Molecular analysis of 12 Chinese patients with 11β‐hydroxylase deficiency and in vitro functional study of 20 CYP11B1 missense variants
Molecular analysis of 12 Chinese patients with 11β‐hydroxylase deficiency and in vitro functional study of 20 CYP11B1 missense variants
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DOI：
10.1096/fj.202201398rr
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
B. Sun;Lin Lu;Shaowei Xie;Wei Zhang;Xiaoxiang Zhang;A. Tong;Shi Chen;Xueyan Wu;J. Mao;Xi Wang;L. Qiu;M. Nie
中科院分区：
文献类型：
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作者：
B. Sun;Lin Lu;Shaowei Xie;Wei Zhang;Xiaoxiang Zhang;A. Tong;Shi Chen;Xueyan Wu;J. Mao;Xi Wang;L. Qiu;M. Nie
类固醇11β-羟化酶缺乏症（11β-OHD）是一种罕见的常染色体隐性遗传病，由CYP 11B 1基因的致病性变异引起。本研究旨在对中国11β-OHD系列进行分子分析，并对20种CYP 11B 1错义变体进行体外功能研究。12例临床诊断为11β-OHD的中国患者被纳入研究，以分析其分子病因。提取患者基因组DNA，测序CYP 11B 1基因所有编码外显子和内含子侧翼序列。对沿着在12例患者中发现的14个错义突变体和本课题组先前报道的6个错义突变体进行了功能评价。使用计算程序分析氨基酸取代，以确定其对CYP 11B 1蛋白三维结构的影响。记录18例携带上述18种错义突变的患者的临床特征和基线激素水平，进行基因型-表型相关分析。在12例患者中共鉴定出21种罕见变异，包括9种新变异和12种复发变异，其中17种为错义变异，2种为无义变异，1种为剪接位点变异，1种为缺失-插入变异。体外功能研究结果表明，20个错义突变体中有3个（p.Leu3Pro，p.Gly267Ser和p.Ala367Ser）具有部分酶活性，其余17个几乎没有酶活性。体外功能研究中酶活性的受损程度也反映在三维模型中野生型/突变型氨基酸与其相邻氨基酸之间相互作用变化的严重程度上。总之，增加9种新变体扩大了CYP 11B 1致病性变体的谱。我们的研究结果表明，20种CYP 11B 1变体导致体外11β-羟化酶活性受损。在CYP 11B 1蛋白的三维模型结构中可视化这些变体可以为体外测量的结果提供合理的解释。
Steroid 11β‐hydroxylase deficiency (11β‐OHD) is a rare autosomal recessive disorder caused by pathogenic variants of CYP11B1 gene. This study aimed to perform molecular analysis of a Chinese 11β‐OHD series and in vitro functional study of twenty CYP11B1 missense variants. Twelve Chinese patients with clinical diagnosis of 11β‐OHD were included in the study to analyze their molecular etiology. Genomic DNA of patients was extracted to be sequenced all coding exons and intronic flanking sequences of CYP11B1. Fourteen missense variants found in 12 patients mentioned above along with 6 missense variants previously reported by our team were evaluated functionally. Amino acid substitutions were analyzed with computational program to determine their effects on the three‐dimensional structure of CYP11B1 protein. Clinical characteristics and hormone levels at baseline of the 18 patients carrying 18 missense variants aforementioned were recorded to perform genotype–phenotype correlation. A total of 21 rare variants including 9 novel and 12 recurrent ones were identified in 12 patients, out of which 17 were missense, 2 were nonsense, 1 was a splice site variant, and 1 was a deletion–insertion variant. Results of in vitro functional study revealed that 3 out of 20 missense mutants (p.Leu3Pro, p.Gly267Ser, and p.Ala367Ser) had partial enzyme activity and the other 17 had little enzymatic activity. The impairment degree of enzymatic activity in vitro functional study was also reflected in the severity degree of interaction change between the wild‐type/mutant‐type amino acid and its adjacent amino acids in three‐dimensional model. In conclusion, the addition of 9 novel variants expands the spectrum of CYP11B1 pathogenic variants. Our results demonstrate that twenty CYP11B1 variants lead to impaired 11β‐hydroxylase activity in vitro. Visualizing these variants in the three‐dimensional model structure of CYP11B1 protein can provide a plausible explanation for the results measured in vitro.