Rare mutation in the SLC26A3 transporter causes life-long diarrhoea with metabolic alkalosis

Abstract

SLC26A3, a chloride/bicarbonate transporter mainly expressed in the intestines, plays a pivotal role in chloride absorption. We present a 23-year-old woman with a history of congenital chloride diarrhoea (CCD) and renal transplant who was admitted for rehydration and treatment of acute kidney injury after she presented with an acute diarrhoeal episode. Laboratory investigations confirmed metabolic alkalosis and severe hypochloraemia, consistent with her underlying CCD. This contrasts with most other forms of diarrhoea, which are normally associated with metabolic acidosis. Genetic testing was offered and revealed a homozygous non-sense mutation in SLC26A3 (Gly-187-Stop). This loss-of-function mutation results in bicarbonate retention in the blood and chloride loss into the intestinal lumen. Symptomatic management with daily NaCl and KCl oral syrups was supplemented with omeprazole therapy. The loss of her own kidneys is most likely due to crystal-induced nephropathy secondary to chronic volume contraction and chloride depletion. This case summarises the pathophysiology and management of CCD.

Background

Chronic diarrhoeal diseases cause significant morbidity and mortality in affected patients. They can be classified into inflammatory, malabsorptive, osmotic, secretory and motility disorders. Congenital chloride diarrhoea (CCD) is a secretory diarrhoea that was first described in 1945 as congenital alkalosis with diarrhoea.^{1 2} It is an early onset disease that presents in utero with polyhydramnios and dilated intestinal loops, and with continuous chloride-rich diarrhoea soon after birth. If untreated, it can be fatal. Family studies elucidated an autosomal recessive mode of inheritance,³ and recent molecular advances have pinned down the disease to damaging mutations in the SLC26A3 gene that codes for the chloride/bicarbonate exchanger.^{4 5} This protein is responsible for most of the chloride absorption in the gastrointestinal tract. While previously thought to be a very rare entity, CCD is prevalent in Finland (1/10 000),⁴ Poland (1/200 000)⁶ and the Arabian Peninsula (1/5500).⁷ The atypical nature and presentation of this disease may take the clinician by surprise. In this report, we review the pathophysiology of CCD in comparison with that of common diarrhoeal diseases.

Case presentation

A 23-year-old Qatari woman from the Bedouin subpopulation presented to the emergency room reporting worsening diarrhoea in the previous 5 days, and loss of appetite. She had a known history of CCD and end stage renal disease (ESRD) status postkidney transplant 9 years ago. She is on immunosuppressive medication, and is in chronic rejection.

The patient was in her usual state of health with moderate diarrhoea of four loose bowel motions (BM) per day until 5 days ago when it had doubled to eight BM/day. However, the volume per BM did not increase, and the description of the stool did not change. She described it as watery, non-bloody, non-mucoid and non-greasy. The new symptoms were associated with fatigue.

Her medical history was significant for CCD diagnosed clinically at 7 months of age in the setting of failure to thrive; she had been on daily oral KCl and NaCl syrups since then. She developed renal failure at 14 years of age for which she received a kidney transplant from an unrelated live donor 1 year later. In the previous year at the age of 22, a kidney biopsy showed chronic antibody-mediated allograft rejection. Her immunosuppressive medications included: prednisolone (5 mg once daily), tacrolimus (3.5 mg once daily) and mycophenolic acid (180 mg twice daily). It is worth noting that the patient needed admission at least once a year because of her diarrhoea and electrolyte disturbances.

As shown in the genogram (figure 1), the patient is the product of a consanguineous union where the parents are first-degree cousins. One of her brothers also suffers from CCD, and is on daily KCl and NaCl oral syrups. However, his symptoms are less severe, requiring less hospital admissions than his sister, and without any renal complications. There are no other family members with CCD, and there are no other diseases that run in the family.

Figure 1.

Congenital chloride diarrhoea pedigree. Individuals III.e and III.f are first-degree cousins, and are the parents of the patient (IV.d). Individual IV.f is her 17-year-old brother. He is the only additional family member diagnosed with congenital chloride diarrhoea, albeit a milder form than his sister (IV.d) and without renal complications. There are no other family members with congenital chloride diarrhoea and no other diseases that run in the family.

Investigations

On examination, the patient was afebrile (temperature=35.6°C), and breathing normally (respiratory rate=18 per minute). However, she was tachycardic (heart rate =134 bpm), and had orthostatic hypotension. She looked cachectic and younger than her stated age (height=141 cm, weight=37 kg). A thorough physical examination was only significant for decreased skin turgor, cold extremities and dry oral mucosal membranes. An abdominal examination was normal, with no costovertebral angle tenderness.

Laboratory investigations revealed hyponatraemia (Na⁺=122 mM), hypokalaemia (K⁺=2.9 mM), severe hypochloraemia (Cl⁻=63 mM) and metabolic alkalosis (HCO₃⁻=48 mM, pHa=7.55).

Moreover, her creatinine (Cr) climbed from a baseline of 1.9–4.8 mg/dL, indicating an acute kidney injury (AKI). The blood urea nitrogen (BUN) to Cr ratio was also elevated (BUN/Cr=25), indicating a pre-renal state. Urine studies showed low Na⁺ (UNa <10 mM) and low osmolality (Uosm=122 mOsm), consistent with extrarenal losses of Na⁺.

Interestingly, the serum tacrolimus levels were subtherapeutic, and the myocophenolate levels were within normal range, thus ruling out medication toxicity.

Stool samples were negative for ova, parasites, leucocytes, occult blood and Clostridium difficile toxins. Cytomegalovirus (CMV) titres were negative. Ultrasound of the kidney showed normal perfusion of the transplanted organ.

Differential diagnosis

When formulating the differential diagnoses, we thought her acute diarrhoea could be due to medication toxicity (tacrolimus and mycophenolate), or due to an acute diarrhoeal infection.

It is very important here to distinguish the key features of inflammatory versus non-inflammatory diarrhoeal infections. Non-inflammatory infections usually affect the small intestine, and result in voluminous diarrhoea. Inflammatory infections have a predilection for the colon, thus resulting in low volume diarrhoea, and they also cause invasion of the mucosa and bleeding.

Hence, a non-inflammatory infection was more likely. Viruses are the most common culprits and these include noroviruses, rotaviruses and adenoviruses. Bacterial pathogens such as cholera usually result in more severe forms of diarrhoea. Parasitic pathogens such as cyclospora and cryptosporidium were also considered but excluded.

We also considered inflammatory aetiologies such as CMV colitis and C. difficile infections, due to her immunosuppression and recurrent hospitalisations, respectively.

Treatment

Acute management consisted of intravenous fluids with potassium replacement, increasing the oral electrolyte syrups to help correct the electrolyte imbalance, and monitoring urine output and kidney function.

In addition, the patient was offered and accepted genetic testing that was not done before. It revealed a homozygous mutation in the SLC26A3 gene that converts a glycine at position 187 to a stop codon as shown in figure 2. This results in a truncated, non-functional Cl⁻/HCO₃⁻ transporter.

Figure 2.

Congenital chloride diarrhoea (CCD) variant of the SLC26A3. SLC26A3 is a 37.8 kb gene composed of 21 exons located on the long arm of chromosome 7. The gene encodes a 764 amino acid transmembrane protein. After translation, the mature protein is trafficked to the apical surface of the epithelial cell. It has 12 transmembrane domains, and a permease domain thus functioning as an ion transporter. There are over 55 mutations in the SLC26A3 gene reported worldwide that cause congenital chloride diarrhoea (autosomal recessive). One of them is in exon 5, resulting in Gly187Stop that accounts for more than 90% of CCD cases in the Arab population. This non-sense mutation truncates the protein at the level of transmembrane region 4. The resultant transporter is non-functional.

The long-term management goals are first to reduce the severity of the diarrhoea and its complications. There is evidence that proton pump inhibitors (PPI) reduce the severity of hypochloraemia and metabolic alkalosis via inhibition of HCl secretion from the gastric parietal cells.⁸ The patient was started on omeprazole (20 mg). Moreover, some case reports suggest that butyrate can be helpful in reducing the severity of the CCD.⁹ It is postulated that it does so via activation of the Cl⁻/butyrate transporter in the intestines, thereby promoting chloride and water absorption. However, the evidence is limited, conflicting and with no studies on the side effects of the treatment in patients with renal transplant.^9–11 Therefore, this treatment was withheld.

The second long-term goal is to conserve the renal allograft and obtain as much life from it as possible by avoiding nephrotoxic substances.

Lastly, genetic counselling was provided to the patient and her family who belong to the Bedouin subpopulation. The Bedouins are known for their high rates of consanguinity where up to 50% of their marriages are consanguineous.¹² Both parents are heterozygous for the mutation and thus have a 25% chance of conceiving an affected child. Her other siblings have a 2/3 chance of being carriers and might opt to test themselves or their potential spouses before marriage. Family members were made aware of the natural history of the disease, and the necessity of early diagnosis to sustain the life of affected infants and minimise complications.

Discussion

Most diarrhoeal diseases result in normal anion gap metabolic acidosis due to the net loss of alkaline fluid in the intestinal lumen. However, there have been a few exceptional cases of acute¹³ or chronic¹⁴ diarrhoea leading to hypokalaemic metabolic alkalosis without any stool electrolyte abnormalities. High volume output in patients with ileostomies can also precipitate metabolic alkalosis.¹⁵ CCD is one of the very few causes of diarrhoea that primarily result in metabolic alkalosis. The pathophysiology of the disease is best understood by examining events on the cellular level. As shown in figure 3, the Cl⁻/HCO₃⁻ transporter is located on the apical surface of the intestinal epithelial cell; it absorbs Cl⁻ in exchange for HCO₃⁻. Another key player is the Na⁺/H⁺ transporter that absorbs Na⁺ in exchange for H⁺. Most diarrhoeal diseases result from disruption of neutral NaCl absorption in the absorptive intestinal villus cells, or from increased electrogenic secretion of Cl⁻ from intestinal crypt cells, or both.¹⁶ Dysregulation of the Na⁺/H⁺ transporter accounts for most of the changes in neutral NaCl absorption observed in many diarrhoeal diseases.¹⁶ As a result, H⁺ is retained in the blood, leading to a normal anion gap metabolic acidosis, and Na⁺ is lost in the lumen leading to diarrhoea. The Cl⁻/HCO₃⁻ transporter is rarely involved.

Figure 3.

The chloride bicarbonate transporter SLC26A3 in an intestinal epithelial cell. The SLC26A3 transporter is located on the apical surface of intestinal epithelial cells. It exchanges Cl⁻ from the intestinal lumen with HCO₃⁻. The transporter is indirectly coupled to the sodium hydrogen exchanger (NHE3) along the gastrointestinal (GI) tract. The Na⁺/K⁺ ATP pump establishes the necessary electrochemical gradient that eases this exchange. In congenital chloride diarrhoea, the SLC26A3 protein is dysfunctional; therefore, the intestinal epithelial cell cannot absorb Cl⁻ from the GI lumen and HCO₃⁻ is retained in the blood. This leads to a watery and Cl⁻ rich diarrhoea. The end result is severe hypochloraemia, hyponatraemia, metabolic alkalosis and hypokalaemia due to contraction alkalosis and activation of the renin-angiotensin-aldosterone system.

In CCD, however, the Cl⁻/HCO₃⁻ transporter is disrupted due to deleterious genetic mutations in the SLC26A3 gene. Consequently, HCO₃⁻ is retained in the blood instead of H⁺, and Cl⁻ is lost in the lumen. The end result is chloride-rich diarrhoea, and hypochloraemic metabolic alkalosis. Potassium is lost not only in the intestines, but also in the kidneys. The latter is due to chloride depletion,¹⁷ and in part due to volume contraction and hyperactivation of the renin angiotensin aldosterone system. This high-renin, high-aldosterone state can also be seen in Bartter syndrome that presents with hypokalaemia and metabolic alkalosis.¹⁸ In fact, CCD can be misdiagnosed as Bartter¹⁹ or pseudo-Bartter syndrome.²⁰ In Bartter syndrome, however, the defect is in the transporters in the loop of Henle. Hence, Bartter patients suffer from polyuria, and chloride is lost in the urine instead of in the intestine. In contrast to CCD, Bartter patients do not have chloride-rich diarrhoea, and their urinary electrolytes panel is very different from that of patients with CCD.

CCD is diagnosed clinically, and is based on high chloride content in the stool, more than 90 mM (more than 60 mM in newborns) after fluid and electrolyte correction.²¹ However, with the advent of molecular diagnostics, the genetic and molecular basis of the disease has been elucidated. Over 55 different mutations are now known to cause CCD, all in an autosomal recessive fashion.²² The most common mutation in the Arabian Peninsula is Gly-187-Stop, and it accounts for more than 90% of cases.²³ Interestingly, the Arabian Peninsula is mostly inhabited by people of Bedouin descent who have high rates of consanguinity, a distinct genetic make-up, and are at risk for different diseases compared to other subpopulations in the Peninsula.^{24 25} The most common mutation in Finland or Sweden is V317del,^{22 23} which leads to the retention of the transporter in the endoplasmic reticulum and failure to reach the plasma membrane. In Poland, the most common mutation is I675dup^{6 14} that results in a non-functional protein.

The SLC26A3 gene is expressed not only in the villi of the duodenum, ileum and colon,^{5 26} but also in the sweat glands,²⁷ epididymis²⁸ and kidneys.²⁹ Therefore, the CCD phenotype is not limited to intestinal symptoms. It also manifests as high sweat chloride, male subfertility and chronic kidney disease.

Our patient did develop ESRD. Although the aetiology of her renal failure could not be determined, it could be a consequence of the mutation, as the SLC26A3 gene is expressed in the distal nephrons.²⁹ There have been several case reports on patients with CCD who develop renal failure; three cases were reported in Saudi Arabia,³⁰ one in Finland²⁹ and one in Poland.²⁹ Moreover, in a case series of 35 Finnish patients with CCD, 9 developed chronic kidney disease.²² A causal effect of the mutation has not been established yet. Other more likely hypotheses suggest that the renal failure is due to repeated AKI.³¹ Others suggested that the severe hypovolaemia, metabolic alkalosis and sodium chloride depletion leads to increased renal absorption of NaCl. This in turn impairs renal calcium absorption in the thick ascending loop of Henle, as Ca⁺² is absorbed passively via a paracellular route and its absorption is dependent on the presence of Cl⁻ and Na⁺ in the urine.²⁹ Consequently, this would cause calcium crystal precipitation in cortical distal nephrons even in the absence of hypercalciuria. According to this hypothesis, crystal induced epithelial injury results in renal failure.²⁹

Learning points.

• Most diarrhoeal diseases result in normal anion gap metabolic acidosis via loss of alkaline intestinal content. The Na⁺/H⁺ exchanger (NHE3) is affected in most diarrhoeal diseases, thus leading to Na⁺ loss into the intestinal lumen and H⁺ retention in the blood.

• Congenital chloride diarrhoea is a rare type of diarrhoea that is prevalent in Northern Europe and the Arabian Peninsula. It results in hypochloraemic metabolic alkalosis and acidic stool rich in chloride, >90 mM (more than 60 mM in newborns).

• Congenital chloride diarrhoea, unlike most other diarrhoeal diseases, results from dysfunction of the Cl⁻/HCO₃⁻ exchanger. It leads to Cl⁻ loss into the intestinal lumen and HCO₃⁻ retention in the blood as the main pathology. Although the SLC26A3 gene is also expressed in the distal nephrons, the renal involvement is believed to be due to crystal-induced nephropathy as a result of chronic volume and chloride depletion.

• Congenital chloride diarrhoea is a lifelong disease with daily symptoms that is treated with daily NaCl and KCl oral replacement syrups. Proton pump inhibitors help counter the electrolyte abnormalities by inhibiting HCl excretion in the stomach. Butyrate may offer some benefit to patients but the evidence is inconclusive.