The pronounced genetic heterogeneity of our results reflects the ethnic diversity in our patient series; this is in line with previous reports that mutations are distributed throughout the SLC26A3 gene (4–12). Three novel missense mutations are considered pathogenic because they affect highly conserved amino acid residues in SLC26A3 (Fig. 1A), and were not present in a panel of samples from 300 control individuals. The pathogenicity of p.H714R is further emphasized by its occurrence in compound heterozygosity with a nonsense mutation in that patient. Histidine-714 is predicted to localize within a short alpha-helix of the highly conserved C-terminal domain of the SLC26A proteins (24). This so-called sulfate transporters and anti-sigma-factor antagonists (STAS) domain is considered important both for ion transport activity and protein–protein interactions such as an intracellular binding with CFTR (20). Mutations p.A216 V and p.G379A localize to 2 of 12 predicted transmembrane domains supposed to participate in anion binding and pore formation (19). Whether these mutations impair protein folding, cause protein instability and degradation, or abrogate the Cl−/HCO3- exchange at the plasma membrane remains to be determined.
A total of 41 missense, deletion/insertion, and nonsense mutations are now known to impair SLC26A3 function, as shown by a compilation of reported SLC26A3 mutations (Table 2). An in silico analysis of missense changes to assess their effect on protein function based on phylogenetic and/or structural conservation represents a complementary approach to mutation segregation analyses in affected families, the determination of population allele frequencies, and functional studies. The good agreement between conventional criteria and PolyPhen scores supports the current classification of reported sequence variants as missense mutations or polymorphisms. Some lack of agreement is observed with SIFT predictions, in line with other studies (25): 2 of 12 patient missense changes were scored as benign (“tolerated”), including p.G379A and p.H714R, and 2 of 9 polymorphic changes emerged as “not tolerated” for protein function (Table 2). Both algorithms do not consider posttranslational modifications, protein interactions, or DNA binding or splicing motives. Robust comparison of the results from the prediction model used with in vivo and in vitro studies (13,20,24,26–28) on several SLC26A3 mutants is hampered by the rarity of such data and by the fact that the actual protein structure is not known. However, the human missense mutations p.I544N and L496R were shown to abolish protein function both by in vitro and in silico analyses (24,26,27). Our predicted model appears to serve as a valuable tool to differentiate between disease-associated mutations and polymorphisms found in healthy controls.
The frequent Arab and Polish founder mutations (p.G187X and p.I675dup) were present in 3 of our patients from Libya, Morocco, and Poland. The knowledge of the causal founder mutations in different populations allows for restricting SLC26A3 mutation analysis on affected exons in patients from such populations (4,9). Although a whole coding sequence analysis as described here can be accomplished within 1 week for any small series of samples in a diagnostic setting, targeted sequencing for founder mutations to confirm or exclude a diagnosis of CLD with high probability can be even faster and more cost-efficient. The amount of DNA needed for complete SLC26A3 sequencing can be extracted from a partial blood spot (Guthrie card) or a buccal swab. Clinical molecular testing enables rapid confirmation or high-probability exclusion of CLD while being less burdensome for the patient as compared to stool sampling or taking a small intestinal biopsy.
Watery stools in CLD can be mistaken for urine, and juxtaglomerular hyperplasia, hyperreninemia, and hyperaldosteronism, leading to hyperkaluria and hypokalemia, may mimic Bartter syndrome. This mistaken diagnosis is particularly likely at the time of acute presentation: after restoration of volume and electrolyte balance, urinary electrolyte levels can be appropriately high (when in balance, net intake and output are equal) but mistakenly interpreted as inappropriate, and volume loss from the urinary tract versus the gastrointestinal tract may not be immediately discriminated in infants if no simultaneous, separate 24-hour collections of urine and stool are performed under balanced fluid and electrolyte conditions. Bartter syndrome refers to a group of disorders that are unified by autosomal recessive transmission of impaired salt reabsorption in the thick ascending loop of Henle with pronounced salt wasting, hypokalemic metabolic alkalosis, and hypercalciuria. Clinical disease results from defective renal reabsorption of sodium chloride (29). A misdiagnosis of Bartter syndrome was made in 1 of our patients (Table 1, ID 09D0297), and has been reported in the literature (12).
Other forms of congenital secretory diarrhea may be considered initially in patients with CLD, and include MVID, which is caused by mutation in the MYO5B gene (30); nonsyndromic congenital sodium diarrhea (CSD) (31); a syndromic form of CSD, caused by mutation in the SPINT2 gene (32); malabsorptive congenital diarrhea, caused by mutation in the NEUROG3 gene (33); and congenital tufting enteropathy, caused by mutation in the TACSTD1 gene (3,34).
Electron microscopy (EM) of a rectal biopsy in a neonate of mixed European descent was performed in lieu of a small bowel biopsy because the infant was considered too unstable to undergo anaesthesia, and interpreted locally as demonstrating MVID. The child died shortly thereafter (09D0917, Table 1); however, subsequent MYO5B gene analysis did not reveal a mutation, and a second opinion of the original EM pictures did not confirm the initial interpretation. Although fecal electrolyte analysis had not been performed, mutation analysis for other types of secretory diarrhea was initiated revealing compound heterozygosity for 2 novel SLC26A3 missense mutations. A third patient with CLD (09D1512) underwent ileostomy at 2 weeks of age because her severely dilated intestinal loops and watery stools were thought to result from bowel obstruction, and the diagnosis of CLD was delayed until 6 months of age.
The outcome in the clinical courses of these 3 late diagnosed patients emphasizes the importance of both 24-hour fluid- and electrolyte-balance studies, including separate measurements of electrolyte excretion in urine and feces and/or molecular confirmation in patients with secretory diarrhea. Our results in addition to the previous studies demonstrate that early diagnosis is essential to avert life-threatening complications in patients with CLD and CSD who can lead an almost normal life under relatively simple treatment regimens (1,4,22,23,32,35–37).
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