Endocrinol Metab.  2018 Sep;33(3):413-422. 10.3803/EnM.2018.33.3.413.

Functional Identification of Compound Heterozygous Mutations in the CYP17A1 Gene Resulting in Combined 17α-Hydroxylase/17,20-Lyase Deficiency

• Mo EY
• Lee JY
• Kim SY
• Kim MJ
• Kim ES
• Lee S
• Han JH
• Moon SD

• KMID: 2447032
• DOI: http://doi.org/10.3803/EnM.2018.33.3.413

Abstract

BACKGROUND
We previously reported a patient with congenital adrenal hyperplasia (CAH) with compound heterozygous mutations in the cytochrome P450 17A1 (CYP17A1) gene. One allele had a p.His373Leu and the other a new p.Glu383fsX36 mutation. The aim of this study was to investigate the functional properties of a new allele present in a compound heterozygote of CYP17A1.
METHODS
To understand how p.His373Leu and p.Glu383fsX36 affect P450c17 enzymatic activity, wild type and mutant CYP17A1 cDNAs were cloned into flag-tagged pcDNA3 vector and introduced into human embryonic kidney cells 293T (HEK293T) cells. Protein expression levels of CYP17A1 were then analyzed. And the activities of 17α-hydroxylase and 17,20-lyase of CYP17A1 were evaluated by measuring the conversion of progesterone to 17α-hydroxyprogesterone and of 17α-hydroxypregnenolone to dehydroepiandrosterone, respectively. In addition a computer model was used to create the three-dimensional structure of the mutant CYP17A1 enzymes.
RESULTS
Production of the p.His373Leu mutant protein was significantly lower than that of the wild type protein, and the p.Glu383fsX36 protein was hardly produced. Similarly the enzymatic activity derived from the p.His373Leu mutant vector was significantly lower than that obtained from the wild type vector, and little activity was obtained from the p.Glu383fsX36 vector. Three-dimensional modeling of the enzyme showed that p.His373 was located in region important for heme-binding and proper folding. Neither the p.His373Leu nor the p.Glu383fsX36 mutant protein formed a heme-binding structure.
CONCLUSION
Enzyme activity measured in both mutants disappeared completely in both 17α-hydroxylase and 17,20-lyase. This result accounts for the clinical manifestations of the patient with the compound heterozygous CYP17A1 mutations.

Keyword

Adrenal hyperplasia, congenital; 17-Alpha-hydroxylase; 17,20-Lyase; Steroid 17-alpha-hydroxylase

MeSH Terms

Adrenal Hyperplasia, Congenital
Alleles
Clone Cells
Computer Simulation
Cytochrome P-450 Enzyme System
Dehydroepiandrosterone
DNA, Complementary
Heterozygote
Humans
Kidney
Mutant Proteins
Progesterone
Steroid 17-alpha-Hydroxylase
Cytochrome P-450 Enzyme System
DNA, Complementary
Dehydroepiandrosterone
Mutant Proteins
Progesterone
Steroid 17-alpha-Hydroxylase

Figure

• 

  Fig. 1 Schematic representation of the constructs used to measure P450c17 enzyme activity produced in human embryonic kidney cells 293T (HEK293T) cells. The entire coding regions of c.1118A>T, c.1148delA, and wild type (WT) cytochrome P450 17A1 (CYP17A1) complementary DNAs (cDNAs) were subcloned into the HindIII and KpnI site of pcDNA3 vector with a C-terminal flag tag. The positions of restriction enzymes, start (ATG) and stop (tga) codons, and HindIII, KpnI, and XhoI sites, are shown. Exons are indicated with arabic numerals and the mutation sites (c.1118A>T and c.1148delA) are indicated. The 1 bp deletion (bp position 1148 in c.1148delA, indicated by the arrow) results in a shift of the reading frame and a termination codon after 36 amino acids. pCMV, promoter cytomegalovirus; SV40, simian virus 40; MT1, mutant type 1; MT2, mutant type 2.

• 

  Fig. 2 The compound cytochrome P450 17A1 (CYP17A1) heterozygote. (A) Pedigree of 17α-hydroxylase/17,20-lyase deficient family. Individuals are represented as follows: male (square); females (circles). The black symbol represents the c.1148delA allele and the gray symbol represents the c.1118A>T allele. The arrow indicates the compound heterozygous proband. The proband inherited mutations c.1118A>T and c.1148delT from her mother and father, respectively. (B) Subcloning of the compound heterozygous CYP17A1 gene. Polymerase chain reaction products of RNA from the proband's leucocytes were subcloned and sequenced to confirm heterozygous mutations. The c.1148delA mutation was found to be in exon 7 and the c.1118A>T mutation in exon 6. WT, wild type; A/T, A and T.

• 

  Fig. 3 Expression and enzyme activities of P450c17 protein produced by cytochrome P450 17A1 (CYP17A1) complementary DNA (cDNA) overexpressed in human embryonic kidney cells 293T (HEK293T) cells. (A, B) Analysis of P450c17 proteins by Western blotting (WB) and immunofluorescence, respectively. Expression of c.1118A>T and the doubly transfected clone (p.H373L and p.E383fsX36) in HEK293T cells is significantly lower than that of wild type (WT) and no expression of c.1148delA mutant is detected. (C) Enzyme activity. Production of 17α-hydroxyprogesterone (17-OHP) in the presence of various concentrations of progesterone (P) (0, 1.0, and 2.0 µM) and production of dehydroepiandrosterone (DHEA) in the presence of various concentration of 17α-hydroxypregnenolone (17-PREG) (0, 1.0, and 2.0 µM). HEK293T cells were transfected with WT, c.1118A>T, c.1148delA, and co-transfection. Compared to the enzymatic activity against WT (744 nmol/L), the 17α-hydroxylase activity for the p.His373Leu mutant was reduced to 21.9 nmol/L and the 17,20-lyase activity for the p.Glu383fsX36 mutant was almost zero (1.3 nmol/L). Data are mean values of P450c17 activity assayed in triplicate. Data are expressed as mean±SD (n=3). DAPI, 4',6-diamidino-2-phenylindole.

• 

  Fig. 4 Computer model of cytochrome P450 17A1 (CYP17A1) (PDB ID; 3SWZ). A, a ribbon diagram of the protein backbone, with heme (red) and highlighting p.His373, p.Glu383, p.His373Leu, p.Glu383Gly. Also shown are residues p.His373 and p.His373Leu in green and p.Glu383 and p.Glu383Gly in yellow. Substitution of His by Leu at amino acid position 373 results in loss of catalytic activity. Mutation of the His-deficient mutant at position 373 or substitution of Glu with Gly at position 383 prevents the incorporation of a heme group, rendering the mutant enzyme inactive. The imidazole side chain of His is a common ligand for the metal protein and is part of the catalytic site of the enzyme. His can participate in acidic catalysis by proton and non-proton states, but Leu does not bind to heme because there is no imidazole branch. Images were created in Swiss PDB viewer (Swiss Institute of Bioinformatics) and UCSF Chimera version 1.11.2 (University of California).

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