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Splicing analysis of CYP11B1 mutation in a family affected with 11?-hydroxylase deficiency: case report.

ABSTRACT: Congenital adrenal hyperplasia (CAH) due to steroid 11?-hydroxylase deficiency (11?-OHD) is a rare form of CAH associated with low renin hypertension, hypokalemia, hyperandrogenemia and ambiguous genitalia in affected females. Herein we describe the clinical, hormonal and molecular characteristics of two Uzbekistan siblings with 11?-OHD and analyze the effects of a splicing mutation.A 46,XX girl presented with genital ambiguity and low renin hypertension; her 46,XY brother presented with precocious puberty. Hormonal studies suggested 11?-OHD. Mutation analysis was performed by PCR followed by Sanger sequencing of the entire coding regions and their flanking introns of the CYP11B1 gene. Mutation analysis showed that both patients were compound heterozygous for IVS7?+?1G?>?A, and c.421C?>?T. Although the identified mutations have been previously described, this is, to our knowledge, the first report of these mutations in compound heterozygotes. A minigene assay was used to determine the effects of the splicing mutation. The constructs containing either the wild-type or the splice-site mutant CYP11B1 genomic DNA of exons-introns 6-9 were transfected into COS-7 cells; subsequently, RNA splicing was assessed by reversed transcribed-PCR of CYP11B1 complementary DNA. The minigene assay revealed that the IVS7?+?1G?>?A mutation resulted in two shorter incorrectly spliced products; one skipping the exon 7 and the other skipping the exons 7-8. The c.421C?>?T mutation leads to the introduction of a premature stop codon at residue 141 (p.R141X). These mutations are expected to code non-functional proteins.Compound heterozygous mutations (IVS7?+?1G?>?A and p.R141X) in the CYP11B1 gene were found to cause 11?-OHD. The IVS7?+?1G?>?A mutation causes aberrant splicing of CYP11B1 leading to exon skipping. This finding could facilitate the future novel therapies targeted on splicing modulation to treat human disease.

SUBMITTER: Charnwichai P

PROVIDER: S-EPMC4912772 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Splicing analysis of CYP11B1 mutation in a family affected with 11β-hydroxylase deficiency: case report.

Charnwichai Pattaranatcha P Yeetong Patra P Suphapeetiporn Kanya K Supornsilchai Vichit V Sahakitrungruang Taninee T Shotelersuk Vorasuk V

BMC endocrine disorders 20160617 1

<h4>Background</h4>Congenital adrenal hyperplasia (CAH) due to steroid 11β-hydroxylase deficiency (11β-OHD) is a rare form of CAH associated with low renin hypertension, hypokalemia, hyperandrogenemia and ambiguous genitalia in affected females. Herein we describe the clinical, hormonal and molecular characteristics of two Uzbekistan siblings with 11β-OHD and analyze the effects of a splicing mutation.<h4>Case presentation</h4>A 46,XX girl presented with genital ambiguity and low renin hypertens ...[more]

PMID: 27316665

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Project description:Unlabelled11β-hydroxylase deficiency (11β-OHD), an autosomal recessive inherited disorder, accounts for 5-8% of congenital adrenal hyperplasia. In Greece, no cases of 11β-OHD have been described so far. The patient presented at the age of 13 months with mild virilization of external genitalia and pubic hair development since the age of 3 months. Hormonal profile showed elevated 11-deoxycortisol, adrenal androgens and ACTH levels. ACTH stimulation test was compatible with 11β-OHD. DNA of the proband and her parents was isolated and genotyped for CYP11B1 gene coding cytochrome P450c11. The girl was found to be compound heterozygous for two CYP11B1 novel mutations, p.Ala386Glu (exon 7), inherited from the father and p.Leu471Argin (exon 9) from the mother. Hydrocortisone supplementation therapy was initiated. Four years after presentation she remains normotensive, her growth pattern is normal and the bone age remains advanced despite adequate suppression of adrenal androgens.Learning points11β-hydroxylase (CYP11B1) deficiency (11OHD; OMIM +202010) is the second most common cause of CAH accounting for approximately 5-8% of cases with an incidence of 1:100 000-1:200 000 live births in non-consanguineous populations.Two CYP11B1 inactivating novel mutations, p.Ala386Glu and p.Leu471Arg are reportedRegarding newborn females, in utero androgen excess results in ambiguous genitalia, whereas in the male newborn diagnosis may go undetected. In infancy and childhood adrenal androgen overproduction results in peripheral precocious puberty in boys and various degrees of virilization in girls.Accumulation of 11-deoxycorticosterone and its metabolites causes hypertension in about two thirds of patients.Diagnosis lies upon elevated 11-deoxycortisol and DOC plus upstream precursors, such as 17α-hydroxyprogesterone and Δ4-androstenedione.The established treatment of steroid 11β-OHD is similar to that of steroid 21-hydroxylase deficiency and consists of glucocorticoid administration in order to reduce ACTH-driven DOC overproduction resulting in hypertension remission and improvement of the virilization symptoms.

Project description: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.

Dataset Information

Splicing analysis of CYP11B1 mutation in a family affected with 11?-hydroxylase deficiency: case report.

Publications

Splicing analysis of CYP11B1 mutation in a family affected with 11β-hydroxylase deficiency: case report.

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