Vol 21, No.

11;Nov 2014

Mutation Of Congenital Chloride Loosing Diarrhea In Saudi Children

Abdulla A. Alharthi1, 2, 3, Naglaa M. Kamal2, 4, Samer E. Ismail1, Gaber M. G. Shehab5,6,
Hamdi M. Youssef7 and Yousri M. Hussein8,9
1
2
3
4
5
6
7
8
9

Biotechnology and Genetic Engineering Research Unit, Deanship of Scientific Research, Taif University, Taif, KSA.
Pediatrics Department, Alhada Armed Forces Hospital, Taif, KSA.
Pediatrics Department, College of Medicine, Taif University, Taif, KSA.
Pediatrics Department, Faculty of Medicine, Cairo University, Cairo, Egypt.
Medical Biochemistry Department, College of Medicine, Taif University, Taif, KSA.
Biochemistry Department, Faculty of Agriculture, Cairo University, Egypt.
Microbiology Department, College of Medicine, Taif University, Taif, KSA.
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
Medical Laboratories Department, Faculty of Applied Medical Sciences, Taif University, Taif, KSA

Corresponding Author: Abdulla A. Alharthi, B.O. Box 689, P. Code: 21944, TAIF, KSA.
E-mail: aharthy@gmail.com

Abstract:
Congenital Chloride Diarrhea (CLD) is a watery diarrhea with metabolic alkalosis and excess
chloride in feces. It is an autosomal recessive inherited disease caused by mutations in
SLC26A3 gene with higher incidence in Arab countries. Due to Arab consanguineous
marriages, only one founder mutation (Gly187Ter)was reported in exon5.We sequenced exon5
to study the molecular background in 27 CLD children from Taif, Saudi Arabia. Interestingly,
the mutation (NG_008046.1:g.17175G>T, ss904491498) was consistent in all children. These
results will support developing CLD early detection kits and specific treatments. Adding it to
the Saudi pre-marital check-up program would greatly decrease CLD burden. We are looking
forward to include screening for the reported founder mutation in the Saudi pre-marital checkup program hoping to decrease the burden of this inherited lifelong disease and with the
challenge of developing specific treatment
Key words: Chloride Diarrhea (CLD), Congenital Chloride Loosing Diarrhea (CCD), SNP, founder mutation.

Introduction:
In 1945, Darrow 1 and Gamble et al.2 simultaneously described a congenital watery
diarrhea with alkalosis and they named that syndrome Congenital Alkalosis with
Diarrhea. The affected children have metabolic alkalosis and their feces have an
excess chloride over the sum of sodium and potassium. The disease results from a
defect in the ileal transport of chloride, which results in high stool chloride
concentration, selective chloride depletion, and secondarily metabolic alkalosis 3, 4and
the metabolic alkalosis is a consequence of potassium and chloride deficiency.
Accordingly, it was suggested to name it alternatively Congenital Chloride Diarrhea
(CLD, and sometimes CCD) 3. It is recommended to consider CLD in any patient with
severe resistant diarrhea to prevent its irreversible and long term organ
damage5.Later, CLD was described as a rare autosomal recessive inherited disorder
due to an intestinal absorption defect in the intestinal chloride/bicarbonate exchanger,
which lasts for life 5-7.
Some studies reported the rarity of CLD in some countries like: Japan 6, Iran 5, UK
7
,Bangladesh 8, and Hong Kong 9. Other studies stated the high incidence of CLD in
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other countries like: Finland 10 and Arab countries including Saudi Arabia due to
consanguineous marriages 10-13.
CLD resembles the renal disorder Bartter syndrome, but in Bartter syndrome the
patients lose chloride mainly in urine 14, 15. Due to the confusion and misdiagnosis with
other diarrheas and syndromes, some patients were not diagnosed with CLD during
infancy 7, 9, 15.
To avoid such confusion, scientists used molecular tools to study the molecular
background of CLD (OMIM: 214700) in order to have a better understanding of the
etiology of CLD and in turn facilitate its treatment. At the beginning, a cDNA was
isolated in 1993 that was called DRA (stands for down-regulated in adenoma) 16. Its
mRNA is expressed exclusively in normal colon tissue and its expression is
significantly decreased in adenomas and adenocarcinomas of the colon. The CLD
gene appeared to be a single copy gene present on chromosome 7 and it was
suggested that it is a transcription factor or protein that nay interact with transcription
factors 16. Using fluorescence in situ hybridization (FISH), the DRA gene was localized
to chromosome band 7q22-q31.1 with a full-length (2.9 kb) cDNA as probe 17. A fine
mapping of the congenital chloride diarrhea gene was carried out by studying linkage
disequilibrium in 24 Finnish and 4 Swedish families and the CLD region was mapped
to ~0.37 cM from the marker D7S496 (ref.18). Later, in 1996, a physical map was
constructed and refined based on a 2.7-Mb YAC contig around D7S496 and identified
2 candidate genes: SLC26A3 (126650) and PRKAR2B (176912). Both genes map
within 450 kb of D7S496 (which is linked to CLD), making them functionally and
positionally relevant candidates for the site of the mutation in CLD 19.
SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain. It
was suggested that mutations in the STAS domain of SLC26A3, which functions as a
coupled Cl-/HCO3-colonic exchanger, cause SLC26A3 transporter misfolding and/or
mistrafficking, both ultimately leading to loss of functional protein at the plasma
membrane, which in turn cause CLD20.
The genomic structure of CLD gene (SLC26A3) was determined using DNA from
several sources including multiple large-insert libraries and genomic DNA from Finish
CLD patients and controls. The CLD gene spans approximately 39 kb and comprises
21 exons. All exon/intron boundaries conformed to the GT/AG rule21.
Many studies have reported the expected mutations in the SLC26A3 in congenital
chloride diarrhea patients (CLD; 214700) in several countries, showing higher
incidence in Finland, Poland and Arab countries and rare cases in other countries
such as Korea 22-28.
By using mRNA in situ hybridization, the expression of SLC26A3 was found to occur
preferentially in highly differentiated colonic epithelial cells and is low in
undifferentiated
(including neoplastic) cells. The demonstration of office@multidisciplinarywulfenia.org
the relationship
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between SLC26A3 mutations and chloride diarrhea indicated that SLC26A3 is an

intestinal transport molecule22.
The delay of diagnosis and treatment of the disease, usually worsen the condition and
leads to further complications. In addition, consanguineous marriages would increase
the incidence of autosomal recessive inherited syndromes such as CLD. The
diagnosis of one child in a consanguine family, helped to predict the incidence of CLD
in younger siblings and with proper treatment they have escaped further complications
because they were diagnosed soon after birth and immediately received therapy 23, 24.
So, there is a need for accurate diagnosis, early detection, and understanding the
molecular background of CLD will facilitate its treatment and prevention of further
complications. Hence, we are seeking to employ sequencing of the exon5 of
SLC26A3 gene for investigating the molecular background of the CLD, in order to
have a better understanding of CLD etiology in Taif, Saudi Arabia, in order to facilitate
its treatment and prevention of further complications. Also, to support decision makers
in the premarital health check-up program, adopted by the Saudi government to limit
the spread of inherited syndromes.
Patients and Methods:
Patients. In the current work, we have diagnosed 27 children to have CLD based on
the clinical phenotype of all patients, which is characterized by a profuse diarrhea with
high chloride content (190 mmolLiter -1) in watery stools, justifying the clinical diagnosis
of CLD. We collected peripheral blood samples from the 27 CLD children into
ethylenediaminetetraacetic acid (EDTA) tubes and stored at -20C until DNA
extraction step . Later, we collected peripheral blood samples from 4 of their parents
that are clinically normal and do not show the clinical characteristics of CLD into
ethylenediaminetetraacetic acid (EDTA) tubes and stored at -20C until DNA
extraction.
DNA extraction. Genomic DNA was extracted from blood samples according to
instructions of Blood DNA Preparation Kit (Thermo Fisher; USA). Quality of extracted
DNA was assessed via electrophoresis on agarose gel 1%. Concentration of DNA
was estimated by UV Spectrophotometry.
DNA amplification via PCR. We carried out the amplification of the exon 5 of
SLC26A3 gene according to Hglund et al.10. We used a total reaction volume of 20 ul
of 2X superhot PCR Master Mix (Bioron; Germany) which contained 10 pmol of each
intronic primer: i49 5`TGG TCT TAT TCT GTG GTT GAA AGA3` and i52 5`AGT CAA
GAT GAA CAG ATT GAG GTG3` primers for SLC26A3 gene and 50 to 60 ng of
genomic DNA. The primer was designed to produce a 315pb fragment that spans the
whole exon5 and parts of the flanking introns. We performed the PCR amplification
reactions in Eppendorf thermal cycler using the following PCR program: 1 cycle at
94C 10 min; 35 additional cycles consisting of 94C 45 sec, 57C 45 sec and 72C
45 sec
236 and finally 1 cycle of 72C 10 min. After the amplification, we electrophoresed
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the PCR reaction products with 100 bp ladder (Fermentas, Germany) on 10 x 14 cm

1.5%-agarose gel (Bioshop; Canada) for 30 min using Tris-borate- EDTA Buffer. The
gel was stained with 0.5 gml -1 of ethidium bromide (Bioshop; Canada).
Gel visualization and documentation. All gels were visualized on a UV
Transilluminator and documented using a GeneSnap 4.00- Gene Genius Bio Imaging
System (Syngene; Frederick, Maryland, USA).
DNA sequencing. We purified the specific fragment for SLC26A3 gene approximately
315 bp via PCR purification kit (Jena Bioscience) according to the manufacturers
instructions. We directly sequenced the specific fragments using the same primers
employed in the PCR amplification process on both forward and reverse directions
and it was repeated to ensure reproducible results. The products were sequenced
using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (ABI Applied
Biosystems, now Life Technology) on a 3130 Genetic Analyzer (Applied Biosystems)
and we used the Data collection software version 3.1 from ABI Applied Biosystems
(Life Technologies) to collect the raw sequencing results.
Sequence analysis and mutation detection. We carried out sequence analysis and
base calling using Sequence Analysis Software version 3.1 from ABI Applied
Biosystems (now Life Technology). After studying the sequence quality and
consistency, we carried out sequence alignment to the SLC26A3 gene on NCBI
database. Following that, we Employed SeqScape software version 2.7 from ABI
Applied Biosystems (now Life Technology) for base-calling and mutation detection.
SNP submission. We have submitted the single nucleotide substitution detected in
the CLD children (NG_008046.1:g.17175G>T) to the Human Variation SNP database
under submission number: ss904491498.
Informative consent. The study was approved by the research and ethical
committees of Alhada Armed Forces Hospital, Taif, Saudi Arabia.Written informed
consents were obtained from the parents of the contributing children for the
participation of their children in the current study.
Results:
We diagnosed 27 Saudi children from Taif Governorate to have CLD based on the
clinical characteristics of that syndrome. They are descendants of different Saudi
clans. Sex ratio is almost 2 female: 1 male. Their age at investigation ranged from 2
month to 12 years, some of them were diagnosed to have CLD at birth.
We collected peripheral blood samples from them. Following that we have extracted
genomic DNA from blood and sequenced the exon number 5 of the SLC26A3 gene
(MIM: 214700) that is reported to have the founder mutation (rs121913032) of Arab
people:Gly187Ter (ref.10). All the 27 children exhibited a Nonsense single base
substitution (NG_008046.1:g.17175G>T) causing a termination signal instead of
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amino acid Glycine (Fig. 1), which would have a serious impact on the solute carrier
function involved in chloride transport in gut membrane. We have submitted the
mutation to the Human Variation Database of the National Center for Biotechnology
Information (NCBI) under submission number: ss904491498.
On the other hand, we collected peripheral blood samples from 4 healthy parents that
we have diagnosed their children as CLD patients through sequencing and clinical
features and we sequenced their genomic DNA after extraction at the same locus and
they showed heterozygous pattern at that locus having G and T together (Fig. 2)
instead of G alone.

Figure1. gDNA sequence electropherogram of children with CLD showing the base
substitution of T instead of G.

Figure2. gDNA sequence electropherogram of carrier parents showing the base

substitution of K (G and T) instead of G.

Discussion:
Previous studies showed several mutations related to congenital chloride diarrhea in
some countries: Finland 12, 22,25,28, Korea 26, 27, and Japan 6. No previous studies
addressed the genetic basis of CLD from KSA apart from a single study carried on
CLD genetics from different countries and included 4 Saudi children 10. In our study we
detected a single nucleotide substitution (NG_008046.1:g.17175G>T, ss904491498)
complying with the majority of mutation types as previously stated 25.
At least 55 mutations that causes CLD, of which 21 (38%) novel were reported 25.
Majority of mutation are single nucleotide substitutions (n 30; 55%) with 18
missense, 7 nonesense, and 5 splice-site mutations. Additional mutations are minor
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deletions/insertions or their combination (n 21; 38%), major deletions (n 3; 5%), and

a major insertion (n 1; 2%). Distinct founder mutations appear in Finland, Poland,
and Arab countries, whereas patients from other countries carry rare homozygous or
compound heterozygous mutations 25.
As employing exon-specific primers facilitated mutation screening studies of CLD
patients10, 14, 26, 27; genetic analysis by direct sequencing were performed on the 20
exons and parts of exonintron boundaries of the SLC26A3 gene in eight Korean
patients, the most common mutation in Korea was c.2063-1G>T, which was found in
at least one allele of all patients it was suggested as a founder mutation in Korean
population so that the targeting sequencing for the mutation would be a cost-efficient
screening method to confirm a diagnosis of CLD in patients of Korean descent 26.Using
direct sequencing analysis, a Korean infant with clinical features of CLD was identified
to have 2 sequence variants: a missense variant of unknown significance (c.525GC;
p.Arg175Ser) and a splicing mutation (c.2063-1GT) in the SLC26A3 gene; these
have been inherited from father and mother, respectively 27.The ability of wholeexome sequencing to capture approximately 95% of the targeted coding sequences
with high sensitivity and specificity for detection of homozygous and heterozygous
variants was demonstrated 14. This approach was utilized to make an unanticipated
genetic diagnosis of CLD in a Turkish infant referred with a suspected diagnosis of
Bartter syndrome. Sequencing this gene in 39 additional patients referred with a
suspected diagnosis of Bartter syndrome, 5 of them who did not have mutations in the
known genes for this disease had homozygous deleterious mutations in SLC26A3.
The results emphasized the clinical utility of whole-exome sequencing for pathogenic
mutation discovery and clinical diagnosis 14. Authors described the geographic and
population distributions of 3 founder mutations: the Finnish V317del mutation
(126650.0001), the Polish I675-676ins mutation (126650.0005), and the Arabic
gly187-to-ter mutation (G187X; 126650.0006) (ref.28).Hence, employing exon5-specific
intronic primers for forward and reverse sequencing facilitated the screening of the 27
CLD children and some of their parents and we found the mutation
(NG_008046.1:g.17175G>T, ss904491498) consistently in all the children. Our results
were in concordance with a previous study10 showing only one base substitution
mutation in that locus (rs121913032) Gly187Ter, that is assigned as the founder
mutation of CLD in the Arab region due to long time of consanguineous marriages.
The affected children were descendants of 9 old Saudi clans with higher incidence in
two clans over the others in agreement with other studies that confirmed the high
incidence of CLD in Arab countries including Saudi Arabia due to consanguineous
marriages 10-13. In addition, there was bias towards female patients over the male
ones (~2:1, respectively) which contradicts previous report of equal distribution of the
syndrome between males and females 10.
The heterozygousity of carrier parents proves that it is an inherited autosomal
recessive syndrome as it was reported before 5-7. The persistence of the
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NG_008046.1:g.17175G>T mutation in the affected children proves that it is the

main founder mutation in Saudi Arabia in concordance with Hglund study 10 in 1998.
As CLD is one of the important inherited syndromes in Saudi Arabia due to the
consanguineous marriages in our region. Our results will open the door in front of
further research to develop early detection kits that will confirm the incidence of CLD
without any doubt or confusion. It will help in the program for health check-up before
marriage adopted by the Saudi government to limit the spread of inherited syndromes.
Also, it will participate to the knowledge required for developing a suitable treatment
for this important condition in our region.
Acknowledgments
Funds: The study received fund from Taif University, Taif, KSA in a call for research fund
grants managed by the Vice-president for scientific Research and Postgraduates Affairs in
2012.
Web Resources:SNP Database, http://www.ncbi.nlm.nih.gov/SNP/
Accession Numbers: The submission number at the SNP database for the single nucleotide
substitution (NG_008046.1:g.17175G>T) reported in this paper is ss904491498.
References:
1- Darrow, D. C. Congenital alkalosis with diarrhea. J. Pediat. 26, 519-532 (1945).
2- Gamble, J. L.; Fahey, K. R.; Appleton, J.; and MachLachlan, E. Congenital
alkalosis with diarrhea. J. Pediat. 26, 509-518 (1945).
3- Evanson, J. M. and Stanbury S. W. Congenital chloridorrhoae or so-called
congenital alkalosis with diarrhoea. Gut 6, 29-38 (1965).
4- Holmberg, C.; Perheentupa, J.; and Launiala K. Colonic electrolyte transport in
health and in congenital chloride diarrhea. Journal of Clinical Investigation 56,
302-310 (1975).
5- Nickavar, A. Congenital chloride Diarrhea: a case report. Iran J Ped 17(2), 179182 (2007).
6- Yoshikawa, H.; Watanabe, T.; Tokinari, A.; Sato, M. and Oda, Y. Japanese
siblings with congenital chloride diarrhea. Pediatrics International 42, 313-315
(2000).
7- Litos, M.; Michala, S.; Brown, R. Congenital chloride diarrhea in pregnancy: a
case report. European J Obstetrics & Gynecology and Reproductive Biology
136, 126-133 (2008).
8- Islam, S. B.; Chisti M. J.; Shahrin, L.; and Alam, N. H. Congenital chloride
diarrhea: first time diagnosis in Bangladesh and its management difficulties. J
Clin Case Rep 3(7), 283-286 (2013).
240

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Vol 21, No. 11;Nov 2014

9- Lok, K-H; Hung, H-G; Li, K-K; Li, K-F; and Szeto, M-L. Congenital chloride
diarrhea: a missed diagnosis in an adult patient. Am J Gastroenterol 102, 13281329 (2007).
10- Hglund, P. et al. Genetic background of congenital chloride diarrhea in highincidence populations: Finland, Poland, and Saudi Arabia and Kuwait. Am. J.
Hum. Genet. 63, 760768 (1998).
11- Kagalwalla, A. Congenital chloride diarrhea, a study in Arabchildren. J
ClinGastroenterol 19, 36-40 (1994).
12- Al-Hamad, N. M. and, Al-Eisa, A. A. Renal abnormalities in congenital
chloridediarrhea. Saudi Med J 25(5), 651-655 (2004).
13- Elrefae, F.; Elhassanien, A. F.; and Alghiaty, H. A. Congenital chloride diarrhea:
a review of twelve Arabian children. Clin& Experimental Gastroenterol. 6, 71-75
(2013).
14- Choi, M., et al. Genetic diagnosis by whole exome capture and massively
parallel DNA sequencing. Proc. Nat. Acad. Sci. 106, 19096-19101 (2009).
15- Hosnut, F. O.; ncel, E. K.; ncel, M. Y.; and zkay, F. A Turkish case of
congenital chloride diarrhea with SLC26A3 gene (c.2025_2026insATC)
mutation: diagnostic pitfalls. Turk J Gastroenterol 21(4), 443-447 (2010).
16- Schweinfest, C. W.; Henderson, K. W.; Suster, S.; Kondoh, N.; and Papas, T.
S. Identification of a colon mucosa gene that is down-regulated in colon
adenomas and adenocarcinomas. Proc. Nat. Acad. Sci. 90, 4166-4170 (1993).
17- Taguchi, T.; Testa, J. R.; Papas, T. S.; and Schweinfest, C. Localization of a
candidate colon tumor-suppressor gene (DRA) to 7q22-q31.1 by fluorescence
in situ hybridization. Genomics 20(1), 146-147 (1994).
18- Hglund, P., et al. Finemapping of the congenital chloride diarrhea gene
bylinkage disequilibrium. Am. J. Hum. Genet. 57, 95-102 (1995).
19- Hglund, P., et al. Positional candidate genes for congenital chloride diarrhea
suggested by high-resolution physical mapping in chromosome region 7q31.
Genome Res. 6, 202-210 (1996).
20- Dorwart, M. R., et al. Congenital chloride-losing diarrhea causing mutations in
the STAS domain result in misfolding and mistrafficking of SLC26A3. J. Biol.
Chem. 283, 8711-8722 (2008).
21- Haila, S., et al. Genomic structure of the human congenital chloride diarrhea
(CLD) gene. Gene 214, 87-93 (1998).
22- Hglund, P., et al. Mutations of the down-regulated in adenoma (DRA) gene
cause congenital chloride diarrhoea. Nature Genet. 14,316-319 (1996).

241

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23- Mohsen, A. M.; Al-Mutairi, H. F.; and Abou Shanab, O. A. Congenital chloride
diarrhea associated with sensorineural deafness, and epilepsy in Kuwaiti Girl.
Kuwait Medical Journal 40(3), 248-250 (2008).
24- Hglund, P.; Holmberg, C.; Sherman, P.; and Kere, J. Distinct outcomes of
chloride diarrhea in two siblings with identical genetic background of the
disease: implications for early diagnosis and treatment. Gut 48, 724-727
(2001).
25- Wedenoja, S.; et al. Update on SLC26A3 mutations in congenital chloride
Diarrhea. Human Mutation 32(7), 715-722 (2011).
26- Hong, J.; et al. Congenital chloride diarrhea in Korean children: novel mutations
and genetic characteristics. Eur J Pediatr 172, 545550 (2013).
27- Lee, E-S.; Cho A. R.; and Ki, C-S. Identification of SLC26A3mutations on a
Korean patient with congenital chloride diarrhea. Ann Lab Med 32, 312-315
(2012).
28- Mkel, S., Kere, J., Holmberg, C., Hoglund, P. SLC26A3 mutations in
congenital chloride diarrhea. Hum. Mutat. 20, 425-438 (2002).

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