Department of Women's and Children's Health, Karolinska Institutet, Pediatric Endocrinology Unit (Q2:08), Karolinska University Hospital, Stockholm, Sweden.
Department of Women's and Children's Health, Karolinska Institutet, Pediatric Endocrinology Unit (Q2:08), Karolinska University Hospital, Stockholm, Sweden; Laboratório de Genética Molecular Humana, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Clin Biochem. 2019 Nov;73:50-56. doi: 10.1016/j.clinbiochem.2019.07.009. Epub 2019 Jul 22.
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.
先天性肾上腺皮质增生症（CAH）是一种先天性代谢缺陷，也是一种常见的性发育障碍，其中>90%的病例是由于 21-羟化酶缺乏引起的。新型和罕见的致病性变异占所有临床病例的 5%。在这里，我们试图研究四个新型（p.Val358Ile、p.Arg369Gln、p.Asp377Tyr 和 p.Leu461Pro）和三个 CYP21A2 变异（即一个等位基因包含两个变异 p.[Ile172Asn;Val358Ile]、p.[Val281Leu;Arg369Gln] 或 p.[Asp377Tyr;Leu461Pro]）在 CAH 患者中的功能和结构影响。
通过体外定点诱变重建所有变异，瞬时表达在 COS-1 细胞中，并测定两种天然底物（17-羟孕酮和孕酮）的酶活性。同时，基于人类 CYP21 X 射线结构对变异的致病性进行了计算机预测。
新型变异 p.Val358Ile、p.Arg369Gln、p.Asp377Tyr 和 p.Leu461Pro 表现出非经典（NC）CAH 变异范围内的残余酶活性（40-82%）。当两个变异一起表达时，观察到酶活性降低（1-17%）的累加效应，如在几个患者中发现的那样，导致 NC 或更严重的表型。计算机预测与体外数据一致，除了 p.Leu461Pro。
总之，临床数据、计算机预测和体外研究数据的结合对于在 CAH 患者中建立正确的基因型和表型相关性非常重要。
