Bartter syndrome (BS) is a rare autosomal recessive disease caused by pathogenic variants in the genes coding for proteins related to salt transport and regulation in the thick ascending limb of Henle's loop in the nephrons. It was first described in 1962, characterized by hyperplasia and hypertrophy of the juxtaglomerular apparatus, hyperaldosteronism and hypokalemic alkalosis, associated with impaired growth and development (1,2). Later studies divided BS into classic, as described by Bartter et al (1), and antenatal (3). Both types were further divided into subtypes, according to the affected gene: SLC12A1 (antenatal, type I) (4-6), KNJ1 (antenatal, type II) (7,8), CLCNKB (classic, type III) (9,10), BSND or CLCNKA and CLCNKB at the same time (antenatal, type IV) (11-14).

BS type IVa (OMIM 602522) is used to refer to BS type IV caused by variants in the gene BSND. BS type IV usually presents with severe clinical manifestations and is the only subtype which incurs sensorineural deafness. It is typically presented by premature birth, polyhydramnios, hyper-PGE2, severe hypokalemic hypochloremic metabolic alkalosis, iso- or hyposthenuria, and previously mentioned sensorineural deafness (12,15-18). The BSND gene, located in 1p32.3, is composed of 4 exons, and encodes for Barttin, the β-subunit of chloride channel (CLC)-kidney a (Ka) and CLC-Kb channels. These largely similar channels are part of the CLC family of voltage-gated chloride channels and were named after appearing to be two different channels of this family that were kidney-specific (chloride channel-kidney; letters ‘a’ and ‘b’ being used solely to differentiate the two). Further studies demonstrated that they are expressed in basolateral membranes of renal tubules, in the thick ascending limb of Henle's loop, and also the stria vascularis of the inner ear (11,19-21).

Barttin is composed of 320 amino acids: A short N-terminus (aa1-8) which affects CLC-K trafficking and activation, two transmembrane helices (aa9-26 and aa35-54), an extracellular linker between them (aa27-34), and a cytoplasmatic C-terminal tail (aa 55-320) of which 17 amino acids (aa55-72) are needed for CLC-K conductivity (11,19,22-24). Barttin is essential for ClC-Ka and ClC-Kb function and altered or loss of Barttin function will alter or impede function of these channels. This will in turn cause an ion imbalance in the thick ascending limb (TAL) of Henle's loop, where all proteins associated with BS act in conjunction with one another to promote ion reabsorption in the nephron (18,25-27). The differentiation of BS type IV from the other types is associated with the expression of Barttin along with ClC-Ka and ClC-Kb in the inner ear, where these channels help maintain K+ levels in the endolymph. Therefore loss of Barttin function will also cause hearing impairment (11,20).

Several pathogenic variants are described in the BSND gene and most of them are located in exon 1, which is the portion that codifies the N-terminus and transmembrane helices (Table I). These variants have significant effects on the structure and function of the Barttin protein, potentially interfering with its ability to interact with CLC-K channels or impairing its stability within the cell membrane (11,12,28-43).

In the present study, an uncommon association between a novel variant in exon 4 of the BSND gene (c.784delG;p.(Ala262Profs*68)) together with the known pathogenic variant in exon 1 (c139G>A, rs74315289) was described. The variant was found in a 10-year-old Brazilian girl diagnosed with typical BS type IV. DNA sequencing revealed that this is the unique alteration found in this gene and that the compound heterozygous variant was potentially responsible for the development of BS type IVa in this patient.

Clinical diagnosis of BS type IV guided the genetic analysis to the BSND gene, as this is the most common gene related to BS with sensorineural deafness, with a few reported cases of recessive pathogenic variants affecting both CLCNKA and CLCNKB genes (47,48). Sequencing analysis revealed that in our patient, the BSND gene was indeed the reason for the present syndrome, caused by the compound heterozygous variants. In exon 1, analysis revealed the pathogenic variant c.139G>A;p.Gly47Arg (rs 74315289), which has been previously described in patients withBS type IV (28-32,42,43,49). In exon 4, the novel variant c.784delG;p.(Ala262Profs*68)was found (Fig. 1A). Variant analysis on the patient's mother, revealed that she is heterozygous for c.139G>A;p.Gly47Arg, but the c.784delG;p.(Ala262Profs*68) variant was not found (Fig. 1B). It was not possible to determine if the novel variant c.784delG;p.(Ala262Profs*68) is a de novo mutation or was inherited from the father, since he was deceased when the present study was carried out and variant analysis was not possible.

In silico analysis of deletion of c.784delG;p.(Ala262Profs*68) in MutationTaster2(39) classified it as ‘disease causing’ and showed that the resulting frameshift modifies 58 residues in the C-terminal region. It then causes the loss of the original stop codon, adding another 9 residues in comparison to the wildtype protein. CADD analysis was also performed (46), obtaining a scaled (PHRED-like) score of 18.80. Barttin's structure has not yet been established empirically. However, prediction models indicate the cytoplasmatic C-terminal region is unstructured. The variants found in the BSND gene including the new variant found in the present study are described in Fig. 2.

The present case represents the first description of the pathogenic variant c.784delG;p.(Ala262Profs*68). To the best of our knowledge, it is the first reported variant affecting exon 4 exclusively. Homology analysis showed that there is conservation in this region with other primates (Fig. 3), suggesting it may have a significant function. The CADD scaled score of 18.8 also indicates the likelihood of pathogenicity. It is not advisable to use hard cut-off values for CADD scores as evidence of pathogenicity. However, the developpers of the tool suggest that a value between 10 and 20 would be adequate, as a score of 10 would indicate the variant is among the 10% most deleterious, and a score of 20 represents the top 1% in this parameter (46). The CADD score for the novel deletion variant is comparable to the score of 26.7 for c.139G>A;p.Gly47Arg.

There are currently 21 BSND variants described in patients with BS type IV, including the aforementioned c.139G>A;p.Gly47Arg, SNPs, small indels and gross deletions (Table I and Fig. 2) (11,12,28-43). Of the 21 variants, three cause loss of START codon, eight are missense SNPs (three located in the N-terminal stretch, one in the first helix, four in the second helix), three affect splicing regions, three result in premature STOP codons, two are gross deletions involving at least two entire exons, and two cause frameshift which alter or truncates the major part of the cytoplasmatic C-terminal.

A total of 14 out of 18 described pathogenic variants of BSND alter the protein prior to aa72. This is not surprising since the C terminal region has little function according to the literature. Studies have shown that truncations of the protein anterior to residue 72 result in the loss of the stimulatory function over ClC channels; however, truncations beyond this residue have little to no influence in membrane insertion and activation of CLC-K channels (19,22,49,50). Some studies question this assumption, as they show the C-terminus region is important for membrane localization and normal CLC-K channel function (22,51). Frameshift variants are reported in several genetic disorders, and usually result in significant truncations (52,53) and/or loss of important motifs (54). In a study presented by Al Salmani et al (55), however, it was shown how a frameshift in the largely disordered cytoplasmatic C-tail of a potassium channel affects its function through several different mechanisms.

The present study revealed pathogenic variant c.784delG;p.(Ala262Profs^*68). This frameshift substitutes the final 58 residues of the wild-type protein for 68 different residues. The variant c.452delC (rs765135576) is similar to the deletion found in the present study, as it is also a single base deletion resulting in frameshift, affecting solely the C-terminal tail (beyond aa262). The patient in that study was homozygous for the variant but no hypothesis for the mechanism responsible for its pathogenicity was discussed (28). A naturally occurring pathogenic truncation in Glu88 was described in 2006(36), and further investigations of this variant showed that even if expression and function were maintained, proteolysis was increased and membrane distribution of ClC-K channels was affected, reducing transepithelial transport (51). This suggests some trafficking regulation function beyond Glu88. Since truncation studies rely solely on electrophysiology observations, they would not note the effects of improper epithelial sorting. This could explain the pathogeny of c.784delG;p.(Ala262Profs*68), if it is assumed that the trafficking regulation region lies beyond Ala262. Another possibility is the impact of the stretch of 68 residues produced by the frameshift that affect proper protein folding, leading to increased proteolysis. For more robust conclusions, these hypotheses must be thoroughly tested in further studies involving the expression of the mutated protein in order to evaluate expression levels, proteolysis and channel function. The lack of western blot experiments is a limitation of the present study.

The current patient appeared with a typical clinical presentation, but late diagnosis despite her clinical history. The diagnosis of BS usually occurs before early childhood, especially in antenatal BS cases (28,36,38,41,56,57). Some cases of late diagnosis of BS type IV have been reported (30-32,43). However in those cases, clinical presentation was markedly milder than in our patient, who had moderately inadequate growth and an epilepsy diagnosis at the age of 7. Interestingly, in all the other late diagnosis cases aforementioned, patients were homozygous for the c.139G>A;p.Gly47Arg variant.

This variant was shown to decrease conductance of ClC-Ka when co-expressed in Xenopus oocytes in a similar manner to other missense variants reported as pathogenic. There was no analysis of co-expression with ClC-Kb (49), which Brum et al (31) pointed out as possibly retaining some function in relation to ClC-Kb, allowing for a milder presentation. In the sole study in which the c.139G>A;p.Gly47Arg variant was found as a heterozygous compound variant with c.94C>T (a nonsense variant), the patient had a mild presentation in the perinatal period but had severe and rapid kidney deterioration in early adolescence. In that case, c.139G>A;p.Gly47Arg was compound heterozygous with c.94C>T; (p.Gln32*), which results in a significant truncation (42). Therefore, the possible function retention of c.139G>A;p.Gly47Arg may not be enough to present as a mild version of BS type IV when in heterozygosity.

In conclusion, the novel c.784delG;p.(Ala262Profs*68) variant in the cytoplasmatic C-terminal region was presented. While caution must be taken as this is a single patient study, the presence of a variant expected to elicit lesser clinical manifestations, combined with the novel variant in the C-terminal region in a patient with typical clinical presentation, suggests important functional features in this region. There is also evidence that the variant c.139G>A;p.Gly47Arg, known to lead to a milder phenotype in homozygosity, may not have the same lessening effect when in heterozygosis. The current lack of functional evidence for the novel variant is a limitation of the present study. Further studies are being performed to better understand the pathological mechanism of this novel variation.