A novel mutation of KCNJ1 identified in an affected child with nephrolithiasis

Nephrolithiasis is not common in children compared to adults, but the incidence of pediatric nephrolithiasis is gradually increased in these years [1]. Urinary tract malformations, urinary infection, dietary habits, geographic region and genetic factor are involved in the etiology of nephrolithiasis [2, 3]. There are at least 30 genes shown to cause monogenic forms of nephrocalcinosis or nephrolithiasis in autosomal-dominant, autosomal-recessive, or X-linked transmission patterns [4]. For the affected child, it is especially important to detect the exact causative mutation of monogenic disease and know the etiology, which may provide an accurate diagnosis, personalized therapy and effective follow-up strategy.

In the present study, we described the clinic features of one 10-year old child who was incidentally found with nephrolithiasis and detected with the genetic mutation, which suggests that NGS is very important and efficient to identify the etiology of nephrolithiasis, which may not be easily diagnosed by manifestation or chemical laboratory tests.

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. KCNJ1 gene is one of the five types of genes involved in the etiology of Bartter syndrome type II (BS II) 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 [8]. 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 function of the Na-K-2Cl cotransporter which is involved in salt reabsorption. 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 BS II in autosomal recessive pattern [9].

In this study, two mutations of KCNJ1 c.89G > A (p.C30Y) and c.65G > T (p.R22M) were detected in the proband. The distribution of the identified variations in KCNJ1 is shown in Fig. 5. ROMK is responsible for K⁺ secretion and control of NaCl absorption in the kidney. The channel is gated by intracellular pH in the neutral range and reach half-maximal activation at a pH of 6.8. The gating is driven by the protonation of lysine within KRR [10], which is assembled by amino acid residues at positions 22, 61, and 292 in the transmembrane region [7]. Structural disturbance of KRR shifts the pKa of the lysine residue away from the neutral pH range and leads to channel inactivation. The predicted model of p.R22M revealed that the distance to Arg292 (11.8 Å) and Lys61 (30.3 Å) changed into 13.1 Å, and 31.2 Å, respectively, when the mutation replaced Arg22 with Met22. The p.R22M mutation could disrupt the conformation of KRR and produce steric clashes with spatially adjacent residues, causing structural destabilization. A three-dimensional structural analysis also revealed that Arg22 formed H bonds with Glu299, Ser294, and Val24. When the mutation replaced Arg22 with Met22, these H bonds were destroyed. In summary, the p.R22M mutation was able to influence KRR in two ways, either shifting the pKa of the lysine residue off the neutral pH range or influencing the tertiary geometry to further change the integrity of the structure and function of ROMK. The p.C30Y mutation caused a side-chain change of residue, which may affect protein structure and function. Clinical reports indicated that the p.C30Y mutation was pathogenic. Through the symptoms and genetic test, the proband was confirmed with type II Bartter syndrome (BS-II).

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. All the symptoms 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, 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 [11, 12]. Rare disease shall also be differentiated from Rabson-Medndenhall syndrome caused by INSR [13].

The other equally important feature in antenatal BS is hypercalciuria. Continuous loss of calcium results in nephrocalcinosis, nephrolithiasis and osteopenia [14, 15], usually medullary nephrocalcinosis is seen [16, 17]. Hypercalciuria and associated nephrocalcinosis are present in approximately 85% of infants with this neonatal BS [18]. 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 [19, 20], 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 [21]. 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. [22], 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 transport [23]. 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 [24]. 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 [25, 26]. 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 [27]. 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 [28].

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.