A Novel Frameshift Mutation of SCNN1G Causing Liddle Syndrome with Normokalemia.

BACKGROUND: Liddle syndrome (LS) is an autosomal dominant disorder caused by single-gene mutations of the epithelial sodium channel (ENaC). It is characterized by early-onset hypertension, spontaneous hypokalemia and low plasma renin and aldosterone concentrations. In this study, we reported an LS pedigree with normokalemia resulting from a novel SCNN1G frameshift mutation.
METHODS: Peripheral blood samples were collected from the proband and eight family members for DNA extraction. Next-generation sequencing and Sanger sequencing were performed to identify the SCNN1G mutation. Clinical examinations were used to comprehensively evaluate the phenotypes of two patients.
RESULTS: Genetic analysis identified a novel SCNN1G frameshift mutation, p.Arg586Valfs*598, in the proband with LS. This heterozygous frameshift mutation generated a premature stop codon and deleted the vital PY motif of ENaC. The same mutation was present in his elder brother with LS, and his mother without any LS symptoms. Biochemical examination showed normokalemia in the three mutation carriers. The mutation identified was not found in any other family members, 100 hypertensives, or 100 healthy controls.
CONCLUSIONS: Our study identified a novel SCNN1G frameshift mutation in a Chinese family with LS, expanding the genetic spectrum of SCNN1G. Genetic testing helped us identify LS with a pathogenic mutation when the genotypes and phenotype were not completely consistent because of the hypokalemia. This case emphasizes that once a proband is diagnosed with LS by genetic testing, family genetic sequencing is necessary for early diagnosis and intervention for other family members, to protect against severe cardiovascular complications.

Over the past several decades, with the development of numerous genetic studies on hypertension, the heritability of hypertension has been widely recognized and mainly separated into three forms, monogenic, polygenic, and epigenetic hypertension.[1]–3 Genetic factors are thought to contribute to estimated 30% of blood pressure (BP) variance.[4] As an important part of secondary hypertension, monogenic hypertension disorders follow Mendelian inheritance patterns because of single-gene mutations, most of which are associated with volume expansion, electrolyte abnormalities, and low serum renin levels.[5,6]

Liddle syndrome (LS, OMIM #177200), an autosomal dominant form of monogenic hypertension, is caused by a gain-of-function mutation in the epithelial sodium channel (ENaC) genes, SCNN1A, SCNN1B, and SCNN1G, which encode three homologous subunits, α-ENaC, β-ENaC, and γ-ENaC, respectively. These mutations can increase the membrane density of the ENaC or the open probability of the ion channel being open, leading to extensive sodium reabsorption and subsequent hypertension.[7] Therefore, LS patients typically present with early-onset hypertension, hypokalemia, low renin and aldosterone levels, and metabolic alkalosis.[8] However, phenotypic diversity allows severe phenotypes and mild forms to coexist among LS patients, even those carrying the same causative mutation.[9]

In this study, we describe the clinical, biochemical, and genetic features of a de novo LS family. A novel SCNN1G frameshift mutation was identified in three family members and confirmed by genetic screening.

METHODS

Subjects

The proband (Figure 1), a 17-year-old male, suffered from uncontrolled hypertension for 3 years. From the age of 14, he was found high BP, with a maximum BP as high as 190/120 mm Hg. He received medication of bisoprolol and a calcium antagonist, but this failed to control his BP (140/90 mm Hg). Biochemical examination showed low plasma renin concentration (PRC) and plasma aldosterone concentration (PAC) in the upright position (Table 1). There were no other significant symptoms. On investigation of his family members, we found that his elder brother (subject III-3) had similar abnormalities as the proband. He was also diagnosed as hypertension (maximum BP: 180/110 mm Hg) for 3 years with symptoms of dizziness. Although he was treated with irbesartan and nifedipine, his BP was still uncontrolled (160/100 mm Hg) with unrelieved symptoms. Low PRC and PAC were detected for him (Table 1). While his parents showed no abnormal symptoms, this situation aroused a suspicion of secondary hypertension, particularly monogenic hypertension. Most of the family members were enrolled in this study. Every participants underwent clinical evaluation, biochemical examination, and genetic testing (Table 1).

Figure 1.

Family pedigree. Black filled symbols, subjects carrying the identified SCNN1G mutation (c.1756delC, p.Arg586Valfs*598); empty symbols, subjects without identified mutation; grey filled symbols, not sequenced; black arrow indicates the proband.

Table 1.

Clinical and laboratory features of all participants

								Follow-upc
Subjects	Gender	Age, years	Onset age of hypertension, years	BP, mm Hga	Serum K+, mmol/l	PRC, µIU/ml	PAC, ng/dl	BP, mm Hga	Serum K+, mmol/l	Genotypeb
II-3	F	46	-	120/86	5.25	14.8	3.0	-	-	1756delC/-
III-3	M	23	19	180/110	4.38	3.0	2.9	124/78	4.67	1756delC-
III-4	M	17	14	190/120	3.68	2.0	2.2	130/83	4.05	1756delC/-
I-1	M	73	66	165/78	4.72	8.0	1.5	-	-	-/-
I-2	F	74	63	178/86	4.93	7.7	4.0	-	-	-/-
II-2	M	50	48	143/102	4.37	8.0	1.5	-	-	-/-
II-4	M	46	-	130/90	4.57	3.0	8.0	-	-	-/-
III-1	F	17	-	124/80	4.15	11.3	9.5	-	-	-/-
III-2	M	6	-	114/74	4.43	8.5	10.3	-	-	-/-

The features of two patients with Liddle syndrome are shown in bold.

BP, blood pressure; F, female; M, male; serum K+: reference value, 3.5–5.3 mmol/l; PRC, plasma renin concentration (upright): reference value, 4.4–46.1 µIU/ml; PAC, plasma aldosterone concentration (upright): reference value, 3.0–35.3 ng/dl.

aMean of three measurements.

b1756delC/-: subjects had identified heterozygous mutation; -/-: subjects had no identified mutation.

cFollow-up results were obtained one month after treatment with compound amiloride.

The specific treatment of LS relies on ENaC blockers, such as amiloride and triamterene. In our study, only a compound amiloride containing 2.5 mg amiloride and 25 mg hydrochlorothiazide in each tablet were available for LS patients. Hydrochlorothiazide may diminish the efficacy of amiloride by increasing the sodium delivery to the collecting duct where it competes with amiloride for ENaC binding.[10] Both of LS patients (subjects III-3 and III-4) took two tablets of compound amiloride every day. During therapy, it is worthwhile monitoring serum electrolytes, especially to prevent hyperkalemia.[11]

This study was approved by the Ethics Committee of Fuwai Hospital and has been performed in accordance with the Declaration of Helsinki. All participants in this family offered Informed consent.

Imaging and laboratory examination

Imaging examinations were performed including echocardiography, abdomen ultrasound, and computed tomography (CT) of the kidneys, adrenal glands, and renal arteries. Laboratory testing covered general biochemical measurements associated with hypertension, such as serum electrolytes, urine protein, microalbuminuria, and blood gas analysis. PRC and PAC were measured at Fuwai Hospital by means of a chemiluminescence immunoassay using the LIAISON Direct Renin kit (DiaSorin S.p.A, Vercelli, Italy) and the LIAISON Aldosterone kit (DiaSorin Inc., Stillwater).

Genetic analysis

All participants provided venous blood samples for genetic testing. Genomic DNA was extracted from peripheral blood leucocytes using the QIAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany) by standard protocols. Next-generation sequencing of 41 monogenic hypertension-related genes was performed for the proband (Supplementary Table 1). All coding exons were enriched using custom-made SureSelect Target Enrichment System (Agilent Technologies, Inc., Santa Clara, CA). Captured DNA library were sequenced on Illumina Hiseq X Ten according to standard protocol for paired-end 150bp reads. Paired-end reads were aligned to the reference genome (hg19) using the Burrows–Wheeler Aligner and duplicated reads were marked by Picard.[12] SNVs and indels were detected by SAMtools, and Annovar[13] was used for annotation. A mutation in γ-ENaC was identified by polymerase chain reaction (PCR) using gene-specific primer pairs: SCNN1G: (GenBank accession number NM_001039): forward primer: 5′-CTTGGGAATCAGGGTTCCTGTG-3′, reverse primer: 5′-AAGCAGGCTTTTTGGTCAGAGT-3′.[14] The PCR products were sequenced bidirectionally using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA).

RESULTS

Clinical features

The proband and his elder brother were admitted to the hypertension center of Fuwai Hospital for further evaluation. On admission, the proband did not complain of any symptoms. Biochemical measurements (Table 2) showed low PAC (supine position: 2.8 ng/dl; upright position: 2.2 ng/dl) and PRC (supine position: 1.8 µIU/ml; upright position: 2.0 µIU/ml), elevated microalbuminuria (42 mg/l) and uric acid (459 µmol/l), and decreased aldosterone concentration (0.74 µg/24 h) and sodium (103.03 mmol/24 h) in urine. Fundus examination indicated mild retinal arteriostenosis. Serum potassium was measured several times within the normal range. Echocardiogram and x-ray showed a normally sized heart, and a CT scan excluded renal and adrenal abnormalities. Laboratory results of the proband’s elder brother were similar to the proband (Table 2) in terms of low PAC (supine position: 2.6 ng/dl; upright position: 2.9 ng/dl) and PRC (supine position: 0.5 µIU/ml; upright position: 3.0 µIU/ml), elevated microalbuminuria (33.1 mg/l) and uric acid (459.36 µmol/l), decreased urine sodium (129.87 mmol/24 h), and mild retinal arteriostenosis. Echocardiogram detected left atrial enlargement (left atrial diameter, 41 mm.), and no abnormalities were found in serum potassium or CT scan of the kidney and adrenal glands. After a month of medication, their BP was successfully controlled, with a normal serum potassium level (Table 1). Both of their parents were healthy without any symptoms or signs. Another three hypertensives (subjects I-1, I-2, and II-2) were diagnosed with primary hypertension.

Table 2.

Laboratory examinations compared between the proband and his elder brother in hospitalization

	Normal reference value	Proband	Elder brother
Renal function
Blood urea nitrogen	2.86–7.90 mmol/l	4.95	4.92
Creatinine	44–133 µmol/l	78	97.17
Uric acid	148.8–416.5 µmol/l	459a	459.36a
Serum electrolytes
Sodium	137–147 mmol/l	139.88	141.3
Potassium	3.5–5.3 mmol/l	3.68	4.38
Chloride	99–110 mmol/l	103.57	103.7
Calcium	2.2–2.75 mmol/l	2.27	2.43
Phosphorus	0.97–1.60 mmol/l	1.33	1.09
Blood gas analysis
pH	7.350–7.450	7.419	7.401
HCO3-	21.0–27.0	25.7	26.2
Actual base excess	−3.0–3.0	1.7	1.6
Standard base excess	−3.0–3.0	1.7	1.9
Urine electrolytes
Sodium	130–260 mmol/24 h	103.03a	129.87a
Potassium	25–125 mmol/24 h	31.55	51.74
Urinalysis
Red blood counts	0–25/µl	0.1	1.3
Proteinuria	0.03–0.14 g/24 h	0.04	0.09
Microalbuminuria	<30 mg/l	42a	33.1a
Aldosterone	1.19–28.1 µg/24 h	0.74a	-
Plasma hormone levels
Renin, supine	2.8–39.9 µIU/ml	1.8a	0.5a
Renin, upright	4.4–46.1 µIU/ml	2.0a	3.0a
Aldosterone, supine	3.0–23.6 ng/dl	2.8a	2.6a
Aldosterone, upright	3.0–35.3 ng/dl	2.2a	2.9a

aAbnormal laboratory examination values.

Genetic findings

A heterozygous frameshift mutation in exon 13 of SCNN1G, p.Arg586Valfs*598, was found in the proband (Figure 2), resulting from the deletion of a single cytosine residue from a sequence of four consecutive cytosines in codons 585–586. This not only generates a premature stop codon at position 598, but also directly deletes the PY motif. This new stop codon results in a truncated γ-ENaC protein lacking the remaining 52 amino acids. This mutation was only detected in three family members (subjects II-3, III-3, and III-4). The identified mutation was also did not found in 100 hypertensives or 100 healthy controls. This variant was not found in the Exome Aggregation Consortium (http://exac.broadinstitute.org/), the 1000 Genomes Project database (http://browser.1000genomes.org), or the Human Gene Mutation Database (http://www.hgmd.org). In silico analysis predicted that it would be a probably deleterious mutation using MutationTaster2. Furthermore, there were no mutations in SCNN1A or SCNN1B in this family.

Figure 2.

Sanger sequencing identifying the heterozygous de novo mutation in SCNN1G. The arrow indicates the mutation site (c.1756delC, p.Arg586Valfs*598), which generated a premature stop codon at position 598.

DISCUSSION

In this study, we report a novel frameshift mutation (p.Arg586Valfs*598) in γ-ENaC with LS. Family screening showed that the identified mutation arose de novo and originated from the proband’s mother. Genetic analysis demonstrated that this pathogenic mutation generates a premature stop codon and deletes the PY motif.

Amiloride-sensitive ENaCs are expressed in various tissues, such as the apical portion of the epithelial cells of the distal nephron, distal colon, lung, and ducts of exocrine glands.[15] Under physiological conditions, ENaC expression and activity in the distal nephron is essential to maintain electrolytes homeostasis, together with the renal outer medullary K+ channels and Na+ /K+ ATPase.[16] The ENaC is composed of three subunits, α-ENaC, β-ENaC, and γ-ENaC, which share 30–40% homologous identity.[17] Each subunit passes through the membrane twice, creating two transmembrane regions separated by a large glycosylated extracellular loop with short intracellular N- and C-terminus.[18] A proline-rich sequence called the PY motif is completely conserved in all three subunits, located at the C-terminus.[19] The E3 ubiquitin-protein ligase Nedd4-2 catalyzes the ligation of ubiquitin to the ENaC subunit, leading to ENaC internalization and eventual degradation in a proteasome or lysosome.[20,21] Mutation or deletion of the PY motif reduces the rate of ENaC ubiquitylation and consequent internalization, leading to an increase in the number of ENaCs in the membrane, and consequently enhancing Na+ reabsorption and increasing water and sodium retention and potassium loss.[22,23] Therefore, this suggests that the PY motif plays a crucial role in the activity of the ENaC.

SCNN1G mutations are malignant and rare in LS. To date, only six different mutations in SCNN1G have been identified from different countries. Compared with the genetic spectrum of SCNN1B, most of SCNN1G mutations are nonsense mutations. In 1995, Hansson et al. firstly identified SCNN1G, in which the nonsense mutation c.1718G>A caused LS.[24] In the following decades, another three nonsense mutations in SCNN1G were reported.[25-27] All these nonsense mutations in SCNN1G truncated the carboxy terminus of γ-ENaC and directly deleted the PY motif. Wang et al. found a frameshift mutation caused by deletion of AGCTC at codon 583 in SCNN1G, resulting in a new termination site at codon 585 of the γ-ENaC and the deletion of the PY motif.[28] In this study, a novel frameshift mutation in SCNN1G, c.1756delC, was regarded as arising de novo, because the mutation came from the proband’s mother and was not detected in her parents. This mutation, with a single cytosine nucleotide deletion, generated a new stop codon at position 598 and caused loss of the PY motif. It was predicted that a dysfunctional truncated γ-ENaC protein would be translated.

The families with LS show marked phenotypic variation in BP, serum potassium, and other clinical manifestations.[9] A total of 54 genetically confirmed LS probands with 26 different mutations were summarized in terms of 100% hypertension, 94% hypokalemia, 84% suppressed PAC, and 94% suppressed plasma renin activity or PRC.[29] Systematic review of the reported LS cases revealed that 92.4% hypertension, 71.8% hypokalemia, and 58.2% hypoaldosteronemia were present in a total of 200 individuals affected by LS with genetic diagnosis, belonging to 72 different families.[7] The LS symptoms in the proband presented more typical than in all LS cases. Hypertension and hypokalemia are common phenotypes in LS subjects. Usually, hypertensives seek medical treatment for uncontrolled elevated BP, and then recurrent spontaneous hypokalemia will lead to a suspicion of LS after excluding common hypokalemic reasons,[30] such as primary aldosteronism, congenital adrenal hyperplasia, low potassium intake, gastrointestinal potassium loss, and licorice ingestion.[31] In our research, the proband and his elder brother manifested with early-onset hypertension and low PAC and PRC. Their mother, with an identified mutation, did not present any abnormalities. Their serum potassium levels were within the normal range. It has previously been reported that two LS patients with a de novo SCNN1G mutation suffered from hypertension[27,28] and hypokalemia.[28] Therefore, LS should be considered even in patients without spontaneous hypokalemia. As a result of the genetic sequencing, subject II-3 was identified as an asymptomatic SCNN1G mutation carrier. It will be necessary for her to be followed up for the rest of her life, as well as early interventions can be made to achieve a better prognosis, such as keeping a healthy lifestyle and reducing salt intake.[8]

The mechanisms leading to variable LS phenotypes remain unclear, although there are several hypotheses that might explain this. Dietary habits favoring a low salt intake could influence the phenotypic consequences.[32] Genetic factors such as gene polymorphisms could affect the phenotypic manifestation of the disease.[33] Epigenetic factors, including methylation, post-translational histone modification, and noncoding microRNAs, have been indicated to be significantly associated with hypertension.[34,35] Gene environment interactions also play an important part in modulating BP.[6] In the future, exploring these mechanisms may allow alteration of the progress and prognosis of LS by interventions for these factors.

In summary, we identified a novel pathogenic mutation of SCNN1G in a LS pedigree. Our study has expanded the genetic spectrum of SCNN1G and enriched the association between genotype and phenotype in LS. Genetic sequencing helps to identify causative mutations for LS cases, especially for those mutation carriers who manifest atypical clinical features. Combined with genetic and clinical characteristics, intervention as early as possible is beneficial to improve prognosis.

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