背景

Liddle综合征（Liddle syndrome）是一种单基因遗传性高血压，由编码上皮钠通道（epithelial sodium channels, ENaC）α、β及γ亚基的SCNN1A、SCNN1B和SCNN1G基因突变所导致，主要表现为顽固的早发高血压、低钾血症及低醛固酮血症。但由于患者临床表现不典型，缺乏系统规范的诊断策略，Liddle综合征早期误诊率极高，很多患者无法在发病早期及时确诊并接受特异性药物阿米洛利的治疗，中青年期即可发生缺血性脑卒中、脑出血及心力衰竭等严重的心脑血管事件。因此，如何实现早期诊断、及时治疗并进行规律随访是Liddle综合征诊治的重点。

基因检测是诊断Liddle综合征的金标准，准确识别Liddle综合征的疑诊患者并进行基因检测可提高Liddle综合征的诊断率。然而，目前国内外尚无Liddle综合征系统的诊断策略，因此，制定系统的诊疗策略将有助于临床医师对Liddle综合征的早期识别，提高Liddle综合征的诊断率及控制率，预防Liddle综合征患者严重心脑血管事件的发生。此外，不断完善Liddle综合征致病突变谱是提高基因检测准确性的基础。由于目前位于SCNN1A、SCNN1B及SCNN1G基因的部分突变位点的致病性还未阐明，基因检测结果解读存在困难，影响诊断。因此，通过细胞及动物实验确定新突变位点的致病性，并对突变位点的功能进行研究将有助于临床中基因检测结果的快速解读。

本研究将通过分析Liddle综合征患者的临床特征建立起临床诊疗策略，并进行诊断效能检验。在收集诊疗资料的过程中，发现一个编码ENaC的β亚基的SCNN1B基因新突变c.1691_1693delinsG，拟通过细胞实验进一步鉴定该突变位点的致病性。同时，通过转基因动物实验验证SCNN1G基因Glu571*新突变的致病性，并构建SCNN1G基因突变的Liddle综合征动物模型，探究该突变位点的功能及具体致病机制，以丰富Liddle综合征致病突变谱。

第一部分 Liddle综合征临床诊疗策略构建与长期随访研究

目的

通过对Liddle综合征患者的临床特征及治疗预后进行统计分析，建立起Liddle综合征系统的诊疗及随访策略，并在外部队列中进行验证，以提高Liddle综合征的诊断率，使患者尽早接受靶向药物精准治疗，减少靶器官损害，降低致残率及病死率。

方法

推导队列纳入2009年1月至2019年4月经基因测序诊断为Liddle综合征的59例患者。通过分析Liddle综合征患者的临床特征，建立起Liddle综合征的诊断策略。为验证该诊断策略的效能，建立起一个外部验证队列，纳入2019年10月至2022年10月就诊的206例2级或3级早发高血压（SBP ≥ 160 mmHg和/或DBP ≥ 100 mmHg）患者，并收集患者的人口学数据、实验室检测指标及影像学结果。在对全部验证队列患者进行基因测序的同时，按诊断策略对患者进行临床分析评估，并进行结果比对，评估诊断策略的效能。此外，采用前瞻性研究设计，通过门诊随访推导队列中的全部患者及验证队列中新确诊的共74例Liddle综合征患者，并根据研究结果进一步完善和细化治疗和随访策略。

结果

第一部分推导队列研究，Liddle综合征的中位发病年龄为19.0（14.5，26.5）岁，平均确诊年龄为33.5 ± 20.2岁。高血压（86.4%）和低钾血症（66.1%）是Liddle综合征最常见的症状，96.6%的Liddle综合征患者存在血清钾、血浆醛固酮和血浆肾素浓度中至少一个指标的降低。通过外部验证队列进行验证，根据Liddle综合征诊断策略结合基因检测，共诊断出7例Liddle综合征患者，敏感性和阴性预测值均为100%，特异性和阳性预测值分别为60.30%和8.14 %。对两个队列中的10个Liddle综合征家系及7例散发病例共74例患者进行随访研究，发现复方阿米洛利治疗效果好，血压达标率为76.9 %，单药或联合其他降压药物治疗后血压控制率从10.8%提高至87.7% (P ＜ 0.001)，血钾正常的患者比例由29.2%上升至95.4% (P＜ 0.001)。复方阿米洛利效果不佳是Liddle综合征患者靶器官损伤的独立危险因素（P = 0.001，OR：40.28，95% CI 4.40-369.06）。

结论

本研究通过Liddle综合征患者临床特征分析及长期随访，建立起了Liddle综合征诊疗及随访策略，为临床提供可借鉴的诊疗流程。此外，长期随访研究表明，Liddle综合征患者复方阿米洛利长期治疗具有理想的安全性及有效性，有效减少了Liddle综合征靶器官损害的发生，为Liddle综合征的诊疗提供了重要的理论指导。

第二部分 Liddle综合征SCNN1B基因新突变的细胞功能鉴定

目的

本研究旨在构建表达野生型及突变型ENaC的非洲爪蟾卵母细胞，通过膜片钳技术检测细胞电流，验证SCNN1B基因新移码突变c.1691_1693delinsG的致病性。同时，对应用复方阿米洛利精准治疗的Liddle综合征患者进行随访，观察临床疗效与预后。

方法

分别构建包含ENaC野生型αβγ亚基及突变型αβ_mutγ亚基的质粒，并通过显微注射使非洲爪蟾卵母细胞表达野生型及突变型ENaC。采用膜片钳技术测定全细胞电流，对比表达突变型与野生型ENaC的非洲爪蟾卵母细胞电流差异。加入阿米洛利后再次测量细胞电流，记录阿米洛利敏感的电流值并进行比较。对于Liddle综合征患者及患病家族成员，给予复方阿米洛利治疗后第6个月及第12个月分别进行随访，评估复方阿米洛利疗效与患者预后。

结果

表达SCNN1B基因c.1691_1693delinsG突变的ENaC的非洲爪蟾卵母细胞全细胞电流相较于表达野生型ENaC的爪蟾卵母细胞显著增加，并可被阿米洛利有效抑制。突变型卵母细胞阿米洛利敏感的电流为野生型卵母细胞电流的2.46倍（P ＜ 0.01）。对接受复方阿米洛利治疗的Liddle综合征患者进行6个月和12个月的随访，所有患者的血压均控制理想，血钾浓度均恢复至正常水平，未发生心血管事件及药物不良反应。

结论

本研究验证了SCNN1B基因新移码突变c.1691_1693delinsG的致病性，拓展了Liddle综合征的致病突变谱。长期随访结果表明，及时个体化治疗对控制患者血压、预防心脑血管并发症的发生具有重要意义。

第三部分 Liddle综合征SCNN1G基因突变小鼠模型的建立

目的

通过CRISPR-Cas9技术，构建与人SCNN1G基因Glu571*突变相对应的小鼠Scnn1g基因Asp577*突变的转基因小鼠，探究人SCNN1G基因Glu571*新突变的致病机制，进一步补充Liddle综合征致病突变谱。同时建立首个SCNN1G基因突变的Liddle综合征小鼠模型，为Liddle综合征的基础研究提供技术支持。

方法

通过CRISPR-Cas9技术构建Scnn1g基因Asp577*截短突变转基因小鼠，继续繁育鉴定获得F2代纯合突变（L/L）、杂合突变（L/+）及同窝野生型（+/+）小鼠，根据基因型分为3组，每组8只小鼠，并单独购置野生型C57小鼠8只作为对照组（WT-ND）。在12周龄前给予各组小鼠普通饲料，12周龄后给予L/L组、L/+组及+/+组小鼠8% NaCl高盐饲料2周，WT-ND组继续给予普通饲料。于各组小鼠6周龄、9周龄、12周龄、13周龄（高盐1周）及14周龄（高盐2周）时进行体重测量、无创血压测量；于12周龄开始高盐饲养前，检测各组小鼠血清钾浓度及血浆醛固酮水平，并在高盐饲养2周后（WT-ND普通饲料饲养）再次检测小鼠血清钾浓度及血浆醛固酮水平，并留取24小时尿测尿钾、尿钠及尿肌酐浓度。最后处死小鼠，取肾脏皮髓交界处组织，应用实时荧光定量逆转录聚合酶链式反应（reverse transcription quantitative Real-time PCR，RT-qPCR）检测SCNN1A、SCNN1B及SCNN1G基因的mRNA表达量，应用Western blot技术对Scnn1g编码的γENaC进行半定量分析，并通过免疫荧光染色观察不同实验组小鼠γENaC在肾小管上皮细胞中的定位。

结果

高盐诱导2周后，L/L组小鼠收缩压及舒张压较+/+组明显升高（SBP P = 0.002；DBP P ＜ 0.001），血钾浓度、血浆醛固酮浓度较+/+组显著降低（P = 0.0125 & P = 0.0216），标化尿钠降低（P ＜ 0.0001）；L/L组及L/+组小鼠标化尿钾均较+/+组显著增加（P = 0.0005 & P = 0.0003）。分子生物学实验发现，L/L及L/+组小鼠Scnn1a、Scnn1b及Scnn1g基因的mRNA表达量均无明显改变，Scnn1g编码的γENaC表达量较+/+组均明显增加（P = 0.0008 & P = 0.0002）。免疫荧光染色提示L/L组及L/+组γENaC广泛分布于顶端膜及胞质内，且表达量较高；而+/+组γENaC主要分布于胞质中。

结论

SCNN1G基因Glu571*（小鼠为Asp577*）突变可造成血压升高并伴有低钾血症及低醛固酮血症，增加肾小管上皮细胞顶端膜γENaC的分布，符合Liddle综合征的病理生理机制，确定为Liddle综合征的致病突变。此外，本研究成功建立起首个SCNN1G基因突变的转基因Liddle综合征小鼠模型，为Liddle综合征的动物在体研究提供了技术支持。

Background

Liddle syndrome is a monogenic hereditary hypertension caused by mutations of SCNN1A, SCNN1B and SCNN1G genes, which encode the α, β and γ subunits of epithelial sodium channels (ENaC). The main clinical manifestations are refractory early-onset hypertension, hypokalemia and hypoaldosteronism. However, due to the atypical clinical manifestations and the lack of systematic and standardized diagnostic strategies of Liddle syndrome, the misdiagnosis rate of Liddle syndrome is pretty high. Many patients are not diagnosed in time and receive specific therapeutic drugs in the early stage, and serious cardiovascular and cerebrovascular events such as ischemic stroke, cerebral hemorrhage and heart failure can occur in young and middle-aged patients. Therefore, early diagnosis, timely treatment and regular follow-up are the key points in the diagnosis and treatment of Liddle syndrome.

Genetic testing is the gold standard for the diagnosis of Liddle syndrome. Accurate identification of patients with suspected Liddle syndrome and getting genetic testing timely can improve the diagnosis rate of Liddle syndrome. However, there is no systematic diagnosis strategy for Liddle syndrome at present. Systematic diagnosis and treatment strategies can help clinicians to identify Liddle syndrome in time, which improve the diagnosis rate and control rate, and reduce the occurrence of severe cardiovascular and cerebrovascular events. In addition, continuous improvement of the pathogenic mutation spectrum of Liddle syndrome is the basis for the accuracy of genetic testing. Because the pathogenicity of some mutations located in SCNN1A, SCNN1B and SCNN1G genes has not been elucidated, it is difficult to interpret the results of genetic testing directly, which affects the diagnosis. Therefore, determining the pathogenicity of the novel mutation sites through cell and animal experiments and studying the function of the mutation sites will help to quickly interpret the results of gene detection in clinical practice.

This study will analyze the clinical characteristics of Liddle syndrome patients to establish clinical diagnosis and treatment strategies, and to test the diagnostic efficacy. A novel mutation c.1691_1693delinsG in the SCNN1B gene, which encodes the β subunit of ENaC was identified in the early work. We aim to further identify the pathogenicity of this mutation by cell experiments. At the same time, transgenic animal experiments were performed to verify the pathogenicity of the novel SCNN1G Glu571* mutation, and an animal model of Liddle syndrome with SCNN1G gene mutation was constructed to explore the function and specific pathogenic mechanism of the mutation site, so as to enrich the pathogenic mutation spectrum of Liddle syndrome.

Part 1 A clinical diagnosis and treatment strategy and long-term follow-up study of Liddle syndrome

Objective

We aimed to establish the systematic diagnosis and follow-up strategy of Liddle syndrome by summarizing the clinical characteristics and prognosis of patients with Liddle syndrome, and perform the validation in an external cohort. So that the diagnosis rat could be improved and patients could receive the targeted drug therapy as soon as possible, which could reduce the disability rate and mortality rate.

Methods

From January 2009 to April 2019, 59 patients diagnosed with Liddle syndrome by genetic sequencing were enrolled in the derivation cohort of this study. Based on an analysis of the characteristics of Liddle syndrome patients, the diagnosis strategies of Liddle syndrome were established. To validate this strategy, another external validation cohorts included 206 patients with grade 2 or 3 early-onset hypertension (systolic blood pressure ≥ 160 mmHg and/or diastolic blood pressure ≥ 100 mmHg) were enrolled in the second part of this study from 2019 to 2022, and their demographic data, laboratory results, and imaging scans were collected. Genetic sequencing was performed in all patients, and the efficacy of the diagnostic strategy was evaluated on the basis of the results. Simultaneously, a total of 74 Liddle syndrome patients who were diagnosed between January 2009 and December 2022 were prospectively followed up through outpatient visits or telephone consultations in accordance with a prospective study design. The treatment and follow-up strategies were further enhanced and refined based on the findings of the study.

Results

In the first part of the derivation cohort study, the median onset age of Liddle syndrome was 19.0 (14.5, 26.5) years old, and the mean age at diagnosis was 33.5 ± 20.2 years. Hypertension (86.4%) and hypokalemia (66.1%) were the most common symptoms of Liddle syndrome. 96.6% of Liddle syndrome patients had a decrease in at least one of serum potassium, plasma aldosterone and plasma renin concentration. A total of 7 Liddle syndrome patients were diagnosed according to the Liddle syndrome diagnostic strategy. The sensitivity and negative predictive value (NPV) were 100%, and the specificity and positive predictive value (PPV) were 60.30% and 8.14%, respectively. A total of 74 Liddle syndrome patients from 10 families and 7 sporadic cases in two cohorts were followed up. The effective rate of amiloride monotherapy was 76.9%, and the blood pressure control rate increased from 10.8% to 87.7% (P < 0.001). The proportion of patients with normal serum potassium increased from 29.2% to 95.4% (P < 0.001). Poor efficacy of amiloride alone was an independent risk factor for target organ damage in Liddle syndrome patients (P = 0.001, OR: 40.28, 95%CI: 4.40, 369.06).

Conclusion

This study is the largest follow-up study of Liddle syndrome patients in the world, based on which the diagnosis, treatment and follow-up strategy of Liddle syndrome was established. This strategy has high sensitivity and NPV, which can reduce the rate of missed diagnosis. At the same time, it proves the safety and effectiveness of long-term treatment of amiloride, which provides an important theoretical guidance for the diagnosis and treatment of Liddle syndrome.

Part 2 Functional identification of a novel SCNN1B frame-shift mutation in Liddle syndrome

Objective

We aimed to construct Xenopus laevis oocytes expressing wild type ENaC and mutant ENaC respectively, and detect the cellular current by patch clamp technique to verify the pathogenicity of the novel frameshifted mutation c.1691_1693delinsG in SCNN1B gene. The clinical efficacy and prognosis were observed.

Methods

Plasmids containing wild-type ENaC αβγ subunit and mutant αβ_mutγ subunit were constructed and microinjected into Xenopus laevis oocytes to express wild-type and mutant ENaC. The whole-cell current was measured by patch clamp technique to compare the difference of current between Xenopus oocytes expressing mutant ENaC and wild-type ENaC. Cell currents were measured again after the addition of amiloride, and amiloride sensitive current values were recorded and compared. Patients with Liddle syndrome and their family members were followed up at 6 and 12 months after amiloride treatment to evaluate the efficacy and prognosis.

Results

The whole-cell current of Xenopus laevis oocytes expressing the c.1691_1693delinsG mutation of the SCNN1B gene was significantly higher than that of oocytes expressing wild type ENaC, and could be effectively inhibited by amilorides. The current sensitive to amiloride in the mutant oocytes was 2.46 times higher than that in the wild type oocytes (P < 0.01). Liddle syndrome patients treated with amiloride were followed up for 6 months and 12 months. All patients had good blood pressure control, normal renin-angiotensin-aldosterone system related biochemical levels, and no cardiovascular events and serious drug adverse reactions occurred.

Conclusion

The novel frameshift mutation c.1691_1693delinsG in the SCNN1B gene was confirmed to be pathogenic for the first time, which expands the mutation spectrum of Liddle syndrome. Long-term follow-up shows that timely and accurate individualized treatment is of great significance for improving blood pressure and preventing cardiovascular and cerebrovascular complications. This study refines the treatment strategy and provides important theoretical guidance and guidelines for the diagnosis and treatment of monogenic hypertension.

Part 3  A transgenic mouse model of Liddle syndrome and identification of a novel SCNN1G gene mutation

Objective

In this study, the Asp577* mutant transgenic mice corresponding to the Glu571* mutation of human SCNN1G gene were constructed by CRISPR-Cas9 technology to verify the pathogenicity of the novel truncation mutation of human SCNN1G gene Glu571*, and further improve the pathogenic mutation spectrum of Liddle syndrome. At the same time, we plan to establish the first Liddle syndrome mouse model caused by SCNN1G gene mutation and provide technical support for basic research of Liddle syndrome.

Methods

We used CRISPR-Cas9 technology to construct F1 heterozygous transgenic mice carrying the Scnn1g Asp577* truncated mutation, and then obtained F2 generation homozygous mutation (L/L), heterozygous mutation (L/+) and litter wild-type (+/+) mice through further breeding and identification. According to the genotype, the mice were divided into 3 groups, with 8 mice per group. Eight wild type C57 mice were purchased as the control group (WT). After 12 weeks of age, the L/L, L/+ and +/+ groups were given 8% NaCl high-salt diet for 2 weeks, and the wild control group was given normal diet (WT-ND). Body weight and blood pressure were measured at the age of 6, 9, 12, 13 and 14 weeks in each group. Serum potassium concentration and plasma aldosterone level were measured before high-salt feeding at the age of 12 weeks, and again after 2 weeks of high-salt feeding (WT-ND normal diet). 24-hour urine samples were collected to measure the concentrations of potassium, sodium and creatinine. After the mice were killed, the tissue at the junction of the renal cortex and medulla were obtained to detect the mRNA expression levels of Scnn1a, Scnn1b, and Scnn1g genes by reverse transcription quantitative Real-time PCR (RT-qPCR), and the expression of γENaC encoded by Scnn1g through the Western blot. Immunofluorescence staining was used to observe the localization of γENaC in renal tubular cells in different experimental groups.

Results

After 2 weeks of high-salt induction, the systolic and diastolic blood pressure of L/L group were significantly higher than those of +/+ group (SBP P = 0.002; DBP P < 0.001), serum potassium and plasma aldosterone concentration (P = 0.0125 & P = 0.0216) and standardized urine sodium (P < 0.0001) were significantly lower than those in +/+ group. The standardized urine potassium in L/L group and L/+ group was significantly higher than that in +/+ group (P = 0.0005 & P = 0.0003). Molecular biology experiments showed that the mRNA expression of Scnn1a, Scnn1b and Scnn1g genes in L/L and L/+ mice were not significantly different from that in +/+ mice, while the γENaC expression was significantly increased compared with +/+ group (P = 0.0008 & P = 0.0002). Immunofluorescence staining showed that γENaC was widely distributed in apical membrane and cytoplasm in L/L group and L/+ group, while the γENaC was mainly distributed in the cytoplasm of +/+ group.

Conclusion

Glu571* (Asp577* in mice) mutation of SCNN1G gene can lead to salt-sensitive hypertension with hypokalemia and hypoaldosteronism, and increase the distribution of γENaC in the parietal membrane of renal tubular cells, which is consistent with the pathophysiological mechanism of Liddle syndrome. Therefore, this mutation was identified as the pathogenicity mutation of Liddle syndrome. In addition, we successfully established the first SCNN1G mutant Liddle syndrome mouse model, which provides technical support for the in vivo study of Liddle syndrome.