remain in detecting and quantifying retained introns and in determining their effects on cellular phenotype. In this review, we provide an overview of these challenges, and highlight approaches that can be used to address them.
A high rate of novel CYP11B1 mutations in Saudi Arabia
2017, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
Previously reported mutations at this codon include c.1342C>T (p.R448C) and c.1343G>A (p.R448H) [22,23]. This last mutation has been frequently reported in Jews from Moroccan origin [23–25]. Arginine at this location is located in the heme binding domain and is only 2 amino acids proximal to a cysteine residue that functions as a ligand to the heme group [6].
Despite ethnic variation, 11 β-hydroxylase deficiency (11β-OHD) has generally been considered the second most common subtype of congenital adrenal hyperplasia (CAH). We report a high rate of novel mutations in this gene (CYP11B1) in patients from Saudi Arabia. We studied 16 patients with 11β-OHD from 8 unrelated families. DNA was isolated from peripheral blood. The 9 exons and exon-intron boundaries of CYP11B1 were PCR-amplified and directly sequenced. The novel mutations were functionally characterized using subcloning, in vitro mutagenesis, cell transfection and 11-deoxycortisol: cortisol conversion assays. Six mutations were found in these 8 unrelated families. Three of these mutations are completely novel and two have just been recently described as novel mutations from the same population. These include a single nucleotide insertion mutation in codon 18 (c.53_54insT) leading to frameshift and truncation in 4 siblings, a novel mutation (c.1343G>C, p.R448P) in 3 unrelated families, a novel mutation (c.1394A>T, p.H465L) in 2 siblings, a novel mutation (c.617G>T, p.G206V) in 1 patient, and a recently described non-sense novel mutation (c.780G>A, p.W260X) in another patient. Out of the 6 mutations described in this report, only one mutation (p.Q356X) was reported previously. In vitro functional testing of the 3 missense and nonsense novel mutations revealed complete loss of the 11 hydroxylase activity. We conclude that 11 β-OHD in Saudi Arabia has a unique genotype with a high rate of novel mutations. The novel p. R448P mutation is the most common mutation in this highly inbred population.
Congenital adrenal hyperplasia due to 11-hydroxylase deficiency—Compound heterozygous mutations of a prevalent and two novel CYP11B1 mutations
2017, Gene
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The affected male patient presented with precocious puberty and severe hypertention concomitantly with hypokalemia, the latter is known as the result of increased serum concentrations of DOC, 11-deoxycortisol and suppressed plasma rennin concentrations and reported occurring in approximately two-thirds of the patients with 11β-OHD (White et al., 1994). All the reported mutations of CYP11B1 ware located in the entire encoding regions, but they tend to cluster in exons 2, 6, 7 and 8 (Curnow et al., 1993; Geley et al., 1996; Charnwichai et al., 2016; Nguyen et al., 2016). Although previous study showed that in vitro activities < 5% were considered severe and consistent with classic 11β-OHD, the exact genotype-phenotype correlations of 11β-OHD has not been well established (Parajes et al., 2010).
11β-hydroxylase deficiency (11β-OHD) occurs in about 5–8% of congenital adrenal hyperplasia (CAH). In this study, we identified three CYP11B1 (encoding Cytochrome P450 11B1) heterozygous mutations: c.1358G>C (p.R453Q), c.1229T>G (p.L410R) and c.1231G>T (p.G411C) in a Chinese CAH patient due to classic 11β-OHD. His parents were healthy and respectively carried the prevalent mutation c.1358G>C (p.R453Q), and the two novel mutations c.1229T>G (p.L410R) and c.1231G>T (p.G411C). In vitro expression studies, immunofluorescence demonstrated that wild type and mutant (L410R and G411C) proteins of CYP11B1 were correctly expressed on the mitochondria, and enzyme activity assay revealed the mutant reduced the 11-hydroxylase activity to 10% (P<0.001) for the conversion of 11β-deoxycortisol to cortisol. Subsequently, three dimensional homology models for the normal and mutant proteins were built by using the x-ray structure of the human CYP11B2 as a template. Interestingly, in the heme binding site I helix, a change from helix to loop in four amino acide took place in the mutant model. In conclusion, this study expands the spectrum of mutations in CYP11B1 causing to 11β-OHD and provides evidence for prenatal diagnosis and genetic counseling. In addition, our results confirm the two novel CYP11B1 mutations led to impaired 11-hydroxylase activity in vitro.
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Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene

Highlights

Abstract

Introduction

Section snippets

Patients

Gas chromatography-mass spectrometry (GC–MS) urinary steroid profiling

Urinary steroid metabolome in patients with 11β-OHD

Discussion

Acknowledgement

Trends Endocrinol. Metab.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Mol. Genet. Metab.

Gene

J. Inorg. Biochem.

J. Biol. Chem.

Congenital adrenal hyperplasia

N. Engl. J. Med.

Disorders of steroid 11 beta-hydroxylase isozymes

Endocr. Rev.

The CYP11B subfamily