Congenital Chloride-losing Diarrhea Research Articles

“Recurrent Papillary Necrosis and Nephrocalcinosis Induced by Nonsteroidal Anti-Inflammatory Drugs for Gouty Arthritis Associated with Congenital Chloride-Losing Diarrhea: A Major Risk for Kidney Loss”

Congenital chloride-losing diarrhea (CCLD) is a rare genetic disorder due to autosomal recessive mutation in the SLC26A3 gene on chromosome 7. It is characterized with chronic watery diarrhea with high fecal chloride (Cl: >90 mmol/L), low potassium (K), and metabolic alkalosis with low urinary Cl and K. The overall long-term prognosis is favorable with optimal life-long salt and K supplementation. In this case report, we describe a man with progressive renal failure and small kidneys that showed nephrocalcinosis and papillary necrosis. His disease was diagnosed since birth and was confirmed by our tests. He was incompliant with therapy and had developed gout. The latter complication of his disease has led to excessive NSAID use over the past years. Reinstitution of diet, drug therapy, and allopurinol had stabilized his renal disease for 1 year of follow-up. In conclusion, excessive analgesic use is a risk factor for renal failure in CCLD.

Segmental differences in Slc26a3-dependent Cl\u2212 absorption and HCO3\u2212 secretion in the mouse large intestine in vitro in Ussing chambers

The anion exchanger slc26a3 (DRA), which is mutated in congenital chloride-losing diarrhea, is expressed in the apical membrane of the cecum and middle-distal colon but not in the proximal colon of rodent large intestines. To elucidate the functional roles of DRA, we measured unidirectional 36Cl− and 22Na+ fluxes and HCO3− secretion in vitro in each of these segments using DRA-KO mice. Robust Cl− absorption, which was largely abolished after DRA deficiency, was present in the cecum and middle-distal colon but absent in the proximal colon. Na+ absorption was present in all three segments in both the control and DRA-KO mice. The luminal-Cl−-dependent HCO3− secretions in the cecum and middle-distal colon were abolished in the DRA-KO mice. In conclusion, DRA mediates Cl− absorption and HCO3− secretion in the mouse cecum and middle-distal colon, and may have roles in H2O absorption and luminal acid/base regulation in these segments.

Congenital chloride losing diarrhea: A single center experience in a highly consanguineous population.

Congenital chloride losing diarrhea (CCLD) is a rare type of chronic watery diarrhea due to mutations in SLC26A3 gene leading to defective chloride–bicarbonate exchanges with the resultant loss of chloride and retention of bicarbonate.We aim to define pediatric Saudi CCLD patients’ characteristics to achieve prompt diagnosis, management, follow up with good quality of life, and prevention of complications in these patients.We carried retrospective data review of demographic, clinical, laboratory, radiographic, and outcome of all pediatric patients fulfilling the criteria of CCLD over 10 years from 2004 to 2014 from a single center in Taif region, Saudi Arabia.Forty-nine patients fulfilled the criteria of CCLD from 21 families with more than one affected patient in the same family in 90% of them and positive consanguinity in 91% of the cohort. Most patients were born preterm with intrauterine growth restriction and usually neonatal intensive care unit (NICU) admissions with prematurity and its complications. Thirteen patients were discharged without diagnosis of CCLD and 3 were misdiagnosed as intestinal obstruction with unnecessary surgical intervention. Many complications do existed with renal complications being the most common with three patients received renal transplantation.Prematurity with abdominal distension and stool like urine were the commonest presentation of CCLD in Saudi children. Positive consanguinity and more than one affected sibling are present in most of our cohort.High index of suspicion by clinicians is a cornerstone for early diagnosis with subsequent favorable outcome.A multicenter national incidence study of CCLD in KSA and its genetic attributes is recommended. Premarital screening should be implemented specially for consanguineous marriage.

Slc26a3 deficiency is associated with epididymis dysplasia and impaired sperm fertilization potential in the mouse.

Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.

Molecular analysis of human solute carrier SLC26 anion transporter disease-causing mutations using 3-dimensional homology modeling

The availability of the first crystal structure of a bacterial member (SLC26Dg) of the solute carrier SLC26 family of anion transporters has allowed us to create 3-dimensional models of all 10 human members (SLC26A1-A11, A10 being a pseudogene) of these membrane proteins using the Phyre2 bioinformatic tool. The homology modeling predicted that the SLC26 human proteins, like the SLC26Dg template, all consist of 14 transmembrane segments (TM) arranged in a 7+7 inverted topology with the amino-termini of two half-helices (TM3 and 10) facing each other in the centre of the protein to create the anion-binding site, linked to a C-terminal cytosolic sulfate transporter anti-sigma factor antagonist (STAS) domain. A plethora of human diseases are associated with mutations in the genes encoding human SLC26 transporters, including chondrodysplasias with varying severity in SLC26A2 (~50 mutations, 27 point mutations), congenital chloride-losing diarrhea in SLC26A3 (~70 mutations, 31 point mutations) and Pendred Syndrome or deafness autosomal recessive type 4 in SLC26A4 (~500 mutations, 203 point mutations). We have localized all of these point mutations in the 3-dimensional structures of the respective SLC26A2, A3 and A4 proteins and systematically analyzed their effect on protein structure. While most disease-causing mutations may cause folding defects resulting in impaired trafficking of these membrane glycoproteins from the endoplasmic reticulum to the cell surface – as demonstrated in a number of functional expression studies – the modeling also revealed that a number of pathogenic mutations are localized to the anion-binding site, which may directly affect transport function.

Novel SLC26A3 mutation in an infant with congenital chloride-losing diarrhea initially misdiagnosed as Bartter's syndrome

Novel SLC26A3 mutation in an infant with congenital chloride-losing diarrhea initially misdiagnosed as Bartter's syndrome

Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W.

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl−/HCO−3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.

Congenital Chloride Losing Diarrhea

Background: Congenital chloride-losing diarrhea is a medical emergency that become a mostly pediatric problem in many countries including Saudi Arabia. It is requiring early diagnostics and treatment to prevent severe dehydration and infant mortality. Aim of the review: To summarize data on congenital chloride diarrhea including: incidence, pathophysiology and management. Methods: Data are based on MEDLINE search for chloride losing diarrhea in addition to clinical experience in treatment of these cases. Results: Life-long salt substitution with NaCl and KCl stabilizes fluid, electrolyte and acid-base balance. When early diagnosed and properly treated, the long-term outcome is favorable. Conclusions: This review summarizes data on congenital chloride diarrhea and provides guide lines of treatment.

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

Compound Heterozygous Mutations in the SLC26A3 Gene in 2 Spanish Siblings With Congenital Chloride Diarrhea

Compound Heterozygous Mutations in the <i>SLC26A3</i> Gene in 2 Spanish Siblings With Congenital Chloride Diarrhea

Great expectations: using massively parallel sequencing to solve inherited disorders

The mutation discovery process for monogenic disorders is undergoing an exciting revolution. Previously dependent on linkage mapping and carefully selected gene-candidate approaches, identifying disease-causing mutations was both time consuming and frustrating. The advent of the new and emerging massively parallel sequencing technologies is driving unbiased discovery of novel disease-causing mutations at an unprecedented rate. How long will it be before the process is fully automated and the job of molecular geneticist is changed forever? We comment on the mutation-discovery strategies compatible with this technology and demonstrate that while the modern gene jockey might need to trade in the horse for something a bit faster, the finish line for interpreting human genetic variation is still a long way ahead. A recent comprehensive and highly recommended review covers the technical capabilities of massively parallel (also called next-generation) sequencing for the field of genetics [1]. Mutation-discovery applications using massively parallel sequencing survey whole-genome, wholeexome, regionor candidate-gene groupspecific DNA templates. Each strategy has strengths and weaknesses that must be considered and matched with the genetics and biology of the clinical phenotype to be studied. Whole-exome sequencing is already the method of choice for most mutation discovery efforts. Commercially available chip-based and in-solution array enrichment platforms make this protocol amenable to high-throughput automation and standardization. There are companies that offer this protocol as a fee for service, including downstream sequence analysis [101]. Currently, 86% of the 100,000 mutations cataloged in the human genome mutation database affect coding regions [2], which are captured by the whole-exome arrays. Even considering the biases, which have driven the discovery of these mutations so far, it is predicted that exome sequencing will be successful most of the time. Exome sequencing is already having an impact on patient care; in one study, mutations in SLC26A3 that are normally associated with congenital chloride-losing diarrhea (Online Mendelian Inheritance in Man [OMIM] 214700) were identified in a patient with a diagnosis of an atypical Bartter syndrome (OMIM 607364) [3]. These findings led to the re-examination of the patient to confirm the diagnosis of chloride-losing diarrhea and the subsequent identification of five additional mutations in SLC26A3 in a cohort of 39 individuals diagnosed with Bartter syndrome. In a similar study, 11 novel mutations in DHODH associated with the recessive disorder Miller syndrome (OMIM 263750) Mark Corbett

A Practical Approach to Genetic Hypokalemia

Mutations in genes encoding ion channels, transporters, exchangers, and pumps in human tissues have been increasingly reported to cause hypokalemia. Assessment of history and blood pressure as well as the K+ excretion rate and blood acid-base status can help differentiate between acquired and inherited causes of hypokalemia. Familial periodic paralysis, Andersen's syndrome, congenital chloride-losing diarrhea, and cystic fibrosis are genetic causes of hypokalemia with low urine K+ excretion. With respect to a high rate of K+ excretion associated with faster Na+ disorders (mineralocorticoid excess states), glucoricoid-remediable aldosteronism and congenital adrenal hyperplasia due to either 11β-hydroxylase and 17α-hydroxylase deficiencies in the adrenal gland, and Liddle's syndrome and apparent mineralocorticoid excess in the kidney form the genetic causes. Among slow Cl- disorders (normal blood pressure, low extracellular fluid volume), Bartter's and Gitelman's syndrome are most common with hypochloremic metabolic alkalosis. Renal tubular acidosis caused by mutations in the basolateral Na+/HCO3- cotransporter (NBC1) in the proximal tubules, apical H+-ATPase pump, and basolateral Cl-/HCO3- exchanger (anion exchanger 1, AE1) in the distal tubules and carbonic anhydroase II in both are genetic causes with hyperchloremic metabolic acidosis. Further work on genetic causes of hypokalemia will not only provide a much better understanding of the underlying mechanisms, but also set the stage for development of novel therapies in the future.

Congenital Chloride-losing Diarrhea Causing Mutations in the STAS Domain Result in Misfolding and Mistrafficking of SLC26A3

Congenital chloride-losing diarrhea (CLD) is a genetic disorder causing watery stool and dehydration. Mutations in SLC26A3 (solute carrier 26 family member 3), which functions as a coupled Cl(-)/HCO(3)(-) exchanger, cause CLD. SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning alpha-helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain homologous to the bacterial anti-sigma-factor antagonists. The STAS domain is required for SLC26A3 Cl(-)/HCO(3)(-) exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3. Here we investigate the molecular mechanism(s) by which four CLD-causing mutations (DeltaY526/7, I544N, I675/6ins, and G702Tins) in the STAS domain lead to disease. In a heterologous mammalian expression system biochemical, immunohistochemical, and ion transport experiments suggest that the four CLD mutations cause SLC26A3 transporter misfolding and/or mistrafficking. Expression studies with the isolated STAS domain suggest that the I675/6ins and G702Tins mutations disrupt the STAS domain directly, whereas limited proteolysis experiments suggest that the DeltaY526/7 and I544N mutations affect a later step in the folding and/or trafficking pathway. The data suggest that these CLD-causing mutations cause disease by at least two distinct molecular mechanisms, both ultimately leading to loss of functional protein at the plasma membrane.

Slc26a3 (dra)-deficient Mice Display Chloride-losing Diarrhea, Enhanced Colonic Proliferation, and Distinct Up-regulation of Ion Transporters in the Colon

Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.

Genetic diseases of acid-base transporters.

Genetic disorders of acid-base transporters involve plasmalemmal and organellar transporters of H(+), HCO3(-), and Cl(-). Autosomal-dominant and -recessive forms of distal renal tubular acidosis (dRTA) are caused by mutations in ion transporters of the acid-secreting Type A intercalated cell of the renal collecting duct. These include the AE1 Cl(-)/HCO3(-) exchanger of the basolateral membrane and at least two subunits of the apical membrane vacuolar (v)H(+)-ATPase, the V1 subunit B1 (associated with deafness) and the V0 subunit a4. Recessive proximal RTA with ocular disease arises from mutations in the electrogenic Na(+)-bicarbonate cotransporter NBC1 of the proximal tubular cell basolateral membrane. Recessive mixed proximal-distal RTA accompanied by osteopetrosis and mental retardation is associated with mutations in cytoplasmic carbonic anhydrase II. The metabolic alkalosis of congenital chloride-losing diarrhea is caused by mutations in the DRA Cl(-)/HCO3(-) exchanger of the ileocolonic apical membrane. Recessive osteopetrosis is caused by deficient osteoclast acid secretion across the ruffled border lacunar membrane, the result of mutations in the vH(+)-ATPase V0 subunit or in the CLC-7 Cl(-) channel. X-linked nephrolithiasis and engineered deficiencies in some other CLC Cl(-) channels are thought to represent defects of organellar acidification. Study of acid-base transport disease-associated mutations should enhance our understanding of protein structure-function relationships and their impact on the physiology of cell, tissue, and organism.

A normal gastrointestinal motility excludes chronic intestinal pseudoobstruction in children.

Gastrointestinal manometry has gained wide acceptance in the approach to patients with suspected enteric neuromuscular disorders. However, performing gastrointestinal manometry in these subjects without a previous exhaustive diagnostic evaluation is unjustified. Twelve children (median age: 7.0 years; range: 8 months-13 years), with clinical and x-ray features suggesting chronic intestinal pseudoobstruction, were referred to our unit for gastrointestinal manometry. The latter was performed with a perfused catheter for 5 hr in the fasting state and for 90 min after feeding. Data were compared with those recorded in eight age-matched controls. In all patients and controls, interdigestive motor complexes with propagated phases III were detected; a regular postprandial antroduodenal motor activity was also recorded. Patients and controls did not differ for fed antral and duodenal motility indexes, fed antroduodenal coordination, and length of duodenal phase III. Most of the patients showed short or prolonged bursts of nonpropagated activity in the fasting and/or fed states; in four cases fasting and/or fed sustained phasic activity was recorded. Manometric evidence of migrating motor complexes and postfeeding activity did not support the diagnosis of intestinal pseudoobstruction and suggested redirecting the diagnostic evaluation. Final diagnoses were: Munchausen syndrome-by-proxy (four cases), celiac disease (two cases), intestinal malrotation (two cases), Crohn's disease (two cases), multiple food intolerance (one case), and congenital chloride-losing diarrhea (one case). It is concluded that in children with suspected chronic intestinal pseudoobstruction manometric evidence of migrating motor complexes and fed motor activity excludes an enteric neuromuscular disorder and suggests a reassessment of the diagnostic work-up. Furthermore, if gastrointestinal manometry shows migrating motor complexes and postfeeding motor activity, qualitative abnormalities of the manometric tracings do not indicate an underlying enteric neuromuscular disorder and must not be overemphasized. Patients referred for gastrointestinal manometry should previously undergo an extensive diagnostic investigation to exclude disorders mimicking chronic intestinal pseudoobstruction.

Congenital Chloride-Losing Diarrhea in Saudi Children

Four Saudi patients with congenital chloride-losing diarrhea (CCD) are described. The disease was characterized by polyhydramnios, chronic diarrhea, abdominal distention, growth failure, and delay ...