Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia
Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia
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DOI：
10.1530/ec-14-0032
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发表时间：
2014-06-01
影响因子：
2.9
通讯作者：
Husebye, Eystein S.
中科院分区：
文献类型：
--
作者：
Bronstad, Ingeborg;Breivik, Lars;Husebye, Eystein S.
在大约95%的病例中，先天性肾上腺皮质增生（CAH）是由编码类固醇21-羟化酶（21 OH）的CYP21A2基因突变引起的。最近，我们在挪威CAH患者人群中报告了4种新型CYP21A2变异体，其中p.L388R和p.E140K与盐耗（SW）相关，p.P45L与单纯男性化（SV）相关，p.V211M + p.V281L与SV至非经典（NC）表型相关。我们的目的是利用新设计的21 OH酶活性和结构模拟的体外测定法，在功能上鉴定新的变体，并将结果与临床表型进行比较。CYP21A2突变和变体在体外表达。通过液相色谱串联质谱法评估17-羟孕酮向11-脱氧皮质醇的转化来测定酶活性。PyMOL 1.3用于结构模拟，PolyPhen2和PROVEAN用于预测突变体的严重性。CYP21A2突变体p.L388R和p.E140K在体外分别表现出1.1%和11.3%的wt 21OH酶活性。我们在体外未检测到p.P45L变体的任何功能缺陷;尽管预测工具表明p.P45L是致病性的。p.V211M在体外显示出与wt相当的酶活性，这得到了计算机模拟分析的支持。我们发现SW突变体的表型和体外酶活性之间存在良好的相关性，但SV p.P45L变异体的表型和体外酶活性之间没有相关性。p.V211M可能与p.V281L具有协同效应，解释了SV和NC CAH之间的表型。
In about 95% of cases, congenital adrenal hyperplasia (CAH) is caused by mutations in CYP21A2 gene encoding steroid 21-hydroxylase (21OH). Recently, we have reported four novel CYP21A2 variants in the Norwegian population of patients with CAH, of which p.L388R and p.E140K were associated with salt wasting (SW), p.P45L with simple virilising (SV) and p.V211M+p.V281L with SV to non-classical (NC) phenotypes. We aimed to characterise the novel variants functionally utilising a newly designed in vitro assay of 21OH enzyme activity and structural simulations and compare the results with clinical phenotypes. CYP21A2 mutations and variants were expressed in vitro. Enzyme activity was assayed by assessing the conversion of 17-hydroxyprogesterone to 11-deoxycortisol by liquid chromatography tandem mass spectroscopy. PyMOL 1.3 was used for structural simulations, and PolyPhen2 and PROVEAN for predicting the severity of the mutants. The CYP21A2 mutants, p.L388R and p.E140K, exhibited 1.1 and 11.3% of wt 21OH enzyme activity, respectively, in vitro. We could not detect any functional deficiency of the p. P45L variant in vitro; although prediction tools suggest p.P45L to be pathogenic. p.V211M displayed enzyme activity equivalent to the wt in vitro, which was supported by in silico analyses. We found good correlations between phenotype and the in vitro enzyme activities of the SW mutants, but not for the SV p.P45L variant. p.V211M might have a synergistic effect together with p.V281L, explaining a phenotype between SV and NC CAH.