Congenital Adrenal Hyperplasia Due to 11-Hydroxylase Deficiency: Functional Characterization of Two Novel Point Mutations and a Three-Base Pair Deletion in theCYP11B1Gene

Abstract

Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion (DeltaF438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 +/- 0.9% (sd) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 +/- 0.9%, whereas the DeltaF438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the DeltaF438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency

“…We found that 41 compound heterozygotes or homozygotes for select missense or nonsense mutations-namely P49L, R141Q, W260X, G267S, L299P, T318M, T318R, A331V, Q356X, A368D, R374Q, V441G, G444D, G446V, and R448H-present with moderate to severe classic CAH because of 11β-hydroxylase, confirming prior results (3,(14)(15)(16)(17)(22)(23)(24). These patients were mainly from Croatia, Tunisia, Africa, Turkey, and Saudi Arabia.…”

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

“…8 The mutagenesis was performed using the QuikChange XL Site-Directed Mutagenesis Kit according to the manufacturer's protocol (Stratagene, Amsterdam, The Netherlands). The introduction of the mutations was confirmed by sequencing the complete cDNA.…”

“…Western blot analysis using a rabbit antihuman CYP11B1 antibody (kindly provided by Dr H Takemori, Department of Molecular Physiological Chemistry, Osaka University Medical School, Osaka, Japan) was performed on the basis of a standard protocol to ensure the expression and translation of CYP11B1 wild-type and mutant proteins. 8 …”

“…14 Parents were not available for genetic analysis, and thus the status of compound heterozygosity is inferred only from the phenotype. Gene analyses in patients 2 and 3 revealed that they were compound heterozygous for the paternal mutation c.896T4C (p.(Leu299Pro)) 8 and for a novel mutation c.995G4A (p.(Arg332Gln)) inherited from the mother (Figure 1). Gene analysis in patient 4 revealed that she was compound heterozygous for a novel c.928G4A mutation inherited from the father, resulting in the amino-acid change p.(Glu310Lys) in the CYP11B1 protein and for the previously described mutation c.896T4C leading to p.(Leu299-Pro) 8 inherited from the mother (Figure 1).…”

“…Gene analyses in patients 2 and 3 revealed that they were compound heterozygous for the paternal mutation c.896T4C (p.(Leu299Pro)) 8 and for a novel mutation c.995G4A (p.(Arg332Gln)) inherited from the mother (Figure 1). Gene analysis in patient 4 revealed that she was compound heterozygous for a novel c.928G4A mutation inherited from the father, resulting in the amino-acid change p.(Glu310Lys) in the CYP11B1 protein and for the previously described mutation c.896T4C leading to p.(Leu299-Pro) 8 inherited from the mother (Figure 1). The analysis in patient 5 revealed that she was compound heterozygous for the paternally inherited mutation c.1066C4T (p.(Gln356Ter)) and for the mutation c.917C4T (p.(Ala306Val)) inherited from the mother (Figure 1).…”