title: Phenotypic, metabolic, and molecular genetic characterization of six patients with congenital adrenal hyperplasia caused by novel mutations in the CYP11B1 gene.
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.
authors.affiliation: Department of Biochemistry, Saarland University, D-66123 Saarbrücken, Campus B2.2, Germany; Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam.
authors.forename: Huy-Hoang
authors.initials: HH
authors.lastname: Nguyen
authors.affiliation: Department of Biochemistry, Saarland University, D-66123 Saarbrücken, Campus B2.2, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Eiden-Plach
authors.affiliation: Department of Biochemistry, Saarland University, D-66123 Saarbrücken, Campus B2.2, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Departments of Biochemistry and Experimental Medicine, The Children's Memorial Health Institute, 04-730 Warsaw, Poland.
authors.forename: Ewa M
authors.initials: EM
authors.lastname: Malunowicz
authors.affiliation: Steroid Research &Mass Spectrometry Unit, Division of Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen, Germany.
authors.forename: Michaela F
authors.initials: MF
authors.lastname: Hartmann
authors.affiliation: Steroid Research &Mass Spectrometry Unit, Division of Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen, Germany.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Wudy
authors.affiliation: Department of Biochemistry, Saarland University, D-66123 Saarbrücken, Campus B2.2, Germany. Electronic address: ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000209:etiology* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005091:Exons; D005260:Female; D006801:Humans; D007438:Introns; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D010375:Pedigree; D022821:RNA Splice Sites; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013256:Steroids / Q000652:urine
chemical_list: D022821:RNA Splice Sites; D013256:Steroids; D013252:Steroid 11-beta-Hydroxylase
keywords: 11β-hydroxylase; CYP11B1; CYP11B1 mutant; Congenital adrenal hyperplasia; Steroid hormone