A major transport function of the human intestine involves the absorption of chloride in exchange for bicarbonate. We have studied a recessively inherited defect of this exchange, congenital chloride diarrhoea (CLD; MIM 214700). The clinical presentation of CLD is a lifetime, potentially fatal diarrhoea with a high chloride content. The CLD locus was previously mapped to 7q3 adjacent to the cystic fibrosis gene (CFTR). By refined genetic and physical mapping, a cloned gene having anion transport function, Down-regulated in adenoma (DRA), was implicated as a positional and functional candidate for CLD. In this study, we report segregation of two missense mutations, delta V317 and H124L, and one frameshift mutation, 344delT, of DRA in 32 Finnish and four Polish CLD patients. The disease-causing nature of delta V317 is supported by genetic data in relation to the population history of Finland. By mRNA in situ hybridization, we demonstrate that the expression of DRA occurs preferentially in highly differentiated colonic epithelial cells, is unchanged in Finnish CLD patients with delta V317, and is low in undifferentiated (including neoplastic) cells. We conclude that DRA is an intestinal anion transport molecule that causes chloride diarrhoea when mutated.

Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma ( DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl− and[Formula: see text] was assayed. Both Cl− and[Formula: see text] were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl− uptake, consistent with Cl−/OH−exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.

Mutated alleles of the SLC26A2 (diastrophic dysplasia sulfate transporter or DTDST) gene cause each of the four recessive chondrodysplasias, i.e., diastrophic dysplasia (DTD), multiple epiphyseal dysplasia (MED), atelosteogenesis Type II (AO2), and achondrogenesis Type IB (ACG1B). SLC26A2 acts as an Na(+)-independent sulfate/chloride antiporter and belongs to the SLC26 anion transporter gene family, currently consisting of six homologous human members. Although Northern analysis has indicated some expression in all tissues studied, the only tissue known to be affected by SLC26A2 mutations is cartilage. Abundant SLC26A2 expression has previously been detected in normal human colon by in situ hybridization. We have used in situ hybridization and immunohistochemistry to examine multiple normal tissues for the expression of human SLC26A2. As expected, a strong signal for SLC26A2 mRNA and protein immunostaining were detected in developing fetal hyaline cartilage, while bronchial cartilage showed mRNA expression in adult tissues. SLC26A2 expression could also be detected in eccrine sweat glands, in bronchial glands, and in placental villi. In addition, immunoreactivity for the SLC26A2 protein was observed in exocrine pancreas. Our results suggest a more limited expression pattern for SLC26A2 than that found by Northern analysis. However, SLC26A2 expression is also detected in tissues not affected in chondrodysplasias caused by SLC26A2 mutations.

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder of intestinal electrolyte transportation caused by mutations in the anion transporter protein encoded by the down-regulated in adenoma (DRA), or CLD, gene. In this study, in situ hybridization and immunohistochemistry were performed to investigate the expression of CLD in extraintestinal normal epithelia and in intestinal inflammatory and neoplastic epithelia. The expression of the closely related anion transporter diastrophic dysplasia sulfate transporter, DTDST, was also examined and compared with that of CLD in colon. The only extraintestinal tissues showing CLD expression were eccrine sweat glands and seminal vesicles. In inflammatory bowel disease and ischemic colitis, expression of CLD mRNA in colon epithelium was similar to histologically normal colon epithelium, but the protein was found deeper in crypts, including proliferative epithelial cells. In intestinal tumors, the expression pattern of CLD was dependent on the differentiation status of the tissue studied: epithelial polyps with no or minor dysplasia showed abundant expression, whereas adenocarcinomas were negative. The DTDST gene was abundantly expressed in the upper crypt epithelium of colonic mucosa.

Congenital chloride diarrhea affects intestinal transportation of electrolytes, resulting in potentially fatal diarrhea. Linkage disequilibrium analyses have suggested the congenital chloride diarrhea gene (CLD) to lie within 0.37 cM from D7S496 in human chromosome 7q31. To clone the CLD gene, we have constructed and refined a physical map based on a 2.7-Mb YAC contig around D7S496 and identified two candidate genes. The physical positions of 4 known genes (DRA, PRKAR2B, LAMB1, DLD), 7 polymorphic repeat markers, and 13 CpG islands were established. DRA (down-regulated in adenoma) is expressed in the gut and encodes a protein with sequence homology to anion transporters, whereas PRKAR2B encodes a regulatory subunit for protein kinase A. Both genes map within 450 kb from D7S496, making them functionally and positionally relevant candidates for CLD.

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Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by defective intestinal electrolyte absorption, resulting in voluminous osmotic diarrhea with high chloride content. A variety of mutations in the solute carrier family 26, member 3 gene (SLC26A3, previously known as CLD or DRA) are responsible for the disease. Since the identification of the SLC26A3 gene and the determination of its genomic structure, altogether three founder and 17 private mutations have been characterized within miscellaneous ethnic groups. We screened for mutations in seven unrelated families with CLD. The diagnoses were confirmed by fecal chloride measurements. The combined PCR-SSCP and sequencing analyses revealed altogether seven novel mutations including two missense mutations (S206P, D468V), two splicing defects (IVS12-1G>C, IVS13-2delA), one nonsense mutation (Q436X), one insertion/deletion mutation (2104-2105delGGins29-bp), and an intragenic deletion of SLC26A3 exons 7 and 8. Two previously identified mutations were also found. This is the first report of rearrangement mutations in SLC26A3. Molecular features predisposing SLC26A3 for the two rearrangements may include repetitive elements and palindromic-like sequences. The increasingly wide diversity of SLC26A3 mutations suggests that mutations in the SLC26A3 gene may not be rare events.

An inherited defect in intestinal anion exchange, congenital chloride diarrhea (CLD), was recently shown to be caused by mutations in the down-regulated in adenoma (DRA) gene. A three base pair deletion resulting in the loss of an amino acid valine (V317del) in the predicted CLD/DRA protein was shown to be responsible for all CLD cases in a Finnish founder population. Two additional mutations, H124L and 344delT, were found in Polish CLD patients. Here, we screened for additional mutations in a set of 14 CLD families of Polish, Swedish, North American, and Finnish origin using primers that allowed mutation searches directly from genomic DNA samples. We found eight novel mutations in the CLD/DRA gene. The mutations included two transversions, one transition, one insertion, and four small deletions. Of 11 sequence alterations detected so far, nine lie clustered in three short segments that are 49 bp, 39 bp, and 65 bp in size, respectively. These short segments span only 6.7% of the total cDNA length, suggesting functional importance or mutation-prone DNA regions of the corresponding CLD/DRA protein domains.