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

Congenital chloride-losing diarrhea (CLD) is a rare genetic disorder causing watery stool and dehydration (1,2). CLD can be detected by ultrasonography in utero. After birth, the patients show voluminous watery acidic stools with high chloride content causing hypochloremia, hyponatremia, hypokalemia, metabolic alkalosis, and dehydration (3). Other clinical findings are hyperbilirubinemia, failure to thrive, and abdominal distension. The disease is caused by a defect of ileal and colonic apical Cl−/HCO3− exchange, normally providing absorption of Cl− and secretion of HCO3−. Patients with CLD also show sodium loss because the exchange of Cl−/HCO3− is coupled with that of Na+/H+. The loss of electrolytes is osmotically followed by water (4). CLD diagnosis can be verified by analyzing fecal electrolytes, which always show Cl− concentrations >90 mmol/L (3). CLD is caused by mutations in the SLC26A3 gene (Online Mendelian Inheritance in Man: 126650 and 214700; GenBank: AC002467). This gene, mapped at 7q31, was initially identified as a downregulated gene in the human colon adenocarcinoma and was called DRA (5). It has been demonstrated that this gene encodes a protein acting as Cl−/HCO3− exchanger with a 2:1 stoichiometry. SLC26A3 has a fundamental role in Cl− absorption and HCO3− secretion in the colon. More than 30 mutations have been identified in patients with CLD from various countries (6,7). Here we describe 2 novel mutations in 2 siblings with CLD from Andalusia (southern Spain). The absence of other relatives with CLD and parental nonconsanguinity led us to investigate the mutations causing CLD in this family by direct sequencing of the SLC26A3 gene in the child and parents. Case 1 (Girl) Case 1 was the first child of healthy nonconsanguineous parents with no family medical history related to CLD symptoms. The girl was born after a spontaneous delivery at 36 weeks, and had a birth weight of 2740 g (p6) and a height of 48 cm (p26). She showed massive watery stools without mucus or blood since birth. Watery diarrhea, present also in interfeeding periods, was a constant complaint of the parents. A period of lactose and cow's-milk protein exclusion did not change the clinical situation of the patient. A trial with a gluten-free diet was done for 1 year, without any difference in outcome. We started our study when the patient was 3 years 5 months old. Metabolic alkalosis was detected and a fecal sample was analyzed to determine Cl− content. The first determination showed Cl− 83.6 mmol/L, K+ 21.86 mmol/L, and Na+ 28.8 mmol/L. With a high suspicion of CLD, a second test with an improved quality sample was conducted, showing Cl− 160 mmol/L, K+ 41 mmol/L, and Na+ 100 mmol/L, from this moment (3 years and 7 months old) an oral supply treatment with NaCl/KCl improved her clinical condition but catch-up was poor. The suspicion of CLD was confirmed by direct sequencing of the SLC26A3 gene when the patient was nearly 4 years old. A slight delay in language acquisition and march development was appreciated, but she joined elementary school without difficulties. At present, she is 8 years old, her weight is 12 kg (z score −2.61), and her height is 90 cm (z score −3.81). Case 2 (Boy) Case 2 was a boy born after spontaneous delivery at 35 weeks, weighing 2660 g. At the 28th week of gestation, CLD was confirmed in his sister, and obstetricians were warned about this fact. The presence of massively dilated prenatal bowel loops and polyhydramnios were confirmed by prenatal ultrasound tests. The newborn showed visible peristalsis through abdominal skin surface, distended abdomen, and voluminous watery stools (Fig. 1A). Intravenous saline solution was applied. An oral supply treatment with NaCl/KCl (2 mmol · kg−1 · day−1 and 1 mmol · kg−1 · day−1, respectively) was also applied at the first week of life. The growth chart of the boy has been significantly better shaped than his sister's (Fig. 1B). In spite of more serious physical findings at birth, his development has been better and virtually free of inward treatment needs. No delay in language or motor acquisition has been appreciated in spite of a tight follow-up. Sequencing of the SLC26A3 was carried out when the patient was 6 months old and CLD was confirmed.FIGURE 1: (A) Intestinal loops visible through the surface of the abdomen of case 2 (boy). (B) Percentile standard curves (P3, P5, P10, P25, P50, P75, P90, P95, and P97) for weight of the 2 siblings. Weight gain in case 2 (boy) is clearly superior to that in case 1 (girl).METHODS Written informed consent for molecular genetic analysis was obtained from the parents. Genomic DNA was extracted from peripheral venous blood samples using the Ilustra blood genomicPrep Mini Spin Kit (GE Healthcare, Waukesha, WI). After extraction, DNA content was determined by spectrophotometry. The primers and polymerase chain reaction (PCR) conditions used to amplify the whole coding sequence and the intron-exon junction of the SLC26A3 gene were those described previously (8). PCR products were purified by using the Agencourt AMPure (Beckman Coulter Genomics, Danvers, MA) PCR purification system. After purification, PCR products were sequenced using the same primer used for PCR amplification on an ABI3130 DNA sequencer with BigDye terminator mix (Applied Biosystems, Carlsbad, CA). The chromatographs obtained were compared with the genomic reference sequence (Ensembl Genome Browser code ENSG00000091138) using the DNASTAR Lasergene (Madison, WI) software. After mutation identification in affected siblings, parents were investigated as carriers of these mutations by amplifying only exons containing both mutations (VI and X). Multiple sequence alignment was carried out with CLUSTALX (Dublin, Ireland) (9). Protein transmembrane helices were predicted with PHOBIUS (EMBL-EBI, Cambridge, UK) (10). RESULTS Sequence analysis of the SLC26A3 gene in this family (Fig. 2A) revealed that both parents are heterozygous for different mutations. The father is a healthy carrier with a deletion at 1148–1149 of a TA dinucleotide giving a frameshift; the resulting truncated protein product was 455 amino acids long (Fig. 2B). The mother is also a healthy carrier with the missense mutation 659A>C (Fig. 2A); the resulting protein has a H220P change (Fig. 2B). The siblings are compound heterozygous carrying both mutations (Fig. 2A).FIGURE 2: Mutations in the family under study. (A) Sequencing of exons 6 and 10 in the SLC26A3 gene of the parents and 2 siblings under study showing a heterozygous deletion (TA1148–1149, exon 6) in the father (panel 1), a heterozygous missense mutation (659A>C, exon 10) in the mother (panel 2), and the same heterozygous mutations in combination in both siblings (compound heterozygosity, panels 3 and 4). (B) Aligned nucleotide and amino acid sequences of the 2 mutated regions shown in (2A). Mutations shown in bold letters.DISCUSSION We show in this report the presence of compound heterozygous mutations as the cause of CLD in this family. The 2 mutations described here are new and this is the first time that Spanish patients with CLD have been molecularly characterized. Mutations in the SLC26A3 gene cause CLD (5). This gene codes for a protein of 764 amino acids that has the typical structure of anion exchangers. Structurally, SLC26A3 protein can be divided into 4 segments (11). First a cytoplasmic N-terminal segment is followed by 12 hydrophobic transmembrane spanning α-helices (Fig. 3A). This transmembrane segment is followed by a cytoplasmic sulfate transporter and antisigma-factor domain homologous to the bacterial antisigma-factor antagonists (STAS), and, finally, the protein ends with a cytoplasmic C-terminal. The transmembrane segment is predicted to contain a SLC26A transporter family signature and a sulfate transporter family domain that overlaps with a xanthine-uracil-vitamin C permease superfamily domain (Fig. 3A) (7). The STAS domain is required for SLC26A3 Cl−/HCO3− exchange and for the activation of cystic fibrosis transmembrane conductance regulator. The STAS domain has been related to membrane targeting and endoplasmic reticulum (ER) transport to the plasma membrane (11). The cytoplasmatic C-terminal shows a PDZ domain necessary for binding to scaffolding proteins E3KARP and CAP70 (12). SLC26A3 spans 39 kb of genomic DNA arranged into 21 exons. More than 30 nucleotide mutations have been described (6,7). In our patients, the mutations described are new.FIGURE 3: Effects of the mutations on the structure and function of the SLC26A3 protein. (A) The transmembrane helices predicted using the program PHOBIUS (10). Black bars indicate the 12 transmembrane segments. The fifth segment is magnified with the sequence containing the H220P mutation and the predicted secondary structure. C = coil, S = strand, H = helix. The asterisk indicates the TA deletion. At the bottom the predicted functional domains are shown (11). (B) Alignment of the amino acid sequence coded for by the nucleotide region in exon 6, which is mutated in the 2 children with CLD and their healthy father. Amino acid H220 (highlighted) is conserved in all vertebrates shown. CLD = congenital chloride-losing diarrhea.The mother carries a missense mutation that changes a histidine at position 220 for proline. This mutation is located in a well-conserved position. To show this, we aligned SLC26A3 homologues from a variety of vertebrates (Fig. 3B) and in all of them this position shows a histidine; this suggests that it is a key position that may help to maintain the structure or function of the protein. The mutation that we have identified here is an H to P change, the substitution score for membrane proteins for this change is −4 (13), the worst value for any histidine substitution. This means that it is an evolutionary disfavorable change, probably disturbing the structure or function drastically. Proline is unable to occupy many of the main chain conformations adopted by other amino acids and can often be found in tight turns of the amino acid chain (13). Position 220 in the SLC26A3 protein may play an important role because it is located in the middle of the fifth predicted transmembrane α-helix and at the beginning of the predicted transport/permease domains (Fig. 3A). The father carries a TA deletion that produces a frameshift in the reading frame (Fig. 2B). The resulting protein is 455 amino acids long. This mutant protein is 40% shorter than the wild-type and the last 73 amino acids are different. The mutation affects the last transmembrane α-helices and eliminates the STAS-like and PDZ domains (Fig. 3A). It has been demonstrated that the STAS-like domain plays a role in the traffic from the ER to the plasma membrane (11). This mutant protein is probably retained in the ER and does not reach the plasma membrane. It has also been proposed that mRNAs of truncated proteins are not expressed due to nonsense-mediated decay (14). Any of these mechanisms leads to the absence of the SLC26A3 protein in the membrane of intestinal epithelial cells. The children have both mutations; the mother's mutation probably leads to a nonfunctional protein and the father's mutation does not allow the synthesis of the SLC26A3 protein, or if expressed, is retained in the ER. Both types of mutation, but by different ways, lead to a loss of Cl− transport. We think that the boy received an early benefit of salts supplementation because the growing rate was clearly better than his sister's at the same age (Fig. 1B). The parents received instructions to increase the amount of salt supplementation (a 30% plus) during the summer because temperatures >37°C to 42°C can be reached in Seville and significant increases in sweating were expected. Despite both siblings having the same SLC26A3 mutations, the clinical outcome has not been equivalent for both, reflecting the influence of circumstances other than SLC26A3 mutations. The boy has been virtually free of hospital admissions for intravenous treatment due to diarrhea-related dehydration, but the girl needed some. After incidental episodes of acute gastroenteritis the boy presented complete recovery in a few days generally without any significant effect on weight. The girl usually has shown significant weight losses after acute episodes of gastroenteritis, diagnosed in the framework of epidemic gastroenteritis. The idea of the influence of environment over genetic background may be an explanation in these paired cases. Despite both children sharing the same mutation, the modified environment (early salt supplementation) pointed to a different growth pattern (Fig. 1B) and response to viral-induced diarrhea. We propose that an early diagnosis could lead to a better outcome for children with CLD. Acknowledgments We express our gratitude to Dr Pia Höglund, Department of Medical Genetics, Haartman Institute, Helsinki, who helped us to confirm the first case, and to the described family: their faithful cooperation with our medical team allowed the final diagnosis and treatment.