Bartter syndromes (BS) are inherited disorders consisting of renal salt reabsorption in the distal renal nephron which lead to hypokalaemic metabolic alkalosis. Recent genetic studies have found that these diseases are caused by mutations in five genes, which lead, directly or indirectly, to either transporter or channel loss of function.1^–7 In recent years, the BSND gene encoding barttin, a subunit common to ClC-Ka and ClC-Kb, has been identified as being responsible for a combination of antenatal BS and sensorineural deafness (SND), and this phenotype is now known as type IV BS.5 The mutation in the BSND gene leads to the impairment of both ClC-Ka and ClC-Kb activity, which suggests that double mutations in both the CLCNKA and CLCNKB genes which encode ClC-Ka and ClC-Kb, respectively, lead to type IV BS phenotype. Indeed, a recently reported case of BS with SND from a consanguineous family was found to have homozygous mutations in both CLCNKA and CLCNKB.8 This form of digenic inheritance was found to be autosomal recessive. However, the possibility remained that consanguinity could have caused a completely different inherited and simultaneous disease associated with the deafness. Furthermore, only one case with this complex inheritance and no such cases from a non-consanguineous family have been reported.

The fact that mutations in both genes, CLCNKA and CLCNKB, indicate the presence of a severe phenotype with SND is compatible with what has been predicted from the interaction of the gene products of CLCNKA, CLCNKB and BSND,9 and points to the existence of digenically inherited diseases, a quite new concept in Mendelian disorders.10 11 Such a concept can explain the absence of genotype–phenotype correlations in inherited disorders.

In this study, we confirm that mutations which affect both alleles of CLCNKA and CLCNKB lead to severe BS with SND.

SUBJECT AND METHODS

Patient

This report concerns a 2-year-old female from unrelated parents. Polyhydramnios was detected before birth, and she was born at 31 weeks’ gestation with a body weight at birth of 1150 g (small for gestational age). Immediately after birth she manifested polyuria and severe volume depletion (18% body weight loss on day 4). On postnatal day 1, she showed hyponatraemia, but serum potassium concentration was normal (table 1). From day 2, severe hypokalaemia and metabolic alkalosis were noted, necessitating intravenous administration of water and electrolytes. On day 4, however, acute renal failure occurred, most probably because of volume depletion so that continuous water, sodium and potassium supplementation was needed. In spite of treatment with indomethacin which was started at the age of 1 month, she showed only minor clinical and biochemical improvement and needed intermittent intravenous supplementation of fluids and electrolytes. Auditory brainstem response (ABR) testing, performed when the patient was 49 days old, disclosed bilateral sensorineural deafness. Since she was suffering from frequent vomiting and showed severe failure to thrive (−4.1 SD from normal body weight at 9 months of gestation), she was started on an alimentary diet through a duodenal tube. This enabled us to wean the child from intermittent intravenous supplementation fluids and electrolytes and her growth improved to −1.8 SD from average body weight at 18 months of gestation. She is now 2 years old but she cannot stand or walk. Renal ultrasound does not show any evidence of nephrocalcinosis. However, low serum potassium, serum magnesium and urinary calcium excretion is accompanied by hyperreninaemic hyperaldosteronisms and mild renal dysfunction (serum creatinine 0.81 mg/dl (71.7 μmol/l); normal for 2 years of age: 0.45 mg/dl (39.8 μmol/l)). All these clinical and chemical findings indicate that this case represents one of the most severe phenotypes of BS reported to date.

Mutation analysis

Having obtained informed consent from her parents, we performed genetic analysis of the BSND, CLCNKA and CLCNKB genes. The CLCNKA and CLCNKB genes are directly adjacent to each other on chromosome 1. Genomic DNA was extracted from leucocytes with the standard phenol-chloroform extraction method. We selected specific primers for these genes, which amplify all exons and exon–intron boundaries. Polymerase chain reaction (PCR) was followed by purification of the PCR products and direct sequencing on both strands. Direct sequencing of double-strand DNA fragments of all exons of the genes was performed with the aid of the PCR primers and an ABI PRISM Bigdye Thermal Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA) and an ABI model 310 autosequencer.

Semiquantitative PCR amplification

A large heterozygous deletion was detected with semiquantitative PCR amplification using capillary electrophoresis, a recently reported methodology.12 PCR cycling conditions were as follows: initial denaturation at 94°C for 5 min followed by 20 cycles of denaturation at 94°C for 45 s, annealing at 55–60°C for 45 s, extension at 72°C for 2 min, and a final extension at 72°C for 5 min. To quantify the amplified products, 1 μl of each reaction mixture mixed with 5 μl of the loading buffer solution containing size markers (15 bp and 1500 bp) was analysed by capillary electrophoresis (Agilent 2001 Bioanalyzer with DNA 1000 Lab Chips; Agilent Technologies, Palo Alto, California, USA). Each of the PCR products was quantified by measuring the peak area and calculating the ratio of the exons of CLCNKA and CLCNKB to exons of the following genes that are outside the locus as internal controls. Two exon fragments, the one corresponding to CLCNKA or CLCNKB exons and an internal control fragment (primer pairs for SLC12A1 exon 1, 22 or SLC12A3 exon 19), were co-amplified in one PCR reaction. The internal control primers were then selected according to the PCR amplification conditions and PCR product size, since, to co-amplify primers with CLCNKA or CLCNKB exons in the same tube, it is necessary to have exactly the same PCR conditions to get equally amplified PCR products while the products must be of different size for electrophoresis. The parents were analysed at the same time with this method.

RESULTS

Mutational analysis with PCR and direct sequencing

No mutations were detected in any of the exons and exon–intron boundaries in the BSND gene. Figure 1 shows the mutations in the CLCNKA and CLCNKB gene of the patient, a heterozygous nonsense mutation of CLCNKA, p.260Q/X (c.778C/T in exon7), and a heterozygous splicing site mutation of CLCNKB, IVS17+1 g/a (fig 1A,B). The patient’s father was found to also carry these heterozygous mutations. Unexpectedly, with direct sequencing only a heterozygous mutation was detected in both CLCNKA and CLCNKB. The patient’s father also possessed these mutations, proving that these two mutations were of the paternal allele. As BS is a recessive disease, this means that the patient should also have a mutation in the maternal allele of CLCNKA and CLCNKB, but we could not identify these mutations with the most common genetic analysis method of direct sequencing.

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Figure 1 Detection of CLCNKA and CLCNKB mutations by direct sequencing. (A) A heterozygous nonsense mutation of CLCNKA, Q260X (c.778C>T in exon 7), in the patient and her father. (B) A heterozygous splicing site mutation of CLCNKB, IVS17+1 g>a in the patient and her father. (C) Large heterozygous deletion mutation from CLCNKA exon 16 to CLCNKB intron 2 was detected in the patient and her mother by using primer pairs at the positions shown in fig 3. The 5′ breakpoint was the 46 base of the CLCNKA exon16 and the 3′ breakpoint was within intron 2 (exon 3–185 base) of CLCNKB.

Semiquantitative PCR amplification analysis

The CLCNKA and CLCNKB genes are directly adjacent to each other on chromosome 1, and are therefore prone to contiguous deletions. Some reports have described large deletions in the CLCNKB gene4 and we have also reported cases with large heterozygous deletion mutations in this gene.12 To identify such deletions in the case reported here, we performed semiquantitative PCR amplification using capillary electrophoresis with the same method as described previously.12 The results showed that the quantity of PCR products from CLCNKA exon 17 to CLCNKB exon 2 possessed by the patient and her mother was half that of her father and normal control. On the other hand, the quantity of PCR products from CLCNKA exon 1 to 15 and CLCNKB exon 3 to 19 was the same (only the results for CLCNKA exon 15, 17 and CLCNKB exon 2 and 3 are shown in fig 2). This reduction in the copy number from CLCNKA exon 17 to CLCNKB exon 2 of the patient and her mother means that the patient possessed a maternal heterozygous deletion of both neighbouring genes.

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Figure 2 Semiquantitative polymerase chain reaction (PCR) amplification of the CLCNKA and CLCNKB gene. Capillary electrophoretic patterns of PCR products. (A) CLCNKA exon 15 and SLC12A3 exon 19 (internal control). (B) CLCNKA exon 17 and SLC12A1 exon 26 (internal control). (C) CLCNKB exon 2 and SLC12A1 exon 1 (internal control). (D) CLCNKB exon 3 and SLC12A1 exon 19 (internal control). The patient and her mother had half the quantity of PCR products from CLCNKA exon 17 to CLCNKB exon 2 as detected in her father and normal control and the same quantity of CLCNKA exon 1 to 15 and CLCNKB exon 3 to 19 (only the results of CLCNKA exon 15, 17 and CLCNKB exon 2 and 3 are shown).

Detection of the large deletion breakpoint using PCR, electrophoresis and direct sequencing

These findings prompted us to conduct a PCR analysis in order to detect the breakpoint of the large heterozygous deletion within the neighbouring genes CLCNKA and CLCNKB. The forward primer in intron 15 of the CLCNKA gene and the reverse primer in intron 3 of the CLCNKB gene were used for PCR and produced a 522 bp amplicon in the patient and the mother. Findings of semiquantitative PCR with electrophoresis suggested that the mother possessed a large heterozygous deletion mutation (fig 3). Direct sequencing of this PCR product showed that the 5′ deletion breakpoint was located in exon 16 of the CLCNKA gene and the 3′ breakpoint in intron 2 of the CLCNKB gene (fig1C; schematic representation of this patient’s mutations in the CLCNKA and CLCNKB genes is shown in fig 4A). This proved to be exactly the same large deletion mutation as previously identified by us in another patient who had type III BS phenotype (including a compound heterozygous mutation with nonsense mutation in the CLCNKB gene in addition to this large heterozygous deletion mutation).12 This suggests that this deletion mutation may be relatively common, at least among Japanese. Our patient’s parents showed no apparent clinical symptoms or laboratory data abnormalities.

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Figure 3 Detection of large heterozygous deletion mutation and point mutations in both alleles of CLCNKA and CLCNKB. The forward primer within intron 15 of the CLCNKA gene and the reverse primer within intron 3 of the CLCNKB gene were used at the positions indicated in fig 4A. Amplification by PCR with this primer pair generated a 522 bp amplicon, which was detected in both the patient and her mother, in whom the presence of a large heterozygous deletion mutation was suggested by semiquantitative PCR. (The result of the direct sequencing of this PCR product is shown in fig1B).

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Figure 4 (A) Schematic representation of mutations in the CLCNKA and CLCNKB genes of the patient. The primer pairs used to detect the large deletion are indicated by arrows (f and r). The paternal allele possessed a nonsense mutation of CLCNKA, Q260X and a splicing site mutation of CLCNKB, IVS17+1 g/a. The maternal allele possessed a large deletion, with the 5′ deletion breakpoint located in exon 16 of the CLCNKA gene and the 3′ breakpoint located in intron 2 of the CLCNKB gene. (B) Schematic representation of the relationship between phenotype and mutations in the CLCNKA and/or CLCNKB genes. In comparison to the wild type, the ClC-Ka defects may lead to mild diabetes insipidus, ClC-Kb to type III Bartter syndrome (BS), barttin to type IV BS and both ClC-Ka and ClC-Kb defects to BS with sensorineural deafness (SND).

DISCUSSION

Bartter syndrome is a salt wasting disorder originating from defects in sodium chloride reabsorption in the thick ascending limb and/or the distal convolute. This report describes a female patient with neonatal BS accompanied by SND and mild renal dysfunction, the most severe phenotype of BS. From the neonatal period onwards, she showed life-threatening volume depletion and severe failure to thrive, indicating a phenotype very similar to the severe phenotype of type IV BS. Although the genetic test result for the BSND gene of our patient was negative, we succeeded in detecting mutations in both paternal and maternal alleles in both the CLCNKA and the CLCNKB gene. The only other reported case with this phenotype featuring mutations in both chloride channels8 was born from consanguineous parents. In such a case, there is a possibility that consanguinity may lead to a completely different, simultaneously inherited disease resulting in deafness as mentioned in that report.8 In our case, however, the parents are not consanguineous and this confirms that the presence of two loss-of-function alleles in both Cl-channels generates a severe BS phenotype with SND as was predicted by the concept on how ClC-Ka, ClC-Kb, and barttin interact.9

A schematic representation of the relationship between phenotype and mutations in the CLCNKA and/or CLCNKB genes is shown in fig 4B. In contrast to our and the previously reported case,8 type III BS, the sole ClC-Kb disorder, has a milder phenotype. Most type III BS cases are characterised by neither polyhydramnios nor premature delivery, while renal function is always normal and no sensorineural deafness has been detected.4 12 Moreover, no diseases caused by CLCNKA gene mutations in humans have been reported to date, although a mild diabetes insipidus has been detected in CLCNKA knockout mice.13 This phenotype has been attributed to impaired passive chloride reabsorption, which impairs water absorption by reducing inner medullary tonicity.14

Recently, atypical patterns of inheritance, including digenic inheritance, have attracted increasing attention.10 11 The first report of digenic inheritance concerned retinitis pigmentosa caused by a heterozygous mutation in the RDS gene in combination with a heterozygous mutation in the ROM1 gene. Retinitis pigmentosa did not occur with either heterozygous mutation alone.15 This report was followed by several accounts of this form of inheritance in Bardet–Biedl syndrome,16 deafness,17 18 cortisone reductase deficiency,19 early onset Parkinson disease20 and early onset autosomal dominant polycystic kidney disease.21 22 Our case showed sensorineural deafness and severe phenotype of BS, while the CLCNKA gene mutation alone may lead to mild diabetes insipidus and the CLCNKB gene mutation alone generates a mild BS phenotype classified as type III BS (fig 4B). Further studies are needed to elucidate why each of the chloride channel disorders by itself shows a very mild phenotype while disorders of both channels show such severe symptoms.

Mutations in the BSND gene have been reported to be associated with progression of chronic renal failure in the infantile period,5 23^–25 which is an uncommon complication for other types of BS.2^–4 However, recent studies of type IV BS, with the missense mutation G47R detected at least in one allele, have reported a mild phenotype without renal insufficiency,26^–29 while electrophysiologically this mutation showed no impairment of chloride transport.9

The same situation may apply to our and the previously reported case. The latter featured a homozygous missense mutation in the CLCNKA gene and an entire homozygous deletion in the CLCNKB gene.8 The authors confirmed by means of voltage clamp analysis that the missense mutation in the CLCNKA gene caused reduced function of ClC-Ka but did not completely suppress it, and their patient did not show renal insufficiency, at least not during the infantile period. Although we did not conduct functional studies of the mutations detected in our case, mutations were detected that can lead to complete loss of function of the chloride channels. Moreover, she showed mild renal dysfunction from the infantile period. Our case indicates that, in addition to the CLCNKB mutation, the disease severity greatly depends on the genotype of the CLCNKA gene, and that missense mutations will generate a mild phenotype, and nonsense or deletion mutations a severe phenotype.

In our case, hypocalciuria and hypomagnesaemia were detected since the age of 6 months. These biochemical abnormalities are always seen in Gitelman syndrome, which is caused by mutations in the SLC12A3 gene that encode the thiazide sensitive sodium chloride channel (NCCT) in the distal tubules. Cases with type IV Bartter syndrome sometimes show hypomagnesaemia, but hypocalciuria is quite rare.5 23^–29 On the other hand, cases with type III BS caused by the CLCNKB gene mutation alone sometimes show this clinical phenotype.12 The mechanism has not been clarified yet, but it is generally considered that the ClC-kb defects in the distal tubules may affect the NCCT function, resulting in the hypocalciuria seen in Gitelman syndrome. This may also apply to our case.

To summarise, our report describes the second case identified to date with mutations in both the CLCNKA and CLCNKB gene that showed severe antenatal BS accompanied by SND. Moreover, this is the first report of a case from a non-consanguineous family, which confirms that the combined mutations of the two chloride channel genes cause not only deafness but also a much more severe renal condition compared with the disease entities associated with type III BS.