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Significance of Molecular Testing for Congenital Chloride Diarrhea

Lechner, Silvia*; Ruemmele, Frank M; Zankl, Andreas; Lausch, Ekkehart§; Huber, Wolf-Dietrich||; Mihatsch, Walter; Phillips, Alan D#; Lewindon, Peter**; Querfeld, Uwe††; Heinz-Erian, Peter‡‡; Müller, Thomas‡‡; Janecke, Andreas R‡‡

Journal of Pediatric Gastroenterology and Nutrition: July 2011 - Volume 53 - Issue 1 - p 48–54
doi: 10.1097/MPG.0b013e31820bc856
Original Articles: Gastroenterology

Objectives: Autosomal recessive, congenital chloride diarrhea (CLD) is a form of persistent secretory diarrhea, presenting with polyhydramnios and intractable diarrhea from birth. CLD is caused by mutations in the SLC26A3 gene, encoding a Na+-independent Cl/HCO3- exchanger. The diagnosis is generally made on the basis of high fecal chloride concentration in patients with serum electrolyte homoeostasis corrected by salt substitution. We aimed to evaluate the role of diagnostic genetic testing in CLD.

Patients and Methods: Clinical and laboratory data were collected from 8 unrelated children diagnosed as having or suspected to have CLD. The evaluation included physical examination, routine clinical chemistry, and SLC26A3 mutation analysis by direct sequencing of DNA extracted from buccal swabs or peripheral leukocytes.

Results: CLD was initially diagnosed on high fecal chloride concentrations in 7 patients, and by mutation analysis in 1 patient. In 3 of these patients the correct diagnosis was made more than 6 months after birth. We identified SLC26A3 mutations on both alleles in all 8 patients with CLD, including 3 novel missense and 4 novel truncating mutations. We present a compilation of reported SLC26A3 mutations and polymorphisms.

Conclusions: The diagnosis and therapy of CLD were considerably delayed in 3 of 8 patients from this series, highlighting the potential of misdiagnosing CLD. We add 7 novel mutations, including 3 missense changes of highly conserved residues to a total of 41 mutations in this gene.

Molecular analysis is efficient and should be considered as a means of early diagnosis of CLD, especially if the clinical diagnosis remains uncertain.

*Division of Human Genetics, Innsbruck Medical University, Innsbruck, Austria

Université Paris Descartes, Faculté Necker, INSERM U989, Paris, France

Genetic Health Queensland, Royal Brisbane and Women's Hospital, Brisbane, Australia

§Centre for Pediatric and Adolescent Medicine, Freiburg University Hospital, Freiburg, Germany

||Department of Pediatrics, Medical University of Vienna, Vienna, Austria

Deaconry Hospital, Schwaebisch Hall, Germany

#Centre for Paediatric Gastroenterology, UCL Medical School, Royal Free Campus, London, UK

**Royal Children's Hospital, Department of Gastroenterology, Herston, Australia

††Department of Pediatric Nephrology, Charité, Berlin, Germany

‡‡Department of Pediatrics II, Innsbruck Medical University, Innsbruck, Austria.

Received 7 September, 2010

Accepted 15 December, 2010

Address correspondence and reprint requests to Andreas R. Janecke, MD, Department of Pediatrics II, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria (e-mail:

The present study was funded in part by Tiroler Medizinischer Forschungsfond (grant no. 198 to ARJ).

The authors report no conflicts of interest.

Congenital chloride diarrhea (CLD; OMIM 214700) is an autosomal recessive disorder with an estimated incidence of 1:35.000 in Finland and up to 1:3200 in Arabian countries in the Persian Gulf region, and is otherwise rare (1). CLD is a persistent secretory diarrhea presenting with polyhydramnios caused by intrauterine diarrhea, and with mild prematurity. Postnatally, CLD is characterized by prominent abdominal distention and dehydration due to voluminous watery stools containing an excess of chloride. Before treatment, hyponatremia, hypokalemia, hypochloremia, and metabolic alkalosis are present owing to an inability of the gut epithelium to reabsorb chloride and secrete bicarbonate. Hyponatremia in CLD is a temporary change before the renal activation of sodium reabsorption and potassium excretion, leading to an increase in both hypokalemia and metabolic alkalosis in untreated CLD (1–3). The diagnosis of CLD is generally based on a high stool chloride content (>90 mmol/L) exceeding the sum of fecal (4) sodium and potassium, measured after correction of the electrolyte imbalance by salt substitution.

Mutations in the SLC26A3 (solute carrier family 26, member 3) gene cause CLD, and were first reported in 1996 (5). Today, clinical genetic testing is provided by a few laboratories including the Finnish group (1) and ours (4) within 3 working days, and has previously confirmed a diagnosis of CLD in the vast majority of published patients with CLD, implying a high mutation detection rate. To date, 34 SLC26A3 mutations, including the founding mutations of Finland, Poland, and Arabic countries have been associated with CLD (5–12). Despite the various types of mutations and their wide distribution in different regions of the SLC26A3 gene, evidence of genotype-phenotype correlation has not been reported. The gene encodes for a Cl/HCO3- exchanger mainly expressed in the brush border of the duodenal, ileal, and colonic epithelia (13,14). Extraintestinal expression has been found in sweat glands, the male reproductive tract, and kidney (15,16), and it is downregulated in human colon adenomas and adenocarcinomas (17). SLC26A3 is a protein predicted to contain 10 to 14 transmembrane-spanning helices (13) and a C-terminal STAS (sulfate transporters and anti-sigma factor) domain homologous to the bacterial anti-sigma factor antagonists (18,19). The STAS domain is required for SLC26A3 Cl/HCO3- exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3 (18,20).

Early and persistent oral supplementation of chloride, sodium, and potassium using NaCl and KCl solutions has proven effective in patients with CLD to prevent acute and chronic dehydration and to prevent or delay renal complications. Optimal dosage of Cl for normal growth and development has been determined for infants and older patients (21), although a high intake of chloride may initially exacerbate diarrhea. All of the patients diagnosed as having CLD and treated adequately reach adult life and their long-term prognosis is favorable (1,22).

Because CLD is rare, the diagnostics are challenging. Although clinical suspicion arises, fecal chloride may not exceed the proposed cutoff of 90 mmol/L because of either stool dilution by urine or insufficient rehydration and salt replacement at the time of sampling (23). In the present study, we show the efficiency of genetic testing by identifying SLC26A3 gene mutations on all of the disease alleles in a series of 8 patients with CLD.

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Eight unrelated patients with congenital, watery diarrhea were included in the present study (Table 1). CLD was suspected in 6 of these patients based on the presence of 1 or several additional clinical findings: polyhydramnios, lack of meconium, hypochloremia, hyponatremia, hypokalemia, metabolic alkalosis, high fecal chloride concentrations, and low fecal pH. In 1 female patient rectal biopsy led to a diagnosis of microvillus inclusion disease (MVID); negative molecular testing for this disorder prompted genetic testing for CLD. Another neonate received treatment for Bartter syndrome; an unremitting course and progressive failure to thrive prompted fecal electrolyte determination and molecular testing for CLD at 2 years of age. Written informed consent for molecular genetic analysis was obtained from the patients' parents.



Genomic DNA was prepared from leukocytes or buccal cells using a robot (GenoM 48, Qiagen, Vienna, Austria). The complete coding region of the SLC26A3 gene and all exon-intron boundaries were amplified using intronic primers based on the National Center for Biotechnology Information mRNA (NM_000111.1) and genomic (NC_000007.12) references (sequences available from the authors on request), and GoTaq polymerase (Promega, Mannheim, Germany). Thirty-five cycles of 20 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C were applied to all of the sets of primers plus 8 minutes of final extension. The 190- to 504-bp amplicons were cleaned using ExoSap-IT (USB, Vienna, Austria), and subsequently sequenced using the M13 universal primer on an ABI 3130 automated sequencer, with BigDye terminator mix (Applera, Vienna, Austria). Patient chromatographs were analyzed using the Sequence Pilot computer program (JSI Medical Systems, Kippenheim, Germany).

Algorithms, which use phylogenetic and/or structural information, PolyPhen (polymorphism phenotyping,, and SIFT (sorting intolerant from tolerant,, were used to evaluate the effect of reported and novel amino acid substitutions on the function of SLC26A3 (NCBI accession no. NP_000102.1) using program default parameters.

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The patient characteristics and the molecular results are summarized in Table 1. All of the patients presented with the typical clinical features of CLD, including persistent diarrhea. Direct sequencing of all 21 SLC26A3 exons and corresponding splice sites identified mutations on both alleles in each patient. We encountered 7 novel mutations, including 1 nonsense (p.R579X) and 3 missense (p.A216 V, p.G379A, p.H714R) mutations, 2 splicing defects (c.1312–1G>T, c.1514+1G>C), and 1 truncating insertion/deletion (p.A148VfsX24). Four novel mutations encode premature stop codons predicting either protein truncation or, rather, nonsense-mediated mRNA decay.

When applied to SLC26A3, the widely used PolyPhen algorithm predicted all 13 missense variants identified in patients to probably damage protein function, whereas 8 of 9 nonsynonymous polymorphisms were predicted to be benign (6) or possibly damaging (2) (Table 2).



Table 2

Table 2

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The pronounced genetic heterogeneity of our results reflects the ethnic diversity in our patient series; this is in line with previous reports that mutations are distributed throughout the SLC26A3 gene (4–12). Three novel missense mutations are considered pathogenic because they affect highly conserved amino acid residues in SLC26A3 (Fig. 1A), and were not present in a panel of samples from 300 control individuals. The pathogenicity of p.H714R is further emphasized by its occurrence in compound heterozygosity with a nonsense mutation in that patient. Histidine-714 is predicted to localize within a short alpha-helix of the highly conserved C-terminal domain of the SLC26A proteins (24). This so-called sulfate transporters and anti-sigma-factor antagonists (STAS) domain is considered important both for ion transport activity and protein–protein interactions such as an intracellular binding with CFTR (20). Mutations p.A216 V and p.G379A localize to 2 of 12 predicted transmembrane domains supposed to participate in anion binding and pore formation (19). Whether these mutations impair protein folding, cause protein instability and degradation, or abrogate the Cl/HCO3- exchange at the plasma membrane remains to be determined.



A total of 41 missense, deletion/insertion, and nonsense mutations are now known to impair SLC26A3 function, as shown by a compilation of reported SLC26A3 mutations (Table 2). An in silico analysis of missense changes to assess their effect on protein function based on phylogenetic and/or structural conservation represents a complementary approach to mutation segregation analyses in affected families, the determination of population allele frequencies, and functional studies. The good agreement between conventional criteria and PolyPhen scores supports the current classification of reported sequence variants as missense mutations or polymorphisms. Some lack of agreement is observed with SIFT predictions, in line with other studies (25): 2 of 12 patient missense changes were scored as benign (“tolerated”), including p.G379A and p.H714R, and 2 of 9 polymorphic changes emerged as “not tolerated” for protein function (Table 2). Both algorithms do not consider posttranslational modifications, protein interactions, or DNA binding or splicing motives. Robust comparison of the results from the prediction model used with in vivo and in vitro studies (13,20,24,26–28) on several SLC26A3 mutants is hampered by the rarity of such data and by the fact that the actual protein structure is not known. However, the human missense mutations p.I544N and L496R were shown to abolish protein function both by in vitro and in silico analyses (24,26,27). Our predicted model appears to serve as a valuable tool to differentiate between disease-associated mutations and polymorphisms found in healthy controls.

The identification of SLC26A3 mutations in patients with CLD demonstrates that it is a Cl/HCO3- exchanger playing an important role in Cl absorption and HCO3- secretion in the colon, and possibly in the small intestine (5).

The frequent Arab and Polish founder mutations (p.G187X and p.I675dup) were present in 3 of our patients from Libya, Morocco, and Poland. The knowledge of the causal founder mutations in different populations allows for restricting SLC26A3 mutation analysis on affected exons in patients from such populations (4,9). Although a whole coding sequence analysis as described here can be accomplished within 1 week for any small series of samples in a diagnostic setting, targeted sequencing for founder mutations to confirm or exclude a diagnosis of CLD with high probability can be even faster and more cost-efficient. The amount of DNA needed for complete SLC26A3 sequencing can be extracted from a partial blood spot (Guthrie card) or a buccal swab. Clinical molecular testing enables rapid confirmation or high-probability exclusion of CLD while being less burdensome for the patient as compared to stool sampling or taking a small intestinal biopsy.

Watery stools in CLD can be mistaken for urine, and juxtaglomerular hyperplasia, hyperreninemia, and hyperaldosteronism, leading to hyperkaluria and hypokalemia, may mimic Bartter syndrome. This mistaken diagnosis is particularly likely at the time of acute presentation: after restoration of volume and electrolyte balance, urinary electrolyte levels can be appropriately high (when in balance, net intake and output are equal) but mistakenly interpreted as inappropriate, and volume loss from the urinary tract versus the gastrointestinal tract may not be immediately discriminated in infants if no simultaneous, separate 24-hour collections of urine and stool are performed under balanced fluid and electrolyte conditions. Bartter syndrome refers to a group of disorders that are unified by autosomal recessive transmission of impaired salt reabsorption in the thick ascending loop of Henle with pronounced salt wasting, hypokalemic metabolic alkalosis, and hypercalciuria. Clinical disease results from defective renal reabsorption of sodium chloride (29). A misdiagnosis of Bartter syndrome was made in 1 of our patients (Table 1, ID 09D0297), and has been reported in the literature (12).

Other forms of congenital secretory diarrhea may be considered initially in patients with CLD, and include MVID, which is caused by mutation in the MYO5B gene (30); nonsyndromic congenital sodium diarrhea (CSD) (31); a syndromic form of CSD, caused by mutation in the SPINT2 gene (32); malabsorptive congenital diarrhea, caused by mutation in the NEUROG3 gene (33); and congenital tufting enteropathy, caused by mutation in the TACSTD1 gene (3,34).

Electron microscopy (EM) of a rectal biopsy in a neonate of mixed European descent was performed in lieu of a small bowel biopsy because the infant was considered too unstable to undergo anaesthesia, and interpreted locally as demonstrating MVID. The child died shortly thereafter (09D0917, Table 1); however, subsequent MYO5B gene analysis did not reveal a mutation, and a second opinion of the original EM pictures did not confirm the initial interpretation. Although fecal electrolyte analysis had not been performed, mutation analysis for other types of secretory diarrhea was initiated revealing compound heterozygosity for 2 novel SLC26A3 missense mutations. A third patient with CLD (09D1512) underwent ileostomy at 2 weeks of age because her severely dilated intestinal loops and watery stools were thought to result from bowel obstruction, and the diagnosis of CLD was delayed until 6 months of age.

The outcome in the clinical courses of these 3 late diagnosed patients emphasizes the importance of both 24-hour fluid- and electrolyte-balance studies, including separate measurements of electrolyte excretion in urine and feces and/or molecular confirmation in patients with secretory diarrhea. Our results in addition to the previous studies demonstrate that early diagnosis is essential to avert life-threatening complications in patients with CLD and CSD who can lead an almost normal life under relatively simple treatment regimens (1,4,22,23,32,35–37).

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chloride bicarbonate exchanger; intestinal transporter; misdiagnosis; sequence analysis; SLC26A3

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