Najafi et al. Orphanet Journal of Rare Diseases (2019) 14:41 https://doi.org/10.1186/s13023-018-0981-5 RESEARCH Open Access Mimicry and well known genetic friends: molecular diagnosis in an Iranian cohort of suspected Bartter syndrome and proposition of an algorithm for clinical differential diagnosis Maryam Najafi1,2, Dor Mohammad Kordi-Tamandani2*, Farkhondeh Behjati3, Simin Sadeghi-Bojd4, Zeineb Bakey1,8, Ehsan Ghayoor Karimiani5,6, Isabel Schüle8, Anoush Azarfar7 and Miriam Schmidts1,8,9* Abstract Background: Bartter Syndrome is a rare, genetically heterogeneous, mainly autosomal recessively inherited condition characterized by hypochloremic hypokalemic metabolic alkalosis. Mutations in several genes encoding for ion channels localizing to the renal tubules including SLC12A1, KCNJ1, BSND, CLCNKA, CLCNKB, MAGED2 and CASR have been identified as underlying molecular cause. No genetically defined cases have been described in the Iranian population to date. Like for other rare genetic disorders, implementation of Next Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnostics and counseling over the last years. In this study, we describe the clinical, biochemical and genetic characteristics of patients from 15 Iranian families with a clinical diagnosis of Bartter Syndrome. Results: Age range of patients included in this study was 3 months to 6 years and all patients showed hypokalemic metabolic alkalosis. 3 patients additionally displayed hypercalciuria, with evidence of nephrocalcinosis in one case. Screening by Whole Exome Sequencing (WES) and long range PCR revealed that 12/17 patients (70%) had a deletion of the entire CLCNKB gene that was previously identified as the most common cause of Bartter Syndrome in other populations. 4/17 individuals (approximately 25% of cases) were found to suffer in fact from pseudo-Bartter syndrome resulting from congenital chloride diarrhea due to a novel homozygous mutation in the SLC26A3 gene, Pendred syndrome due to a known homozygous mutation in SLC26A4, Cystic Fibrosis (CF) due to a novel mutation in CFTR and apparent mineralocorticoid excess syndrome due to a novel homozygous loss of function mutation in HSD11B2 gene. 1 case (5%) remained unsolved. (Continued on next page) * Correspondence: [email protected]; [email protected]; [email protected] 2Departement of Biology, University of Sistan and Baluchestan, Zahedan, Iran 1Genome Research Division, Human Genetics department, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525KL Nijmegen, The Netherlands Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Najafi et al. Orphanet Journal of Rare Diseases (2019) 14:41 Page 2 of 11 (Continued from previous page) Conclusions: Our findings demonstrate deletion of CLCNKB is the most common cause of Bartter syndrome in Iranian patients and we show that age of onset of clinical symptoms as well as clinical features amongst those patients are variable. Further, using WES we were able to prove that nearly 1/4 patients in fact suffered from Pseudo-Bartter Syndrome, reversing the initial clinical diagnosis with important impact on the subsequent treatment and clinical follow up pathway. Finally, we propose an algorithm for clinical differential diagnosis of Bartter Syndrome. Keywords: Bartter syndrome, Whole exome sequencing, Pseudo-Bartter-syndrome Background milder phenotype and is usually diagnosed during late Bartter Syndrome (BS), firstly reported by Bartter and adulthood. And third, Gitelman syndrome which com- his colleagues in 1962, is a very rare autosomal recessive pared to other variants is marked with hypocalciuria and salt-losing tubulopathy characterized by hypokalemic hypomagnesaemia and is usually diagnosed during late metabolic alkalosis with normotensive hyperreninemia childhood and adulthood. In most cases, BS IVa and IVb and hyperaldosteronism [1] occurring with an estimated subtypes are accompanied by sensorineural deafness incidence of 1.2/million in the population [2]. Based on [12]. However, the BS subtype can often not be deter- the loss of function mutations in the salt reabsorption mined clinically with certainty due to similar presenta- transporters and channels in the thick ascending limb of tion of different forms, rendering diagnostics and precise the loop of Henle, genetically, five variants of this prognosis complex [13]. Despite the need for rigorous syndrome have been described: type I resulting from loss classification of BS phenotypes, currently few practical of function mutations in the Solute Carrier Family 12 indicators exist. Member 1 SLC12A1 gene encoding the apical Making the landscape of BS and BS-like clinical pre- furosemide-sensitive Na-K-Cl co-transporter (OMIM sentations appear even more complex, several renal and #600839), type II caused by mutations in the potassium extra-renal disorders as congenital chloride diarrhea, voltage-gated channel subfamily J member 1 (KCNJ1) Pendred syndrome, Cystic fibrosis as well as some ac- gene encoding the apical renal outer medullary potas- quired conditions associated (for example laxative abuse) sium channel (ROMK) (OMIM # 600359), type III may present clinically in a similar way as BS with caused by mutations in the chloride voltage-gated chan- regards to hypokalemic metabolic alkalosis. However, nel Kb (CLCNKB) gene encoding the basolateral chloride there is only a limited number of reports in the channel Kb (OMIM #602023), type IVa resulting from literature investigating misdiagnosis of BS where in fact dysfunction of the Barttin CLCNK type accessory beta other rare hypokalemic disorders (CF) were causative for subunit (BSND) gene encoding Barttin, a subunit of the phenotype [14–22]. Nevertheless, such clinical mis- chloride-channels Ka and Kb (OMIM #606412) and fi- diagnosis can result in serious health problems due to nally, type IVb caused by co-mutation in the CLCNKA wrong treatment choices [23, 24]. However, novel high and CLCNKB genes (OMIM #602024) [3–7]. Gitelman throughput sequencing technologies nowadays offer an syndrome which shares several clinical characteristics additional diagnostic tool refining clinical diagnostics. with BS type III has been described later in history. In In the present study, we describe 17 patients from 15 contrast to BS type III, Gitelman syndrome is caused by Iranian families with a clinical diagnosis of BS. Imple- mutations in a single gene, SLC12A3 (OMIM #263800), menting WES as additional diagnostic step combined encoding the thiazide-sensitive sodium chloride with long-range PCR screening for CLCNKB, we identi- co-transporter (NCCT) in the distal convoluted tubule [8, fied the underlying genetic cause in 16/17 cases. While 9]. More recently, 2 genes, namely calcium sensing recep- we confirmed the clinical diagnosis of BS in 12 cases, tor (CASR) (OMIM #601198) and MAGE family member our genetic analysis established a diagnosis different D2 (MAGED2) (OMIM #300971, BS type V) have been from BS in 4 cases. Additionally, we propose a identified which cause autosomal dominant and X-linked cost-efficient clinical differential diagnostic algorithm. recessive forms of BS [10, 11]. Currently, from a phenotypical perspective, BS has Results been classified into three different forms according to Clinical and genetics findings are summarized in Table 1. the average age at onset of symptoms: antenatal BS, the Laboratory results of all 17 patients showed severe hypo- most severe form of BS, marked by polyhydramnios, hy- kalemic alkalosis. Comparison of normal ranges of urin- percalciuria, nephrocalcinosis, hypochloremia and failure ary calcium/creatinine ratio for age (hypercalciuria to thrive in infancy; Second, classic BS which has a screening) indicated that case 3 showed hypocalciuria Najafi et al. Orphanet Journal of Rare Diseases Table 1 Clinical characteristics and description of the genetic findings in the cohort Code sex weight Hight Age Cr(mg/dL) Na mEq/L K mEq/L Ca mg/dL PH HCO3 mEq/L UrineCa/ Defective variant Mode of Final diagnosis (nl 0.6–1.0) (nl 135–155) (nl 3.5–5.3) (nl 8.6–10.2) (nl 7.35–7.45) (nl 22–26) Cr > 0.2 gene detection 1 F 4.5 60 0.5 0.5 126 2.4 NA 7.61 31 0.9 CLCNKB c.(?_-1)_(*1_?) del, p.0 WES Bartter syndrome 2 F 3.7 65 0.5 1 124 2.2 10 7.57 48 3.5 CLCNKB c.(?_-1)_(*1_?) del, p.0 PCR Bartter syndrome (2019)14:41 3 F 5.1 64 0.5 0.6 125 2.7 NA 7.58 37 0.06 CLCNKB c.(?_-1)_(*1_?) del, p.0 PCR Bartter syndrome 4 M 4.7 60 0.6 0.5 144 2.1 NA 7.66 35 0.1 CLCNKB c.(?_-1)_(*1_?) del, p.0 WES Bartter syndrome 5 M 10 96 6 0.7 144 2 8 7.56 32 0.5 ? – WES – 6 M 9 83 3 0.5 131 2.8 10 7.47 31 0.8 CLCNKB c.(?_-1)_(*1_?) del, p.0 PCR Bartter syndrome 7 F 8.35 85 2.5 0.6 123 2.5 8 7.51 32 4.25 CLCNKB c.(?_-1)_(*1_?) del, p.0 WES Bartter syndrome 8 F 7.4 76 2.5 0.5 125 2.1 7 7.65 39 4.75 CLCNKB c.(?_-1)_(*1_?)del, p.0 PCR Bartter syndrome 9 M 3.2 63 0.5 0.5 134 3.4 NA 7.47 32 NA CLCNKB c.(?_-1)_(*1_?)del, p.0 PCR Bartter syndrome 10 F 4.6 60 0.9 0.5 138 3.2 10.7 7.52 27 3.6 CLCNKB c.(?_-1)_(*1_?) del, p.0 PCR Bartter syndrome 11 M 5.7 75 0.4 0.5 134 3 NA 7.47 27 NA CLCNKB c.(?_-1)_(*1_?)del, p.0 PCR Bartter syndrome 12 F 7.25 60 3 1 135 2.4 NA 7.53 31 0.88 CLCNKB c.(?_-1)_(*1_?) del, p.0 WES Bartter syndrome 13 M 5.2 67 1.3 0.4 126 2.5 8.2 7.56 32 0.54 CLCNKB c.(?_-1)_(*1_?) del, p.0 WES Bartter syndrome 14 M 7 73 2.3 0.6 132 2.1 7 7.58 34 0.55 CFTR c.473G > A, p.