Novel non-classic CYP21A2 variants, including combined alleles, identified in patients with congenital adrenal hyperplasia

• Published in: Clinical biochemistry Vol. 73; pp. 50 - 56
• Main Authors: Karlsson, Leif, de Paula Michelatto, Débora, Lusa, Ana Letícia Gori, D'Almeida Mgnani Silva, Camila, Östberg, Linus J., Persson, Bengt, Guerra-Júnior, Gil, Valente de Lemos-Marini, Sofia Helena, Baldazzi, Lilia, Menabó, Soara, Balsamo, Antonio, Greggio, Nella Augusta, Palandi de Mello, Maricilda, Barbaro, Michela, Lajic, Svetlana
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2019
• Subjects: 21-hydroxylase; Congenital adrenal hyperplasia; Functional studies; Medicin och hälsovetenskap; Synergistic effects; Synergistic effects; Congenital adrenal hyperplasia; Functional studies; 21-hydroxylase
• ISSN: 0009-9120; 1873-2933; 1873-2933
• DOI: 10.1016/j.clinbiochem.2019.07.009

Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH.
All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed.
The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40–82%). An additive effect on the reduction of enzymatic activity (1–17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro.
Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.
•All novel amino acid changes had mild effects on enzyme function when expressed alone.•In vitro studies confirmed an additive effect between mild variants on the same allele, leading to a more severe phenotype.•In silico predictions were in line with the in vitro data except for the p.Leu461Pro variant.