Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

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.

由21-羟化酶缺乏（21-hydroxylase deficiency）所致的先天性肾上腺皮质增生症（Congenital adrenal hyperplasia, CAH）是最常见的先天性代谢缺陷病，约占所有CAH病例的90%~95%。该缺陷酶P450C21由CYP21A2基因编码，该基因与一段核苷酸序列同源性高达98%的CYP21A1P假基因（pseudogene）共同定位于6号染色体p21.3区域。尽管多数患者携带源自CYP21A1P的突变，但近年来在致病等位基因中发现的新型罕见突变数量持续增多。本研究报道了4例来自阿根廷的非经典型及1例失盐型CAH患者体内检出的5种CYP21A2新型突变，分别为p.R132C、p.149C、p.M283V、p.E431K以及移码突变g.2511_2512delGG。所有新型点突变均位于哺乳动物物种间高度保守的CYP21蛋白残基位点，且在对照个体中均未检测到上述突变。本研究通过计算机模拟（in silico）分析了上述新型变异的潜在致病机制：通过同源建模（homology modeling）构建CYP21的三维结构，并利用蛋白质设计算法FoldX计算CYP21A2蛋白的稳定性变化。分析结果显示，突变酶的蛋白稳定性或表面电荷发生改变，这可能与患者的临床表现存在关联。