背景

17α-羟化酶缺陷症（17α-Hydroxylase/17,20-lyase deficiency，17α-hydroxylase/17,20 carbon chain lyase deficiency，17-OHD）是先天性肾上腺皮质增生症（congenital adrenal hyperplasia，CAH）的罕见类型，约占CAH的1%，其发病率为1:50000 。17-OHD是由CYP17A1基因突变所致，CYP17A1基因编码由508个氨基酸组成的P450c17蛋白。该蛋白同时具有17α-羟化酶和17,20-裂链酶两种酶活性，是糖皮质激素和性激素合成过程中的关键酶。不同的CYP17A1基因突变可导致17α-羟化酶和17,20-裂链酶活性不同程度的丧失。据此可将患者分为17α-羟化酶/17,20-裂链酶联合缺陷症及孤立性17，20-裂链酶缺陷症（Isolated 17,20 lyase deficiency，ILD），前者根据P450c17蛋白酶功能受损程度的不同可进一步分为完全型或部分型17α-羟化酶/17,20-裂链酶联合缺陷症。低肾素性高血压、低钾血症、女性性幼稚、原发性闭经以及男性假两性畸形是17-OHD的典型临床表现。17-OHD患者往往会由于长期高血压而引起严重的高血压介导靶器官损害（hypertension-mediated organ damage，HMOD）。17-OHD的及早发现、准确诊断和规范治疗对于该疾病患者的病情管理乃至远期生活质量尤为重要。

由于17-OHD疾病具有较高的临床异质性，17-OHD患者在疾病早期可无典型的临床表现，往往存在诊断延迟，常于青少年阶段因高血压、低血钾和青春期性发育障碍而就诊。对于CYP17A1基因突变谱和临床表型的研究对于17-OHD疾病的临床诊疗具有重要指导意义。CYP17A1基因突变谱的研究能够从分子遗传学层面更好地分析疾病致病机理，对于疾病的分子诊断和遗传咨询也具有重要意义。到目前为止，国内外尚且缺乏关于17-OHD患者的基因突变谱及临床特征的大样本研究，并且已报道的研究主要关注于17-OHD患者疾病诊断时的临床特征，尚且缺乏关于该疾病由于长期患病而导致的HMOD发生及其相关危险因素的大样本研究。

目的

本研究旨在探究中国大样本17-OHD疾病患者的基因突变谱和临床特征的关系，并进一步分析17-OHD患者中HMOD的发生情况及其相关危险因素。

方法

纳入2003年1月至2021年12月于北京协和医院内分泌科就诊的17-OHD患者79例，收集患者的一般临床资料。提取患者外周血白细胞DNA，利用PCR扩增联合Sanger测序，分别检测患者CYP17A1基因突变，依据美国医学遗传学与基因组学学会(American College of Medical Genetics and Genomics, ACMG)指南对于检出变异位点进行致病性注释，并分析基因型和临床特征的关系。

从以上79例17-OHD患者中筛选出68例具备完整靶器官（心脏、肾脏、视网膜）损害评估资料的患者，分析患者高血压和HMOD发生情况，并采用Logistic回归模型来分析17-OHD患者发生HMOD及其各个器官损害发生的危险因素。

结果

79例17-OHD患者均存在CYP17A1基因突变，共检测出点突变、插入变异、缺失变异及插入/缺失变异37种。其中文献已报道的变异位点27种,

10种，包括:p.S94P, p.R96G, p.L361P, p.H373T

fsX418,p.I394del,p.H407QfsX415,p.L418X,p.W499X,c.436＋1G>T以及c.662_666&c.666+7delTGAAGGTGAGATinsCCACCCTGCTTTCA变异。

p.Y329KfsX418在所有等位基因中的发生率为57.0%（90/158），是本17-OHD研究队列的最常见变异位点，p.D487_F489del的发生率次之，为10.1%(16/158)。检出3次的变异位点有3种：c.297+2T>C,c.298-1G>A，R362H,检出2次的变异位点有p.A82D, p.R96W, p.T101IfsX102,p.I259HfsX274X,

和

。6号外显子和8号外显子为最常发生致病变异的外显子，这两个外显子发生的变异占所有检出变异的81.0%(128/158)。89.9%(71/79)的17-OHD患者可检测出Y329KfsX418或D487_F489del变异。

在68例17-OHD患者中，3例（4.4%）患者血压正常，65例（95.6%）患者被诊断为高血压，36例（52.9%）患者合并存在靶器官损害，被纳入HMOD组。同non-HMOD组相比，HMOD组17-OHD患者年龄更大[分别为26.00(24.00,30.00)岁和20.50(19.00,25.00)岁]，高血压病程更长[分别为8.50(2.50,12.00)年和3.50(0.25,5.75)年]，高血压等级(高血压3级比例分别为87.5%和27.8%)和低血钾分级均更高（重度低钾血症比例分别为56.3%和16.7%）。

多因素logistic回归分析结果表明，高血压病程、高血压等级和低钾血症分级是HMOD发生的独立危险因素。高血压病程每增加1年，HMOD的发生风险增加32%，高血压等级每加重1个等级，HMOD的发生风险增加10.2倍，低钾血症分级每加重1个等级，HMOD的发生风险增加2.3倍。

结论

本研究检测出37种变异，发现10种新报道的变异位点，丰富了CYP17A1 的罕见变异谱。

c.985_987delTACinsAA(Y329KfsX418)和c.1459_1467delGACTCTTTC (D487

是中国17-OHD患者的 “热点变异”位点。

17-OHD患者发生HMOD的机率较高。

HMOD的发生与突变CYP17A1的酶残余活性无相关性，较长的高血压病程、严重的高血压等级与低钾血症等级是发生HMOD的独立危险因素。

关键词：17α-羟化酶缺陷症，CYP17A1基因，基因突变谱，临床特征

Context

17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare type of congenital adrenal hyperplasia and accounts for nearly 1% of the condition. 17-OHD has an estimated incidence rate of 1:50000 and is an autosomal recessive disorder caused by biallelic mutations in the CYP17A1 gene. The gene encodes the P450c17 protein composed of 508 amino acids, with activities of both 17α-hydroxylase and 17, 20-lyase, which is a key enzyme in the biosynthesis of glucocorticoids and sex steroids.

Different CYP17A1 genetic mutations can lead to varying degrees of loss of 17α-hydroxylase/17,20-lyase. Accordingly, the patients suffered can be divided into combined 17α -hydroxylase /17, 20-lyase deficiency and isolated 17,20 lyase deficiency (ILD), the former can be further divided into complete or partial form of combined 17α -hydroxylase /17, 20-lyase deficiency according to the quite different degrees of functional impairment of P450c17 protein.  Low-renin hypertension, hypokalemia, female infantilism, primary amenorrhea, and male pseudohermaphroditism are the typical clinical features of 17-OHD. It has been reported that 88% of the 17-OHD cases are not diagnosed accurately until puberty or even later due to its extremely low incidence and varied clinical phenotypes. Accordingly, some 17-OHD patients may develop HMOD because of poor blood pressure control and inappropriate disease management. For the patients of 17-OHD, early detection, accurate diagnosis and standardized treatment of 17-OHD are particularly important for the management of health status and the maintenance of long-term life quality. 

Due to the pretty high clinical heterogeneity of 17-OHD, the patients suffered often do not seek treatment owing to the lack of obvious clinical manifestations in the early stage and later consult the doctor for hypertension, hypokalemia and sexual development abnormality. The study of CYP17A1 genetic mutational profiles and clinical phenotype can provide important guidance for the clinical diagnosis and treatment of 17-OHD and help to better understand pathogenesis of 17-OHD from the perspective of molecular genetics, which is also of great significance for  accurate  molecular diagnosis and meaningful genetic counseling.  Up to now, large sample studies of genetic mutational profiles and clinical characteristics on the 17-OHD patients are still lacking at home and abroad, the reported studies mainly focused on the clinical characteristics of 17-OHD patients at the first visit, the occurrence of HMOD and its related risk factors based on large clinical cohort has never been analyzed.

Objective

The purpose of this study was to explore the relationship between genetic mutations and the clinical features in a large cohort of 17-OHD patients in China, and the occurrence of HMOD and its related risk factors were also further analyzed.  

Methods

A total of 79 Chinese patients with 17-OHD who were admitted to the Peking Union Medical College Hospital (Beijing) were recruited for this retrospective cohort study between 2003 and 2021. Detailed medical data were collected and analyzed. DNA was extracted from peripheral blood leukocytes of patients, PCR amplification combined with Sanger sequencing was used to detect the CYP17A1 genetic mutations.  The pathogenicity of the loci was annotated according to the American College of Medical Genetics and Genomics (ACMG) guidelines, and the relationship between the genotype and the clinical characteristics was also analyzed. 

68 patients with detailed damage assessment data of target organs (i.e., heart, kidney and retina) were selected to analyze the occurrence of hypertension and HMOD, the Logistic regression model was further employed to analyze the risk factors of HMOD and the respective organ damage.  

Results

37 kinds of genetic mutations (i.e., point mutation, insertion mutation, deletion mutation and insertion/deletion mutation) were detected from 79 patients of 17-OHD. Among them, 27 mutations had been reported in previous literature.10 variants were newly identified in this study, including p.S94P, p.R96G, p.L361P,p.H373TfsX418,p.I394del,p.H407QfsX415,p.L418X,p.W499X,c.436＋1G>Tand c.662_666&c.666+7delTGAAGGTGAGATinsCCACCCTGCTTTCA.

p. Y329KfsX418(57.0%,90/158) and p.D487_F489del (10.1%,16/158)were the top 2 mutations in this Chinese cohort. The mutations detected three times include c.297+2T>C, c.298-1G>A, R362H. The mutations detected twice include A82D, R96W, T101IfsX102, I259HfsX274, p.325/326/327Kdel, R358X, L361FfsX418, L361P, R362C, H373L and P409R.Exon 6 and exon 8 were the most common exons with pathogenic mutations, 81.0% (128/158) of the total detected mutations were from the above two exons. p.Y329KfsX418 or p. D487_F489 del can be detected in 89.9% (71/79) % of the 17-OHD patients.

Among the 68 patients with 17-OHD, 3(4.4%) were normotensive and the remaining 65 (95.6%) were hypertensive. 36 patients (52.9%) were identified to have HMOD. Compared with the patients in the non-HMOD group, the patients in the HMOD group were older [26.00 (24.00,30.00) years and 20.50 (19.00,25.00) years, respectively]. The hypertension duration of the 17-OHD patients in the HMOD was longer (8.50 (2.50,12.00) years and 3.50(0.25,5.75) years, respectively), and the hypertension grade (the proportion of grade 3, 87.5% and 27.8%, respectively) and hypokalemia grade were both higher (the proportion of severe hypokalemia,56.3% and 16.7%, respectively) in the HMOD group. 

Disease duration, hypertension grade, and hypokalemia grade and medication duration were the independent risk factors for HMOD in the patients with 17-OHD, as inferred from multivariate logistic regression analysis. The risk of HMOD increased by 32% for each additional year of disease duration, 10.2-fold for each one-grade increase in hypertension level, and 2.3-fold for each grade of exacerbation of hypokalemia.

Conclusion

In this study, 37 variants (including 10 newly reported variants) were detected, which enriched the rare variation spectrum of CYP17A1 in 17-OHD. 

c.985_987del TAC ins AA (p.Y329KfsX418) (57.0%,90/158) and c.1459_1467del (p. del D487_F489) were ‘hot spot’ mutations in Chinese 17-OHD patients.  

Patients with 17-OHD experience high incidence of HMOD

There was no correlation between the HMOD occurrence and enzyme activity of mutant CYP17A1.Disease duration, hypertension grade, hypokalemia grade and medication duration are independent risks for the occurrence of HMOD.

Keywords: 17α-Hydroxylase/17,20-lyase deficiency, CYP17A1, Genetic profile,clinical characteristics