Genetic spectrum of Brazilian suspected Bartter Syndrome Patients

This paper’s goal is to show the genetic study results of suspected Bartter syndrome (BS) patients followed in a pediatric nephrology reference center in Sao Paulo/Brazil, verify a possible genotype-phenotype correlation and compare the genetic results with those from other regions of the globe. This descriptive study included 22 patients (21 families) with clinical diagnosis of BS. Pathogenic variants in BS-related genes were detected in 19/22 patients. No BS-related genes were detected in three patients (one case of Congenital Chloride Diarrhea and two siblings with clinical Antenal BS that, in fact, had Gitelman Syndrome). We observed that 16/19 BS-conrmed patients had CLCNKB mutations (BS type 3) with a large phenotypical diversity. Among them, the deletion of the entire gene (del 1–20) was the most frequent variant detected. Interestingly, we observed that patients with homozygous or heterozygous del 1–20 presented earlier manifestations than patients with other CLCNKB mutations. They presented no other clinical signicant difference.


Introduction
Bartter syndrome (BS) encompasses a group of genetic tubular renal diseases characterized by hypokalemia, hypochloremia, hyponatremia, metabolic alkalosis, increased blood levels of renin and aldosterone in patients with normal to low blood pressure [1]. Clinically, one can identify 2 types of presentation: Antenatal BS and Classic BS.
Antenatal BS is considered the most severe form, characterized by polyhydramnios, premature birth, lifethreatening episodes of neonatal salt and water loss, failure to thrive, hypercalciuria, and early-onset nephrocalcinosis [2]. Classic BS is supposed to occur in infancy or early childhood and is characterized by remarkable waste of salt and potassium clinically manifested as polyuria, polydipsia, volume contraction, failure to thrive, muscle weakness, growth retardation and, sometimes, nephrocalcinosis [3].
However, before the establishment of BS clinical hypothesis, acquired or genetic pseudo-Bartter conditions, renal or extrarenal, should be ruled out [4][5][6][7]. Among them Gitelman Syndrome (GS; OMIN 263800) is an important differential diagnosis. GS is classically described as a milder BS-like renal tubular disease frequently associated with hypomagnesemia and hypocalciuria that often manifests in teenagers and young adults with cramps, muscle weakness, salt craving, paresthesia, and tetany [8].
It is important to mention the Autosomal Dominant Hypocalcemia (OMIN 601198) that can cause Bartter syndrome-like features. It is a consequence of gain-of-function mutation in CASR which encodes the calcium receptor sensor (CaSR) located in the thick ascending Henle's loop leading to secondary disturbances in NKCC2 and ROMK channels and in Na-K-ATPase pump [9]. This disease was named BS type 5 in the past.
The genetic classi cation of BS is presented in Table 1 and is based on the mutations in the genes that code speci c transporters in renal tubular membranes. Table 1 Genetic classi cation of Bartter Syndrome, specifying the affected genes, the molecule that is involved and its location in the renal tubules.

Subtypes of BS Affected gene / Location
Molecule These mutations lead to dysfunctional proteins determining the clinical and biochemical abnormalities.
Currently, the treatment is just symptomatic, based on potassium, chloride and sodium supplementation, and cyclooxygenase (COX) inhibitors, either nonselective (ex.: indomethacin) or COX2 selective (ex.: celecoxib). [10][11]. A potassium sparing drug such as spironolactone can be associated. These patients can also be treated with renin-angiotensin-aldosterone system (RAAS) blockers replacing the COX inhibitors. However, these drugs can be associated with signi cant and symptomatic hypotension [11].
Even though these treatments can promote complete or partial electrolyte and metabolic recovery leading to growth improvement, they are associated with signi cant side-effects [10]. In addition, the patient would always be prone to metabolic and electrolyte severe imbalance during common pediatric diseases.
Di culties to clinically differentiate the types of BS and GS patients have been recognized and a new name has already been proposed for these diseases, Inherited Salt-Losing Tubulopathies. This group has been classi ed based on genetic ndings [12]. Therefore, the genetic study in this group of patients is necessary to clarify the true pathway of the disease, and serves as a base to implement speci c treatments [13]. In this approach, it is very important to be alert for population differences.
This paper's goal is to show the genetic study results of clinically suspected BS patients followed in a pediatric nephrology reference center in Sao Paulo/Brazil, verify a possible genotype-phenotype correlation and compare the genetic results with those from other regions of the globe.

Patients And Methods
This study included Brazilian patients up to 21 years-old, both genders, with suggestive clinical and laboratory ndings of BS followed at Pediatric Nephrology Unit of the Instituto da Criança, University of Sao Paulo. This protocol was approved by the Local Ethical Review Board. The patients were enrolled after they and/or their parents (or guardians) have signed the informed consent form.
Clinical and laboratory data was collected from the patient's records by the same doctors that had been following the patients. Clinical data included gestational age at birth, familial and consanguinity history, age at presentation, presence or not of polyhydramnios, polyuria, polydipsia, episodes of fever, vomiting, seizures, cramps, neurosensorial deafness and failure to thrive. The main laboratory data studied were venous gas analysis, serum potassium, chloride, sodium, ionic calcium, phosphate and magnesium; the urinary exams included excretion fraction of potassium, chloride and sodium as well as morning urinary calcium/creatinine ratio, microalbuminuria and urinary protein/creatinine ratio. Renal ultrasound was also evaluated.

DNA analysis
The blood was collected, genomic DNA was extracted and the sample kept at -20 Celsius degrees until analysis.  Pathogenic variants in BS-related genes were detected in 19 of the 22 study patients.
No BS-related genes were detected in three patients and these cases are described below.
Patients with no BS-related genes mutations observed in the current study -Patients with Pseudo-Bartter conditions. Case 1 -We detected one case of Congenital Chloride Diarrhea (CCD) (OMIM #214700).
This patient presented in the neonatal period with polyhydramnios and prematurity, polyuria and BS typical electrolyte and metabolic disturbances. Although she was receiving potassium and sodium supplementation, she evolved with failure to thrive. During one episode of acute diarrhea, CCD was suspected, but the diagnosis was not confirmed by fecal and urinary electrolytes measurements. The records were reviewed and showed that the collections were inappropriate, fecal sample was collected during dehydration status resulting in a false low fecal chloride. Likewise, a urinary sample was collected soon after physiologic solution infusion and potassium reposition, resulting in a high urinary chloride content. These findings led to misdiagnosis BS. During follow up, liquid stools were probably misinterpreted as polyuria. Unfortunately, no other urinary or fecal chloride measurement was performed to confirm the diagnosis of BS during follow up.
As patient presented no mutations in the genes panel applied in this study, whole exome sequencing (WES) was performed and detected a homozygous mutation (c.1487 T>G; p.Leu496Arg) in the solute carrier family 26 (SLC26A3) on chromosome 7q31, encoding for a transmembrane Cl /HCO 3 exchanger mainly expressed in the apical brush border of ileum and colonic epithelium [14]. This variant was confirmed by Sanger sequencing. This mutation is considered likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines [15] and was reported at least once related to CCD in a family from Hong Kong [14]. This case has already been reported [6].
Cases 21 and 22-Two young male siblings from non-consanguineous parents presented a homozygous SLC12A3 pathogenic variant, p.Thr648Met, diagnosing GS. They were preterm neonates and after 1 month of age they evolved with vomiting, polyuria, polydipsia, failure to thrive and seizures. Hypocalciuria was observed in both patients and hypomagnesemia was observed in one. Therefore, they presented a completely different clinical syndrome than which is described as classical GS. This finding shows the importance of the genetic evaluation in such confusing diagnosis, especially when faced with a potential specific therapy in next future.
Pathogenic variants in the BS related genes detected in the current study.
Mutations in BS-related genes were detected in 19/22 potential BS Brazilian patients.
All of them are classified as pathogenic variants according to the ACMG [15]. Based on those mutations we were able to genetically classify these patients.  Sensorial deafness can be considered as a consequence of prematurity in this patient [17].
The other CLCNKB variants detected are described in Table 2.  -Bartter Syndrome type 4b -Applying the pathway proposed in the Figure 1 and firstly performing MLPA to verify the presence of CNVs in CLCNKB, we were able to detect a homozygous deletion of the entire CLCNKB and CLCNKA in one girl with suspected BS establishing the diagnosis of BS type 4b. Neurosensorial deafness was later diagnosed in this patient. This BS type 4b is a consequence of a digenic defect in the closely adjacent genes encoding ClC-Ka (CLCNKA) and ClC-Kb (CLCNKB) on chromosome 1p36. [16].
No patient was diagnosed with BS type 2 (ROMK mutations), type 5 or BS-like due to gain-of-function mutation in CaSR.
Based on this data, it was almost impossible to differentiate clinically suspect BS patients due to the clinical overlap.
Comparison of the variants found in this Brazilian cohort with those identified in some other countries.
In the current study, we also evaluated some of the main genetic reports of suspected Bartter Syndrome patients around the globe. The goal is to compare the Brazilian findings with other countries' findings. Table 4 shows the different genetic findings in different regions of the globe and, when is available, the ancestry or ethnic origin is presented.

Discussion
Bartter syndrome is composed by a group of rare salt-losing tubulopathies with an estimated incidence of 1 Seyberth proposed a new classi cation of BS and GS based on the location of the involved channels or transporters, including the subunit Barttin. He named it "Inherited salt-loss tubulopathies" [12].
Therefore, a rational way for investigation is important if we want to achieve the true diagnosis in clinically BS suspected patients. It is noteworthy the importance of the early investigation of pseudo-Bartter conditions in which the treatment would be completely different. Therefore, it is important to measure the urinary sodium, potassium and chloride and to calculate the excretion fraction of these electrolytes and con rm the urinary losses in all suspected BS patients and choose the correct timing to collect the urine sample [6].
After ruling out pseudo-Bartter conditions, one should verify the presence of neurosensorial deafness, a speci c feature that can lead to the diagnosis of BS type 4a or BS type 4b. In this study, we detected neurosensorial deafness in 3 patients. One of them with nal diagnosis of BS type 4a (homozygous BSND mutation), one with nal diagnosis of BS type 4b (performing just MLPA to investigate CLCNKB and CLCNKA), and other patient was genetically diagnosed with BS type 3 (homozygous deletion in CLCNKB and no mutation detected in CLCNKA by NGS or MLPA). In the last patient, deafness could just be related to the prematurity which has been reported in literature [6].
We detected one patient with clinical antenatal BS with genetic BS type 1. Polyhydramnios developed during gestation, he was an extreme preterm baby with remarkable metabolic alkalosis, hypokalemia, hypercalciuria and early nephrocalcinosis. However, clinical antenatal BS was also observed in patients with CLCNKB mutations, especially in cases of homozygous del 1-20.
We observed that most of the cases had CLCNKB mutations (BS type 3) with a large phenotypical diversity. Among these patients, del 1-20 was the most frequent genetic abnormality detected. Interestingly, we observed that patients with del 1-20 presented earlier manifestations than patients with other CLCNKB mutations. No other signi cant difference was detected in the clinical parameters evaluated in this study. However, some authors have already demonstrated that the hypokalemia is more di cult to be corrected in BS type 3 than BS type 1 or type 2 [37][38][39].
Surprisingly, GS was diagnosed in two siblings with a complete different manifestation that is commonly observed in GS patients as they presented a typical clinical picture of Antenatal BS. The typical clinical history of GS is a milder presentation in adolescents or young adults with cramps. Therefore, this nding denotes the importance of the genetic evaluation in such confusing diagnosis, especially if a speci c therapy is to be developed and also emphasizes the phenotypical variation of these patients especially in those with early manifestations. Therefore, we suggest even in young children in which del 1-20 in CLCNKB has been ruled out, the genetic investigation of GS should be performed.
No patient was diagnosed with BS type 2 caused by mutations in the potassium voltage-gated channel subfamily J member 1 (KCNJ1) gene encoding the apical renal outer medullary potassium channel (ROMK) (OMIM # 600359).
Based on these ndings, we suggested a pathway to genetically study Brazilian patients and it is shown in Fig. 1. According to this pathway, performing MLPA for CLCNKB we were able to diagnose a new patient with BS type 4b, as she presented del 1-20 in CLCNKB and a deletion in CLCNKA, using the kit for MLPA to evaluate CLCNKB we could also evaluate the CLCNKA.
Therefore, one can conclude that genetic analysis is the only way to get the correct diagnosis in suspected BS patients.
In this study, the main conclusion is the group of diseases encompassing clinical and laboratory ndings of BS are clinically confusing and the genetic analysis is essential to established the correct diagnosis. Therefore, the clinical classi cation of BS seems to be ine cient and should be replaced by the genetic classi cation. This conclusion has also been achieved by other authors [12]. In addition, based on genetic abnormality a speci c therapy for each defect can be develop in next future [13]. As one knows, population differences have been observed among patients with the same clinical diagnosis [21].

Conclusion
The del 1-20 in CLCNKB was the most frequent variant detected in this group of patients. Evaluating the genetic studies results from different parts of the world we can suppose the genetic background of this Brazilian cohort is more related to African and Portuguese origin and this fact can do one concluded that the genetic BS originated in the early period of immigration (colonization and slavery period).
The study has some limitations as the low number of patients from a single center. However, as a rare disease one must consider that certain points can be concluded even in a small sample population.
Evidence-based medicine seems to be not appropriate in the evaluation of rare diseases and the scientists are looking for more appropriate ways to interpret the results in these conditions. Meanwhile, all data of these patients are important to be reported as it can collaborate for a better understanding of these conditions. Declarations Acknowlegements Not applicable.
Author´s contributions MHV drafted the study design, the manuscript and made substantial contribution to conception and interpretation of the data, revised and supervised critically the work, ACHLM recruited the patients and contributed general patient data, ACSR and FAMF performed the genetic tests and analysis, JCOAF wrote and revised critically the manuscript. The author(s) read and approved the nal manuscript.

Funding
This research received no speci c grant.

Availability of data and materials
Data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate
The study was approved by the Local Ethical Review Board, São Paulo, Brazil (reference number 3.261.257).

Consent for publication
The parents/guardians gave a written consent to publication of their anonymized clinical data.