This descriptive study included 22 patients (12 females) from 21 families with clinical diagnosis of BS.
Table 2 shows the initial clinical diagnosis of BS (Antenatal or neonatal BS, Classic BS) and the final genetic diagnosis. In addition, this table shows the characteristics of the variant found for each enrolled patient and the reference in what the variant was previously described or if this is a novel mutation.
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-/HCO3- 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.
- Bartter Syndrome type 1 - The boy was born to non-consanguineous healthy parents at 28 weeks of gestation. Severe maternal polyhydramnios had made repeated amniocenteses needed during the last weeks of gestation. He had a long hospitalization with respiratory complications. He evolved with hypokalemic hypochloremic metabolic alkalosis, severe polyuria and failure to thrive. The genetic analysis revealed a compound heterozygous mutation in SLC12A1 gene (variant 1- c.1103A>G, p.Glu368Gly; variant 2- c.905G>A, p.Arg302Gln) resulting in loss of function in the Solute Carrier Family 12 Member 1 encoding the apical furosemide-sensitive Na-K-Cl co-transporter, BS type I (OMIM #600839).
- Bartter Syndrome type 4a - A homozygous pathogenic BSND variant (c.G>A, p.Gly47Arg) was found in one female child establishing the diagnosis of BS type 4a. She was a preterm neonate born to consanguineous (first-degree cousins) healthy parents. After 6 months of age she presented polyuria, polydipsia, failure to thrive and dehydration episodes, associated with neurosensorial deafness. She also presented hypocalciuria and nephrocalcinosis was not detected in renal ultrasound.
- Bartter Syndrome type 3 - CLCNKB pathogenic variants were detected in 16 patients. Among them, the deletion of the entire gene (del 1-20) was the most frequent and was observed in homozygosis (5/16 pts) and compound heterozygosis (6/16 pts). Therefore, the frequency of this variant among all BS-related alleles (19 patients) was 42.1 % (16/38).
One premature girl with homozygous del 1-20 in CLCNKB has neurosensorial deafness and was supposed to have an additional mutation in CLCNKA gene. Associated mutations in CLCNKA and CLCNKB can cause BS with neurosensorial deafness, Type 4b BS (Table 1) [16]. However, no pathogenic variant in CLCNKA was detected by neither NGS nor MLPA. Therefore, the final genetic diagnosis of this patient was BS type 3. Sensorial deafness can be considered as a consequence of prematurity in this patient [17].
The other CLCNKB variants detected are described in Table 2.
Table 3 shows the main clinical and laboratory data of BS type 3 patients, comparing patients with homozygous or compound heterozygous del 1-20 and patients with other CLCNKB mutations. From these tables one can conclude, BS type 3 was the most frequent type of BS diagnosed in this Brazilian cohort. The deletion of the entire CLCNKB was the most frequent variant found in this group and its presence was correlated with earlier manifestations.
Table 3- Clinical and laboratory characteristics of Brazilian BS type 3 patients, comparing patients with del 1-20 and those with other CLCNKB variants.
Characteristics
|
1- CLCNKB del 1-20
n= 11
|
2- Other CLCNKB mutations
n= 5
|
1 Versus 2
|
Consanguinity
|
7/11(63,6%)
|
3/5 (60%)
|
|
Polyhydramnios
|
8/11 (72,7%)
|
2/5 (40%)
|
p= 0,22
|
Prematurity
|
5/11 (45,4 %)
|
1/5 (20%)
|
p= 0,3
|
Age at manifestation (months)
X ± DP
med(range)
|
3,4 ± 2,2
3 (0-6)
|
15,6 ± 13,6
18 (2-36)
|
p= 0,009
|
Creatinine Clearance at
presentation (Schwartz formula)
|
152,1 ± 186
(45-317)
|
129,3 ± 139,7 (64-178)
|
p= 0,6
|
Hypocalciuria
|
3/11 (27,3%)
|
3/5 (60%)
|
p=0,22
|
Nephrocalcinosis
|
2/11
|
0/5
|
N/A
|
Neurosensorial deafness
|
1/11
|
0/5
|
N/A
|
N/A not applicable; del 1-20= deletion of the entire gene CLCNKB; GS= Gitelman Syndrome; BS= Bartter Syndrome. Significant p value: p< 0.05
Based on these results, the authors suggested a pathway to investigate BS potential Brazilian patients (Figure 1). The first step is to rule out pseudo-Bartter conditions, acquired or genetic, and this requires a proper anamnesis and exams to locate the waste of sodium, potassium and chloride. In addition, as the deletion of the entire CLCNKB was the most frequent variant detected in BS patients, we suggested to begin the investigation performing MLPA to check for CNVs in CLCNKB. It is important to emphasize that the MLPA kit used to verify CNVs in CLCNKB includes the analysis of CLCNKA.
- 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.