This study was agreed by the Research Ethical Review Committee, Beijing Children's Hospital Affiliated to Capital Medical University (approved protocol no.2017-k-81). 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. All blood samples were obtained after written informed patient consent and were fully anonymized. The participants and legal guardian provided written informed consent to participate in this study.

A 19-day-old neonate suffered an inexplicable electrolyte disturbance for 15 days and was admitted to the neonatal intensive care unit at the Beijing Children's Hospital with a history of poor feeding, hypothermia, lethargy, and bradycardia. She was born by cesarean section after a full-term pregnancy with a birth weight of 3,490 g (10–90^th percentile) and lost 15% of her weight 5 days after birth. She was the first child of her parents, and the mother had no perinatal problems. Neither of the parents had a family history of significant illness or unexplained death.

On physical examination, her weight and height were 3.09 kg (<3^rd percentile) and 50.2 cm (3^rd-10^th percentile), respectively, and her head circumference was 34.1 cm (10–50^th percentile). Routine examination showed a blood pressure of 73/39 mmHg, pulse rate of 148/min, respiratory rate of 44/min, and temperature of 37.2°C. The neonate was pale and lethargic and exhibited hypotonic extremities and severe dehydration. Her external genitals developed normally, and other physical examination results were within the normal range.

The initial serum laboratory examinations results were as follows: hyponatraemia: sodium 111 mmol/L (normal range = 130–150 mmol/L), hyperkalaemia: potassium 9.0 mmol/L (normal range = 3.5–5.5 mmol/L), hypochloraemia: chloride 83.6 mmol/L (normal range = 98–108 mmol/L), urea nitrogen 4.2 mmol/L (normal range = 1.7–7.1 mmol/L), and creatinine 23 μmol/L (normal range = 30–104 μmol/L). Arterial blood gas analysis revealed metabolic acidosis: pH = 7.227 (normal range = 7.251–7.429) and base excess:−14 mmol/L (normal range = −6–2). Plasma adrenocorticotropic hormone < 5–11.9 pg/ml(normal range = 0–46 pg/ml), Plasma cortisol 1.27–9.28 umol/dl (normal range =5–25 μmol/dl), Plasma testosterone < 20 ng/dl (normal range = 0–106.9), Plasma 17-α-hydroxyprogesterone 5.18 nmol/L (normal range = 0.31–38.1). Further laboratory studies subsequently revealed markedly increased plasma renin 5.36 ng/mL/h (normal range = 0.00–0.79 ng/mL/h), serum aldosterone 22.78 ng/dL (normal range = 5.9–17.4 ng/dL), and angiotensin II 398.7 pg/mL (normal range = 40.6–90.0 pg/mL). B-ultrasound of adrenal gland showed that the structure of adrenal gland was normal. Symptoms of hyperkalaemia persisted during the treatment from day 1 to day 4 (Figure 1). Meanwhile, through differential diagnosis, we ruled out renal failure, hypovolemia: shock or dehydration, adrenocortical insufficiency: adrenal hemorrhage, adrenal hypoplasia, etc., congenital adrenal hyperplasia, potassium retention diuretics and other diseases. Based on these clinical tests, a presumptive diagnosis of PHA was made (Table 1), metabolic acidosis was corrected, and blood potassium was lowered using simultaneous high-salt milk feeding (1 g Nacl/day, 20–30 ml/feeding). Because the PHA disease was not confirmed, the type and genetic pattern was also unclear, accurate treatment could not be provided; therefore, whole-exome sequencing and Sanger sequencing were performed on the neonate and her parents who without typical PHA disease symptoms.

We obtained the peripheral blood of the neonate and her parents at Beijing Children's Hospital and extracted the DNA immediately using the QIAamp DNA Blood Mini Kit according to the manufacturer's instructions.

Exome capture was performed using the Agilent SureSelect Human All Exon V6, according to the manufacturer's standard protocols. The Illumina Hiseq 2500 platform was used to detect the DNA sequence, and paired-end sequencing was conducted with a read length of 150 bp (16). More than 10 GB of clean sequence data and more than 100 × read depth were obtained for each DNA sample. All individual sequence reads were aligned to the human reference genome (NCBI build GRH37/hg19) using the Burrows-Wheeler Aligner software (17). The genome analysis toolkit, SAMtools, and Picard tools were used for bioinformatic analysis, and SNPs and insertion-deletions (indels) were identified using the genome analysis toolkit (18, 19).

Amino acid sequence alignment and the 3D structure of SCNN1B were constructed using Swiss-Model software (https://swissmodel.expasy.org/).

In order to identify the PHA disease and the type of PHA, SNPs and indels in the SCNN1A, SCNN1B, SCNN1G, WNK1, and WNK4 genes were investigated. As shown in Table 2, two novel frameshift mutations in SCNN1B (c.1290delA and c.1348_1361del) were revealed, both of which led to the production of the stop codon TGA. We further verified the two new mutations using Sanger sequencing (Figure 2A). Neither mutation had been described previously (http://www.ncbi.nlm.nih.gov/projects/SNP). Other candidate genes were also analyzed, but no significant pathogenic SNPs or indels were found (Table 2). Based on the molecular-level results, PHA type I was preliminarily identified.

Because the neonate's parents do not have typical PHA disease characteristics, next, we explored the genetic patterns of these mutations. As shown in Figures 2A–C, c.1290delA was carried by her mother, whereas c.1348_1461del was carried by her father, and the neonate inherited these two frameshift mutations concurrently. Her parents possessed heterozygous mutations with a normal phenotype, indicating a recessive inheritance pattern (Figure 2C). Thereafter, we confirmed that these two mutations were distributed in different chromosomes in the neonate, so the translation of the SCNN1B gene was terminated prematurely in both chromosomes. Whole-exome sequencing results showed that c.1290delA occurred at chr16:23388505, whereas c.1348_1461del occurred at chr16:23388651, and the distance between these two mutations was 146 bp (Figure 2D). We then counted the number of reads covered in this range, there was no single read containing both mutations, all the reads covered from chr16:23388505 to chr16:23388651 had only one mutation, either c.1290delA or c.1348_1461del, which indicates that the two mutations occurred in different chromosomes. Based on these findings, the neonate suffered a “double-stop” compound heterozygous mutation in the SCNN1B gene, which led to no functional B-subunit protein of ENaC and severe and persistent electrolyte abnormalities.

Further, we want to know whether these two mutations will cause changes in the three-dimensional structure of the protein. We found that the two mutations at the amino acid level were p.Gln431ArgfsTer2 and p.Thr451AspfsTer6. After amino acid sequence alignment, we found that these two frameshift mutations led to the early termination of amino acid synthesis (Figures 2E,F). We also found that both mutations led to an incomplete structure in the protein 3D prediction model (Figure 2G).

After confirmation of recessive hereditary PHA type I using genetic analysis, the parents were asked to go back to the local hospital for treatment after a clear diagnosis, we recommend continuing oral potassium lowering resin (1–2g/kg/day) and high-salt milk feeding (1 g Nacl/day, 20–30 ml/feeding) as therapeutic approach. Serum electrolytes returned to the normal range after initial parenteral hydration, oral sodium supplementation, and kayexalate usage; all clinical indicators were within the normal range after returning to the local hospital, but the development level was slightly slower than that of normal infants. At 3 months of age, the following measurements were obtained: weight 3.8 kg (normal range = 4.4–8.71 kg), height 55 cm (normal range = 54.2–67.5 cm), and head circumference 37 cm (normal range = 37.4–42.2 cm). At the age of 4 months, the infant returned to Beijing Children's Hospital due to convulsions without obvious causes, respiratory infection symptoms, increased blood potassium level, decreased blood sodium level, and ventricular tachycardia. Subsequently, resuscitative efforts were unsuccessful and the infant died.