Systemic hypertension is a commonly recognized health problem affecting over 1.2 billion adults worldwide (1). Chronic elevated blood pressure or hypertensive emergencies can lead to morbid complications such as stroke, heart failure, and renal failure (2). Significantly, 15–20% of cases of hypertension occur secondary to some definable causes, and for those, precise determination of the etiology could help to avoid severe cardiovascular outcomes (3). Monogenic hypertension plays an important role in the development of secondary hypertension, such as Liddle syndrome, apparent mineralocorticoid excess, and congenital adrenal hyperplasia (4).

Liddle syndrome is an autosomal dominant form of hereditary hypertension with early penetrance caused by mutations in the epithelial sodium channels (ENaCs). ENaCs, which are responsible for the rate-limiting step of water and sodium reabsorption in the aldosterone-sensitive-distal nephron, consist of α, β, and γ homologous subunits, encoded by SCNN1A, SCNN1B, and SCNN1G, respectively (5). Any mutation in the subunits can induce the constitutive activation of ENaCs by either accumulative channel density or an increased probability of the channels being open, thereby resulting in excessive sodium reabsorption and volume expansion (6, 7). The disease is classically characterized by severe hypertension, hypokalemia, and metabolic alkalosis in the setting of low/suppressed aldosterone and renin levels (8). However, a variable phenotype of Liddle syndrome is observed in clinical work. Some patients are misdiagnosed and may suffer catastrophic complications because of a lack of awareness of the genotype-phenotype heterogeneity (9). Genetic sequencing, considered a gold standard, is useful and effective for the diagnosis of Liddle syndrome. The blood pressure and potassium levels can be improved greatly by adopting a low potassium diet and tailored therapy including amiloride or triamterene, which are specific antagonists of ENaCs, but patients show no response to spironolactone treatment.

In the present study, we report a Chinese family with Liddle syndrome and identify a novel frame-shift mutation (c.1691_1693delinsG) in SCNN1B by whole-exome sequencing. Sanger sequencing indicated that eight family members also carried the variant in addition to the proband. Intra-familial phenotypic heterogeneity was observed in terms of the blood pressure and serum potassium levels. Tailored therapy is beneficial and cost-effective for Liddle syndrome patients to avoid catastrophic complications.

In this report, we identified a novel frame-shift mutation (c.1691_1693delinsG) in the SCNN1B gene in a Chinese family. Family genetic screening indicated that a total of nine family members were carrying the pathogenic mutation. Intra-familial phenotypic heterogeneity was prominent in this case. Namely, the proband presented with classical Liddle syndrome symptoms, while some members with the same mutation had only mild hypertension and hypokalemia, or even normotension and normokalemia. Tailored therapy, including amiloride and a low sodium diet, play an important role in controlling the disease. Timely genetic testing aids in accurate diagnosis of Liddle syndrome.

In 1963, Liddle syndrome was first described by Liddle et al. in a Caucasian female who presented with hypertension, hypokalemia, and metabolic alkalosis in the absence of hyperaldosteronism (17). In 1994, Shimkets et al. first uncovered the molecular defect of Liddle syndrome by analyzing the β subunit of ENaC in several Liddle syndrome kindreds (18). The disorder results from gain-of-function mutations of SCNN1A, SCNN1B, and SCNN1G, encoding the α, β, and γ subunits of ENaCs, respectively. In the kidney, ENaCs are mainly distributed in the apical membrane of the principal cells located in the aldosterone-sensitive-distal nephron, which play a vital role in maintaining sodium and water reabsorption. Each subunit of ENaC contains a proline-rich segment (PY motif), which is located in the C-terminus and functions by binding the WW domains of the ubiquitin ligase protein Nedd4–2, thereby inducing internalization and proteasomal degradation of the ENaCs. Any deletion or alteration of the PY motif leads to the reduced inactivation of the ENaCs, resulting in an increased density and constitutive activation of ENaCs and consequently followed by excessive sodium reabsorption and plasma volume expansion (19). Additionally, increased sodium reabsorption generates a luminal negative voltage that promotes an elevated secretion level of potassium, leading to abnormal hypokalemia (20).

The worldwide prevalence of Liddle syndrome, irrespective of race or sex, remains unknown. So far, there have been relatively few studies on the prevalence of Liddle syndrome. In 2010, Tapolyai et al. conducted a cross-sectional cohort of 149 patients characterized by hypertension and hypokalemia or metabolic alkalosis and detected a high prevalence (6%, 9/149) of Liddle syndrome biochemical phenotypes (21). In a Chinese early-onset hypertensive population, the prevalence of Liddle syndrome confirmed by genetic testing has been estimated as 1.52% (5/330) and 0.91% (7/766) (22, 23). However, because of the variable penetrance of Liddle syndrome and the incomplete application of genetic testing, the prevalence may be underestimated.

Until now, more than 24 mutations in SCNN1B have been reported worldwide, with the most common variants altering the conserved PY motif in the carboxy-terminal cytoplasmic tail of ENaC. The mutation in this report (c.1691_1693delinsG), which is also near the C-terminus, results in the substitution of an Arg amino acid in place of a Gln at position 564 and a truncated open reading frame that ends at position 592. To date, several mutations have been described that are close to the locus reported herein, including missense and nonsense mutations. R563Q has been identified as related to a hypertensive phenotype, and might alter the three-dimensional structure of the β subunit, resulting in weakened intracellular interactions and upregulated ENaC activity (24, 25). Liu et al. identified a nonsense mutation in SCNN1B gene (c.C1690T) which introduces a new stop codon instead of a Gln at codon 564 (23). In addition, R566X is considered a hot-spot locus which leads to the removal of the cytoplasmic carboxyl tail (18, 22, 23, 26–31). Previously, Cui et al. screened SCNN1B and SCNN1G of 12 patients with Liddle syndrome and detected transition from C to T at various sites of SCNN1B (c.C1690T, c.C1702T), which leads to premature termination of the protein (28). In renal epithelial cells derived from the distal nephron, Snyder et al. found that βR566X coexpressed with wild-type α and γ subunits generated more than twice as much amiloride-sensitive current as did expression of wild-type αβγ hENaC, indicating that truncation of βhENaC led to increased Na⁺ current (32). A quantitative assay was developed by Firsov et al. (7). This method was based on the binding of a monoclonal antibody directed against a FLAG reporter epitope introduced in the α, β, and γ subunits of ENaC, and demonstrated a significant correlation between the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC on the plasma membrane (7). Schild et al. investigated sequential deletions in the C-terminus of the βENaC, and reported that a functional domain, – PY motif regulated channel activity (33). Staub et al. found that mutations in the PY motif of βENaC abolished Nedd4-WW binding in the two-hybrid binding assay, and the protein-protein interaction of ENaC with Nedd4 mediated internalization of ENaC (34).

The effect of a frame-shift mutation of SCNN1B has been verified in a previous expression study in Xenopus laevis oocytes, which is caused by insertion of a cytosine at the codon 594 and truncates the C-terminus at codon 607, disrupting 46 amino acids of βhENaC (32). Snyder et al. introduced serial stop codons into βhENaC and illustrated that deletion of 19 residues was sufficient to reproduce the effect of disease-associated mutations (32). Their data identified that truncation in the C-terminus of the β subunit deleted the conserved PY motif, and induced an increase of transmembranic sodium transport, compared with expression of the wild-type αβγENaC (32). The deletion of the PY motif leads to constitutive activation of the ENaC, and results in Liddle syndrome.

According to previous studies and in silico prediction softwares, the frame-shift mutation identified in this report also leads to the absence of the PY motif, by generating a new stop codon at 592 (original stop codon at 641). As a result, the inactivation of the ENaCs is reduced, consequently followed by increased number of ENaC on the plasma membrane and excessive sodium reabsorption. Increased sodium reabsorption leads to plasma volume expansion and promotes secretion of potassium, causing refractory hypertension with hypokalemia of the proband.

Phenotypic variability is common in the clinical and biochemical characteristics of Liddle syndrome, and this was also observed in this study (35, 36). The blood pressure level, degree of hypokalemia, therapeutic effect of amiloride, and targeted organ damage were the key features in the heterogeneity analysis (22, 27). Previously, genotype–phenotype correlation analysis has been conducted in two Liddle syndrome families with various mutations by Gong et al., which revealed the existence of inter- and intra-familial phenotypic variability (27). Even patients in the same family carrying the same pathogenic mutation may present with mild hypertension or even normotension (22). In terms of the serum potassium condition, hypokalemia was observed in 71.8% of Liddle syndrome patients, and 78% of pediatric Liddle syndrome patients (37, 38). In this study, subjects IV-3 and III-1 presented typically with early-onset severe hypertension, whereas subjects III-7 and III-5 showed mild hypertension, and subjects IV-1 and IV-2 had normal blood pressure. Moreover, the incidence rate of hypokalemia in this family was slightly lower than that previously reported, around 66.7%, which may be due to the limited patient number in the pedigree.

Because some Liddle syndrome patients harbor atypical clinical symptoms and biochemical characteristics, they may be easily misdiagnosed with primary aldosteronism or primary hypertension (23, 39). Long-term uncontrolled blood pressure often results in severe disease complications and targeted organ damage, such as heart failure, renal failure, or even sudden death. To avoid these, clinicians should consider Liddle syndrome when they encounter patients presenting with early-onset hypertension, suppressed PRA, low renin level, and hypokalemia. Genetic testing is considered an essential and rapid tool to ensure that patients with Liddle syndrome can be diagnosed in an accurate and timely fashion. In addition, genetic screening of high-risk family members based on the proband is necessary and efficient for detecting potential Liddle syndrome patients. Once diagnosed, ENaC inhibitors (such as amiloride and triamterene) in combination with a low sodium diet should be individualized. Liu et al. reported that an average follow-up study for 4 years of 17 Liddle syndrome patients showed that blood pressure and potassium levels can be controlled well by the adoption of amiloride therapy (23).

In conclusion, we have identified a novel frame-shift mutation in the β subunit of ENaC in a Chinese family with Liddle syndrome by whole-exome sequencing, further refining the known genetic mutations of SCNN1B. Patients with Liddle syndrome may be misdiagnosed when diagnosis is based on clinical or biochemical characteristics alone because of the existence of phenotypic heterogeneity. Genetic analysis is necessary to ensure timely and accurate diagnosis of Liddle syndrome and to apply tailored treatment to prevent the occurrence of disease complications.

The data presented in the study are deposited in the Sequence Read Archive (SRA) repository, accession number SAMN2886425.

The studies involving human participants were reviewed and approved by the Ethics Committee of Fuwai Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Y-TL, X-CL, FL, and X-LZ designed the study and modified the manuscript. Y-TL, X-CL, Z-MZ, DZ, and LS collected the clinical information and performed the data analysis. YZ, PF, LZ, and Y-XL performed the experiments. Y-TL and X-CL wrote the manuscript. All authors reviewed this work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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