Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients

Abstract

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients.

Figure 1. Representative electropherograms (left panel) and restriction enzyme assays (right panel) of the five novel mutations found.

Left panel: A: Mutation g. 782C>T, in exon 3, changes an R residue to C at position 132. B: Mutation g.940C>T, in exon 4, changes an R residue in position 149 to a C residue. C. Mutation g.1695A>G, in exon 7, predicts a change in residue M283 to V283. D: mutation g.2515G>A, in exon 10, changes E431 to K431. E: mutation g.2511_2512delGG in exon 10 leads to a frameshift in the carboxy-terminal end of the protein. Right panel: A: BtsCI restriction enzyme assay for R132C. B: HhaI restriction enzyme assay for R149C. C: Hin1II restriction enzyme assay for M283V. D: BseDI restriction enzyme assay for g.2511_2512delGG in exon 10. E: BsrbI restriction enzyme assay for E431K. C: control individuals, P: patients. C(-E): control without enzyme. MWM: molecular weight marker.

Figure 2. Partial Clustal W alignment analyses of CYP21A2 proteins from different mammalian species.

The residues of each mutation are highlighted in yellow. A: R residue at position 132. B: R residue at position 149. C. M residue at position 283. D: E residue at position 431.

Figure 3. Structural analysis.

A: Cartoon representation of the structure of CYP21A2. Residues implicated in the novel mutations found are labeled and highlighted by blue spheres. The residue D322 is also represented. Heme cofactor is depicted in sticks. B: Model discrepancies in the surroundings of V281. Detail of the superimposition of the Biskit (green) and 2GEG (orange) models. V281 and M283 are named and depicted in sticks. C: Enhancement of the basic region in the mutant surface. Surface electrostatics of the wild type E431 and mutant K431 are presented. Acidic regions are depicted in red and basic ones in blue.