The girl in this study was the second child in a non-consanguineous Chinese family. Her mother was 27 years old, with gravida 5 para 2. She was born at 37 + 6 gestational weeks. Her birth weight was 2.8 kg and no complications occurred during delivery. Her Apgar scores at 1, 5, and 10 min were (10/10/10), respectively. At 32 gestational weeks of pregnancy, an ultrasound examination revealed a very slight increase in amniotic fluid levels (amniotic fluid index, AFI: 21.9 cm). Her parents were healthy with no family history of disease. The patient's elder sister started showing progressive metabolic alkalosis and hypokalemia at 18 days of life (Figure 1A). The mother's past terminations of pregnancy were not due to medical reasons. On the third day after birth, the patient started to show symptoms of jaundice, and her transcutaneous bilirubinometer reading in the outpatient clinic showed 22.1 mg/mL (mature newborns <12 mg/mL). She was diagnosed with neonatal hyperbilirubinemia (NHB) and admitted to the hospital. After hospitalization, more laboratory tests were conducted: the patient's total bilirubin (TBIL) was 344.9 mumol/L (normal range: 0-23 micromol/L). The patient also revealed an elevated creatine kinase-MB level (CK-MB: 6.2 microg/L) and an elevated high-sensitivity troponin I level (hsTnI: 0.102 microg/L), which indicated possible myocardial damage. An arterial blood gas (ABG) analysis indicated metabolic alkalosis (pH: 7.595, PO2: 134 mmHg, PCO2: 36.7 mmHg, HCO3-: 35.6 mmol/L). A blood electrolyte test revealed hypokalemia (K+: 2.9 mmol/L), hypochloremia (Cl-: 89 mmol/L), and plasma hypoosmolality (mOsm: 271.9). Moreover, the patient showed hyperreninemia (renin: >835.0 microIU/mL), hyperaldosteronism (aldosterone: 1,427.89 pg/mL), and an increased blood Ang II level of 169.84 pg/ml, while her blood pressure was normal (65/44 mmHg) (Table 1). During the physical examination, urine volume was found to be normal, and an ultrasound of the kidneys and adrenal glands showed no sign of nephrolithiasis.
A comprehensive timeline of the patient is presented in Table 2.
Peripheral blood samples of the patient and her parents were used for genetic analysis. We used custom-designed NimbleGen SeqCap probes (Roche NimbleGen, Madison, WI, United States) for in-solution hybridization to amply target sequences. Clinical exon sequencing (CES) candidate genes include more than 5,000 disease-causing genes selected from reports in the OMIM, HGMD, and reliable literature, and known pathogenic variants in deep introns and non-coding regions in targeted genes were also included (Supplementary Table). The DNA samples were indexed and sequenced by using the Illumina sequencer (San Diego, CA, United States). The average coverage depth was about 200 x with over 98% of the target regions covered by at least 20 reads. Nucleotide changes found in the aligned reads were pulled and analyzed using the NextGENe software (Version 2.4.2) (SoftGenetics, State College, PA, United States). Sequence variants were annotated and included population (gnomAD, 1,000 Genomes, dbSNP) and variant databases (Clinvar, HGMD). Online software Polyphen-2 and SIFT were used for in silico analysis of missense variants. The variants were classified as "Pathogenic," "Likely Pathogenic," "Uncertain Significance," "Likely Benign," or "Benign" according to the American College of Medical Genetics and Genomics (ACMG) guidelines. A trio-clinical exome sequencing (AmCare Genomics Lab, Guangzhou) was performed on the patient and her parents. A frameshift mutation c.1257delC (p.M421Cfs*58) in exon 13 of the CLCNKB gene was found in the patient, which was inherited from the patient's asymptomatic father (Figure 1A). Other candidate genes of Bartter syndrome and Gitelman syndrome such as SLC12A1, KCNJ1, BSND, CLCNKA, MAGED2, and SLC12A3 did not reveal any significant variants. Bartter syndrome is an autosomal recessive disease, in which both alleles of the CLCNKB gene are affected by recessive mutations to present symptoms. However, CES detected no variants in the trans allele of the CLCNKB gene at first. Given that both the patient and her elder sister presented typical cBS clinical characteristics, we reviewed the data origin and manually searched for a non-sense mutation c.595G > T (p.E199*) in exon 6 of the CLCNKB gene with a low-variant allele frequency (VAF) of 16.44%. This mutation was also verified in the patient's mother with a VAF of 20.8% (Figure 1B,C). Therefore, we speculated that mutation c.595G > T (p.E199*) in the CLCNKB gene was a low-grade mosaic mutation. Sanger sequencing was used to confirm the low VAF mutation (Figure 1D). Therefore, the diagnosis of Bartter syndrome was confirmed.
The patient was treated with blue light for NHB and potassium infusion therapy to reverse the electrolyte disturbance. After 7 days of blue light treatment, hyperbilirubinemia significantly reduced, and blood TBIL dropped to 94 micromol/L. The patient was followed up for 22 months. She accepted daily oral potassium chloride supplementation and indomethacin (7.5 mg, tid). On the last follow-up, the patient's metabolic alkalosis had significantly improved, but hypokalemia (3.07 mmol/L) and hypomagnesemia (0.46 mmol/L) were still present. No obvious developmental delay or a degeneration of the renal function was observed in the patient.