The present study was approved by the Ethics Committee of Fuwai Hospital, Peking Union Medical College (Beijing, China; approval no. 2020-1321) and was performed in accordance with the Declaration of Helsinki. All study participants provided written informed consent before study enrolment.

The proband (Fig. 1; III-2) was a 28-year-old male patient who was admitted with severe hypertension to the Department of Hypertension, Fuwai Hospital (Beijing, China) in 2021. The hypertension of the patient had been incidentally diagnosed during a medical examination at the age of 16 years. During hospitalization, detailed screening tests, including imaging (echocardiography and computed tomography of the aortic and renal arteries), physical examination, biochemical assessments (including plasma serum, aldosterone, renin and urinary microalbumin concentration measurements) and hormone measurements (including growth hormone, cortisol and adrenocorticotropic hormone concentration measurements), were performed to identify the etiology of hypertension. Moreover, the fundus lesions of hypertension were evaluated by mydriasis followed by fundoscopy. The patient had a family history of early-onset hypertension. Affected family members included the father (II-4) of the patient, two paternal aunts (II-1 and II-5) and a deceased paternal uncle (II-7). Therefore, seven biological relatives of the patient (five females and two males) with a median age of 42 years (interquartile range 35, 60 years) were enrolled in the present study. They underwent full clinical evaluation (including blood pressure measurement, medical history and symptom assessment), biochemical testing (including plasma serum, aldosterone and renin concentration measurements) and genetic analysis.

DNA was extracted from leukocytes in the peripheral blood of all enrolled family members using the QIAamp^® DNA Blood Mini kit (cat. no. 51104; Qiagen GmbH) according to the manufacturer's protocol. The DNA concentration and quality was measured using a Qubit^® 3.0 Fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). Additionally, gel electrophoresis was carried out with a 1% agarose gel to visually inspect the DNA for intact, high molecular weight bands, to ensure that it was not degraded or fragmented. Whole-exome sequencing was performed on DNA from the blood of the proband using the SureSelectXT Human All ExonV6 kit (cat. no. 5190-8864; Agilent Technologies, Inc.) for exome capture and the Novaseq 6000 platform (Illumina, Inc.) was used for genomic DNA sequencing (paired end; 150 bp). The concentration of the library was measured using a Qubit^® 3.0 Fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.) and then the size distribution of the library was analyzed using a NGS 3K reagent kit (cat. no. CLS960013; PerkinElmer, Inc.) and the loading concentration of the final library was 17.56 nM. Except for the proband, Sanger sequencing was performed on the DNA of the family members to evaluate the candidate variant and to perform co-segregation analysis. The forward primer used was 5′-CCCACCCAAGAATCACCTCC-3′ and the reverse primer used was 5′-TCAGGACAGGTAGGGACGAG-3′. To ensure that the mutation was accurately identified, the exon was sequenced forward and backward, and all products were analyzed using Chromas 2.22 software (Technelysium Pty Ltd.).

The whole-exome sequenced data were first checked using in-house quality control software to remove low-quality reads. The sequenced data were then aligned to the reference human genome (GRCh37/hg19) using Burrows-Wheeler Aligner (version 0.7.12) (8). SAM tool (version 0.1.19; http://sourceforge.net/projects/samtools/files/samtools/0.1.19/) and Sambamba (version 0.8.2; http://github.com/biod/sambamba/releases) were used to sort bam files and perform duplicate marking, generating the final bam file (9,10). Single-nucleotide variants and insertion/deletion variants were identified using the SAM tool (version 0.1.19) (10) and copy number variants were detected using CoNIFER (version 0.3) (11). Annotation was performed using ANNOVAR software (version 322; http://annovar.openbioinformatics.org/en/latest/). Mutations with a frequency of ≥1% in the 1000 Genomes Project (1000G_all; http://www.internationalgenome.org/), ESP6500 (ESP6500SIV2_ALL; http://esp.gs.washington.edu/drupal/) and gnomAD (gnomAD_ALL and gnomAD_EAS; http://gnomad.broadinstitute.org/) databases were removed. In silico analysis software, including MutationTaster (MutationTaster2021; http://www.mutationtaster.org/) and Phylogenetic Analysis with Space/Time Models (phast) Cons (version 1.3; compgen.bscb.cornell.edu/phast) were used to predict the pathogenicity of variants. The pathogenicity of the sequence variants was assessed using the method developed by the American College of Medical Genetics and Genomics (ACMG) (12).

The follow-up data of patients with Liddle syndrome were analyzed to evaluate the prognosis of patients with Liddle syndrome after targeted therapy. ‘Liddle syndrome’, ‘Liddle's syndrome’, ‘pseudoaldosteronism’ and ‘SCNN1B’ were used as key words to search publications in English in the MEDLINE (https://www.medline.com/), Cochrane (https://www.cochranelibrary.com/) and Web of Science (https://clarivate.com/products/scientific-and-academic-research/research-discovery-and-workflow-solutions/webofscience-platform/) databases from December 1963 to July 2023. Patients that were genetically diagnosed with Liddle syndrome with available follow-up data were included in the analysis, while those without genetic diagnostic information or follow-up data were excluded. As the prevalence of Liddle syndrome is low, ranging from 0.91–1.52%, the genotype-phenotype association is still unclear (13,14). Therefore, a genotype-phenotype association analysis was performed by reviewing the clinical and biochemical characteristics of patients carrying the same mutation as the participants in the present study.

Statistical analysis was performed using SPSS software (version 16.0, SPSS, Inc.). The data are presented as mean ± standard deviation or frequency (%), as applicable. To assess the differences in blood pressure and serum potassium concentration before and after treatment with an ENaC inhibitor, paired Student's two-tailed t-tests were used. To ensure the statistical robustness of the findings, a minimum of three replicates were conducted for each experiment. P<0.05 was considered to indicate a statistically significant difference.

The proband was 28 years old and had a history of hypertension, which was first diagnosed at the age of 16 years and had remained refractory to a 12-year combination therapy that included telmisartan, benidipine and bisoprolol. In December 2021, the proband was referred to Fuwai Hospital (Beijing, China) for a detailed evaluation of the hypertension of the patient.

Physical examination at the time of hospital admission demonstrated that the patient was overweight (body mass index, 26.2 kg/m²) and had a high blood pressure (200/140 mmHg; normal range, <140/90 mmHg). Biochemical assessments demonstrated a serum potassium concentration of 3.18 mmol/l (normal, 3.50–5.30 mmol/l), a plasma aldosterone concentration of 1.0 ng/dl (normal, 3.0–35.3 ng/dl) in the standing position (Table I), a urinary microalbumin concentration of 56.7 mg/l (normal, <30.0 mg/l) and a plasma chloride concentration of 108 mmol/l (normal, 98–106 mmol/l). The plasma renin concentration (5.4 mIU/ml; normal range, 4.4–46.1 mIU/ml), aldosterone/direct renin concentration ratio [0.19 (ng/dl)/(mIU/ml); normal range, <3.70 (ng/dl)/(mIU/ml)] and results of a low-dose dexamethasone inhibition test (cortisol, 3.00 µg/dl; normal range, <22.45 µg/dl; and adrenocorticotropic hormone concentration, <5 pg/ml; normal range <46 pg/ml) were normal. In addition, the growth hormone concentration (18.40 µg/dl; normal reference, 5.27–22.45 µg/dl), cortisol concentration (<0.1 ng/ml; normal reference, 0.0–3.0 ng/ml) and adrenocorticotropic hormone concentration (10.30 pg/ml; normal reference, 0.00–46.00 pg/ml) were also within the normal range. The result of aortic and renal artery computed tomography was also normal (Fig. 1A and B). Transthoracic echocardiography demonstrated thickening of the left ventricular posterior wall and interventricular septum and widening of the ascending aorta (Fig. 1C and D). Evaluation of the retinal microvasculature suggested bilateral retinal artery stenosis and grade 1 hypertensive retinopathy.

The father (II-4) of the patient and two paternal aunts (II-1 and II-5) also had early-onset hypertension and chronic hypokalemia, for which they intermittently took potassium citrate supplementation. The paternal grandmother (I-1) of the patient was diagnosed with hypertension at the age of ~20 years and died of a stroke at 35 years of age. A paternal uncle (II-7) was reported to have had hypertension at the age of ~30 years, with little compliance to blood pressure control and died of cardiac arrest at the age of 50 years. The mother (II-3) of the proband was unaffected. Clinical and biochemical profiles of family members are presented in Table I. The frequency of early-onset refractory hypertension, hypokalemia and history of sudden mortality on the paternal side of the family of the proband was suggestive of an autosomal dominant disease (Fig. 2).

The results of whole-exome sequencing in the proband demonstrated a frameshift variant in SCNN1B (NM_000336: c.1806dupG, p.Pro603Alafs*5). This mutation led to a heterozygous G insertion at base 1806 of the β subunit of ENaC. This resulted in the substitution of alanine in place of proline at codon 603 and a shortened open reading frame that created a premature stop codon at position 607. It also abrogated the highly conserved PY motif, which conformed to the criterion for protein-altering variant severity (PVS1) (12). This variant has not been reported in the 1000 Genomes Project or ExAC (https://exac.broadinstitute.org/) databases, which matches the criterion for pathogenic moderate 2 (PM2) (12). Sanger sequencing demonstrated that four affected family members (II-1, II-4, II-5 and III-3) had the same mutation as the proband (Fig. 3), indicating that they matched the criterion for pathogenic supporting 1 (PP1) (12). Therefore, it was assumed that the mutation identified in the proband was derived from the paternal lineage and probably from the grandmother who died at the age of 35 years as a result of early-onset severe hypertension. Moreover, the c.1806dupG mutation was predicted to be ‘disease causing’ and ‘1’, determined according to the in silico analysis tools MutationTaster and phastCons, respectively, and aligning to the criterion for pathogenic supporting 3 (PP3) (12). Thus, according to the ACMG guidelines (12), SCNN1B c.1806dupG was assessed as a pathogenic mutation based on the evidence of PVS1, PM2, PP1 and PP3.

All family members with the pathogenic mutation were prescribed amiloride (5 mg daily, oral administration), except for III-3, whose blood pressure was within a normal range. Lifestyle measures, including a low-sodium diet and exercise, were implemented. Combination antihypertensive therapy (50 mg losartan potassium/12.5 mg hydrochlorothiazide daily, oral administration) was prescribed to one family member (II-4), for whom amiloride alone was ineffective. Moreover, genetic counseling was provided to the affected individuals and DNA testing by amniocentesis was recommended. After 3 months of tailored treatment, blood pressure and electrolyte concentrations in the affected individuals were reassessed and these were demonstrated to be within normal ranges (Table II). Subsequently, all of the patients were followed up for ≥1 year and no abnormal blood pressure measurements, new cardiovascular events or serious adverse drug reactions were recorded.

A total of 108 patients with Liddle syndrome from 47 families were identified through the literature search. Follow-up data of these individuals are detailed in Table SI. Median age at hypertension onset was ~18 years (interquartile range, 14, 23.5 years). Incidence of hypertension, hypokalemia, hypoaldosteronism and hyporeninemia was 97.2, 81.3, 65.1 and 80.2%, respectively. In patients without targeted therapy, the most common hypertensive damage was left ventricular hypertrophy (9.6%), followed by stroke (4.6%). A family history of stroke was reported in ~12% of the patients. After treatment with an ENaC inhibitor (amiloride or triamterene), blood pressure significantly decreased from 170.8±23.8/107.6±18.7 to 124.2±9.9/79.4±9.9 mmHg (P<0.001) and serum potassium concentration significantly increased from 3.14±0.5 to 4.3±0.5 mmol/l (P<0.001). During follow-up, only one mortality that was unexplained and no cardiovascular events were reported.

Moreover, 13 patients with Liddle syndrome were identified from three families, all with c.1806dupG in SCNN1B (15,16). The relevant clinical data for all individuals known to have the c.1806dupG mutation, to the best of our knowledge, are summarized in Table II. Individuals carrying this mutation had varying degrees of blood pressure elevation, except for III-3 in the present study (17/18, 94.4%). The median age at hypertension onset was 18 years (interquartile range, 16, 28 years) and the mean arterial blood pressure was 180/120 mmHg. Hypokalemia was identified in 10/17 patients (58.9%), hypoaldosteronism in 9/17 (52.9%) and hyporeninemia in 7/17 (41.1%).