Case reports

SEARCH FOR MUTATIONS IN THE SLC26A3 GENE

Genomic DNA from blood samples from both brothers and their parents was prepared according to standard procedures. Exon specific primers and conditions used in the polymerase chain reaction (PCR) amplification of genomic DNA have been described previously.2 ,7 PCR fragments were recovered and sequenced using an automated sequencer (ABI373A). Sequencing of the whole coding region and exon-intron boundaries of the SLC26A3 gene resulted in identification of a single novel missense mutation (fig 1). Both siblings were found to be homozygous for a T to A change at the nucleotide position 1631 in exon 15, and their parents were heterozygous carriers. The change leads to an isoleucine to asparagine change at codon 544 (I544N) in the predicted SLC26A3 transmembrane protein sequence. The codon 544 resides at a site which is highly conserved among the human, animal, and plant members of the SLC26 family (previously known as “sulphate transporter family”). These proteins share high sequence homology to each other and most act as anion transporters, but structurally they are clearly distinct from the “classical” anion exchanger family. Analysis of healthy individuals2 from Coriell Institute's DNA Polymorphism Discovery Resource (n=44) and anonymous Finnish blood donors from the Finnish Red Cross (n=30) revealed no carriers of this nucleotide change. No other sequence changes in the coding region or in the exon-intron boundaries of the SLC26A3 gene were identified in these patients, and no locus heterogeneity is known for CLD8(table 1). Thus this amino acid substitution was considered to be responsible for the functional abnormality of the Cl⁻/HCO₃ ⁻protein.5

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Figure 1

I544N mutation of the SLC26A3 exon 15 identified by sequencing in both siblings. (Top) Homozygous sequence change from patient No 1. (Bottom) Normal sequence from an unaffected control individual. The arrow shows the T to A mutation, changing an isoleucine residue at 544 to asparagine.

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Table 1

Summary of all mutations reported in the SLC26A3 gene worldwide. The three founder mutations are highlighted

Discussion

Despite the uniform genetic background of CLD in this family, the clinical picture and outcome of the disease were remarkably different in the siblings. A neonate that rapidly develops a severe potentially fatal state of dehydration, hypoelectrolytaemia, and hyperbilirubinaemia is the classical presentation of CLD. They do not pass meconium and Hirschsprung's disease may be suspected. Watery diarrhoea may go unnoticed for some time because it is easily confused with urine. An infant may lose over 10% of its weight in the first day of life.8-11 Early diagnosis is essential as hyponatraemic episodes in infancy may result in mental and psychomotor impairment.12 A family history may provide valuable information. Here, the well documented history of the younger sibling of the proband fits these features of CLD. He had a fulminant hypoelectrolytaemia and dehydration postnatally which, together with the family history, alerted the paediatricians to suspect CLD. Supplementation therapy was started during the neonatal period providing the opportunity for normal growth and development. The optimal dose of chloride varies between 6 and 8 mmol/kg/day in neonates (given as 2:1 NaCl:KCl), and smaller doses (4 mmol/kg/day) are sufficient in older patients, maintaining serum chloride levels within the normal range with chloride excretion in urine.8

Patients who remain undiagnosed in early infancy and survive, like the proband, have a chronic course of the disease with persistent hypovolaemia and hypoelectrolytaemia that leads to growth retardation. Older patients with an undiagnosed and/or untreated disease tend to present more variation in their clinical picture as dietary compensation, such as consumption of salt, varies between patients. Acute worsening of the clinical condition may follow common infections or vomiting. The proband was diagnosed as having CLD at the age of five years after a long period of chronic diarrhoea of unknown origin. Thereafter, compliance with electrolyte and fluid substitution was poor, inhibiting normal growth. In the long term, chronic contraction of the intravascular space predisposes these patients to complications such as renal impairment and gout.8 ,11 ,13 ,14 To date, four patients diagnosed late or receiving suboptimal or no substitution for long periods of time have developed end stage renal failure leading to dialysis and renal transplantation (Holmberg, unpublished).

Therapeutic manoeuvres to reduce diarrhoea have met with little success.11 Cholestyramine reduces stool volumes in the short term.15 This may reflect the ability of cholestyramine to bind bile acids which reach the colon with increased ileal effluent and stimulate an additional chloride secretory response.11 The only other situation in which diarrhoea is reduced is chronic intravascular volume contraction which, in the long term, leads to renal injury. Omeprazole, an inhibitor of the gastric proton pump, was suggested for CLD patients because of its ability to inhibit secretion of chloride by the gastric mucosa. In a patient with CLD and severe diarrhoea, treatment with omeprazole decreased the amount of stool chloride losses and stool volumes.6 In that case, optimisation of oral salt substitution would probably have the same effect because over substitution exacerbates diarrhoea by an osmotic mechanism. Because chloride absorption in CLD patients occurs passively and is determined by the intraluminal concentration of chloride, it may be expected that diarrhoea could be reduced by decreasing the intraluminal Cl⁻ concentration, either by inhibiting Cl⁻ secretion or by limiting oral salt substitution. Consequently, these patients develop Cl⁻deficiency, chronic intravascular volume contraction, and are at high risk of developing end stage renal disease. Ideally, the concentration of electrolytes in the small intestine should be only slightly higher than in plasma, enabling net absorption of both salt and water. Omeprazole therapy had no beneficial effect in either of our patients.

There is no need to over substitute patients with CLD because diarrhoea and incontinence greatly reduce their quality of life. Monitoring hydration, electrolyte balance, pH, and excretion of chloride in urine provides valuable information on the delicate electrolyte balance of these patients, and should be carried out frequently during the first years of life and at regular intervals in adults. Furthermore, families should be provided with detailed instructions for additional substitution and/or contact their paediatrician during intercurrent infectious diseases or episodes of vomiting. In growing children, electrolyte doses need to be regularly increased to meet the requirements of increased weight, but should not exceed 4 mmol chloride/kg/day in the older child and adult. Non-compliant and under substituted patients should have glomerular filtration rate measured at regular intervals.

We conclude that the time of diagnosis, optimal substitution therapy, compliance, and compensatory mechanisms, such as dietary habits and activation of the renin-aldosterone system, are more important modulators of the clinical manifestations of the disease than the specific type of mutation in the SLC26A3 gene. Screening of faecal chloride concentration should be performed in all patients, especially neonates with a family history of congenital chloride diarrhoea or an undefined intestinal absorption defect with a clinical picture including polyhydramnios, prematurity, and chronic watery diarrhoea. Early diagnosis and maintenance of optimal treatment will increase the likelihood of these children growing and developing normally, and avoiding long term complications in adulthood.