In the present study, the proband, a ten-year old boy primarily incidentally detected with bilateral nephrolithiasis was found with compound heterozygous mutations of KCNJ1 gene by NGS. A novel heterozygous missense mutation c.65G>T (p.R22M) identified by Sanger sequencing was inherited from the phenotypically healthy father, which could be discerned from a polymorphism since the harmful predictions by all three bioinformatics softwares were pathogenic and this mutation was not found as SNP in the global SNP database. Through the symptoms and genetic test, the proband was confirmed with type II Bartter syndrome (BS II).
KCNJ1 gene is one of the five types of genes involved in the etiology of BS which is a group of rare tubulopathies. Patients with different type of BS present with overlapping clinical phenotypes as polyuria, polydipsia, volume contraction, muscle weakness and growth retardation induced from hypokalaemia, hyperreninism and hyperaldosteronism. According to the onset and severity of BS, it can be grouped into three types: the hypocalciuric-hypomagnesemic variant described by Gitelman et al., the classic syndrome originally described by Bartter et al., and the antenatal hypercalciuric variant associated with severe systemic manifestations classical type 6. KCNJ1 gene encodes the apical potassium inwardly-rectifying channel (ROMK) in the thick ascending limb of the Henle’s loop (TALH) in the distal nephron to ensure adequate luminal potassium available for the efficient functioning of the Na-K-2Cl cotransporter which function in salt reabsorption 7. Effective chloride reabsorption in the TALH prevents renal salt wasting and is an essential mechanism to maintain tubular concentrating capability. Loss-of-functional mutations in the KCNJ1 gene cause antenatal/neonatal Bartter syndrome type II (aBS II) in autosomal recessive pattern 8.
The Phenotype in most of patients with BS II can begin in utero with marked fetal polyuria presenting polyhydramnios from 24 weeks of gestation and premature delivery. During neonatal period, patients may have life-threatening volume depletion caused by severe renal salt wasting or failure to thrive. During childhood, other secondary symptoms including developmental retardation, fever, vomitting, occasional diarrhea may present resulted from metabolic alkalosis, hyposthenuria, hyperreninaemic, hyperaldosteronism which was stimulated by elevated plasma concentration of prostaglandin E2 (PGE2). The basic deficiency of antenatal BS is the malfunction of mTAL chloride transport, which involves an interaction among the apical Na-K-2Cl cotransporter (NKCC2), the luminal ATP-sensitive potassium channel ROMK (KCNJ1), the basolateral chloride channel (ClC), a basolateral K-CL cotransporter and the Na-K-ATPase. Therefore, any gene encoding or involving in these channels or transporters will result in defective chloride transport. NKCC2, KCNJ1, CLCNKB for chloride channel and BSND gene encoding barttin, a subunit for ClC-Ka and ClC-Kb have been confirmed with antenatal BS 9,10. Rare disease shall also be differentiated such as Rabson-Medndenhall syndrome caused by INSR 11.
The other equally important feature in antenatal BS is hypercalciuria. Continuous loss of calcium results in nephrocalcinosis, nephrolithiasis and osteopenia 12,13, usually medullary nephrocalcinosis is seen 14,15. Hypercalciuria and associated nephrocalcinosis are present in approximately 85% of infants with this neonatal BS 16. The prevalence of nephrolithiasis is high, but the prevalence secondary to BS is not known very well, and may be lower than the prevalence of nephrocalcinosis. Both nephrocalcinosis and nephrolithiasis share a well-recognized heritability 17,18, and around 15% of the patients were detected with causative genes 4. Although low plasma potassium concentration, secondary low urinary citrate, tubulointerstitial damage, chloride deficiency, and increased intracellular chloride activity were also suggested to contribute to the hypercalciuria, the exact pathogenesis of nephrocalcinosis or nephrolithiasis in BS remains unclear 19. Renal function is generally well preserved. In the present study, GFR of the proband was lower than the normal population. According to the ten-year outcome study by Puricelli E et al. 20, 25% of the patients with type I or type II BS had GFR lower than the normal range, which may be resulted from nephrocalcinosis. More than 30 genes have been reported to be with the etiology of nephrolithiasis 4. Two-thirds of the genes currently known to be associated with nephrolithiasis coding for membrane proteins or enzymes involved in renal tubular transport21. The TALH and connecting tubules (CNT) have a central role in maintenance of fluid, electrolytes and acid-base homeostasis. Therefore, mutations of genes involved in TALH and CNT function can result in phenotypically severe disease. 14 of all genes are of paramount importance accounting for 15% of nephrolithiasis or nephrocalcinosis 22. Recessive causes were more frequent among children, whereas dominant disease occurred more abundantly in adults. Therefore, NGS panel including genes involved in functions of TALH, connecting tubules, systemic disorders such as chromic hypercalcemia from vitamin D, primary hyperoxaluria, ARPT deficiency, distal renal tubular acidosis, Dent’s disease, cystinuria and family hypomagnesemia with hypercalciuria shall be applied 23,24. In this study, 248 genes associated with hereditary kidney diseases were all included in the panel, and no other suspicious gene mutations were found except KCNJ1 gene.
KCNJ1 gene mutation associated antenatal BS is phenotypically distinct from the other disease because of prominent polyhydramnios with preterm delivery together with discontinuous fatigue, still phenotypic variability presents in patients with KCNJ1 mutation and absence of enough recognition for this type of disease may exist. The patient in the present study was not gotten accurate diagnosis until he was ten-years old and incidentally found bilateral nephrolithiasis, although he had the previous infant history with polyhydramnios and preterm delivery, and the intermittent cramps, fatigue and muscle weakness during childhood.
There are other causes which could also induce either of these symptoms. The clinicians or parents may ignore the real etiology beneath the manifestations and the clinical misdiagnosis of BS was nearly 25%, especially in developing countries 25. Also the onset of BS type may be late. One adult male patient initially presented with an incidental finding of nephrocalcinosis was diagnosed as a late-onset BS due to detection of a homozygous KCNJ1 missense mutation (c.658C > T, p.L220F) 26.
Our case showed that the presentations in patients with BS may not be unusual, and specific disorders within the spectrum of BS or nephrolithiasis may not easily be diagnosed or differentiated by rigorous clinical manifestations. Genetic test, especially NGS is a very efficient tool to distinguish specific disorder from multiple confusing spectrums.