THEMES
Genetic Disorders of Membrane Transport
III. Congenital chloride
diarrhea*
Juha
Kere1,2,
Hannes
Lohi2, and
Pia
Höglund2
1 Finnish Genome Center and
2 Haartman Institute, University
of Helsinki, 00014 Helsinki, Finland
 |
ABSTRACT |
Congenital chloride diarrhea (CLD) is a recessively
inherited disorder of intestinal electrolyte absorption that involves, specifically,
Cl
/HCO
3
exchange. CLD is caused by mutations in a chromosome 7 gene, first
known as DRA (for
downregulated in adenoma). The disease occurs in all parts of the world
but is more common in some populations with genetic founder effects. More than 20 mutations in the gene are known to date. The
CLD (or
DRA) gene encodes a transmembrane
protein belonging to the sulfate transporter family with three known
members in humans, all associated with a distinct genetic disease.
Members of the gene family can transport other anions as well that may
turn out to be physiologically more important than sulfate transport.
The gene family is well conserved in many prokaryotic and eukaryotic species and is expected to be much larger than presently known.
autosomal recessive gene; anion exchanger; colon; acid-base
balance
 |
INTRODUCTION |
CONGENITAL CHLORIDE DIARRHEA (CLD) was recognized as a
disease entity in 1945 by Gamble et al. (8) and Darrow (5), but it was
not until 1971 that it was shown to be caused by an autosomal recessive
gene [Ref. 24; also see Ref. 25 (MIM #214700)]. The main
clinical feature of CLD is prenatal onset of watery diarrhea that in
utero leads to polyhydramnios and often premature birth. Ultrasound
diagnosis is possible, based on dilated bowel loops (21). Babies have
distended abdomens and absence of meconium, and they fail to thrive.
They develop metabolic alkalosis, hyperbilirubinemia, dehydration, and
severe electrolyte disturbances with hypochloremia, hypokalemia, and
hyponatremia. If suspected, diagnosis can be verified by measuring
fecal Cl
concentration,
which is always >90 mmol/l in a patient with corrected electrolyte
balance. Without treatment, the disease is often fatal; occasionally,
patients survive in a state of chronic contraction, causing early
kidney failure (16). CLD is successfully treated in newborns by
intravenous replacement and in older children and adults by oral
replacement of fluid and electrolyte losses. Detailed guidelines for
therapy have been given by Holmberg (16).
In normal intestine, absorption of
Cl
and other electrolytes
occurs either passively along the concentration and/or
electrostatic gradient or actively against the gradient. Passive
transport predominates in the permeable mucosa of upper small
intestine, but, in the ileum and especially in the colon, where the
intestinal mucosa is tight, active transport is essential for
electrolyte trafficking. In the ileum and colon,
intestinal NaCl absorption is mediated by the operation of two separate
exchangers:
Cl
/HCO
3
and
Na+/H+,
which are indirectly coupled by H+
economy. In CLD, the
Cl
/HCO
3
exchange is absent or defective, causing a severe intestinal
Cl
absorption defect (17).
Massive amounts of Cl
are
lost in the stools, and the patients develop hypochloremia. The
respective defect in HCO
3 secretion
leads to metabolic alkalosis and the acidification of intestinal
content, which further inhibit the absorption of
Na+ through the
Na+/H+
exchanger. In the intestine, the high luminal electrolyte content leads
to diarrhea by osmotic mechanisms.
Na+ and water losses cause
secondary hyperaldosteronism and
K+ wastage, leading to both
hyponatremia and hypokalemia (16).
 |
IDENTIFICATION OF THE CLD GENE |
The first recognized patients were of European descent; altogether, at
least 100 patients have been described in many different populations,
including the United States, Canada, almost all European countries, the
Middle East, and Asian countries such as Japan, Hong Kong, and Vietnam
(see Ref. 18 for references). There are three recognized geographic
areas where the disease is more common: Finland, Poland, and Saudi
Arabia and Kuwait (1, 15, 19, 22, 24, 30). In Finland, the disease
occurs in the eastern provinces, with an approximate incidence of 1 in
20,000. In Poland, the incidence has been estimated at 1 in 200,000 across the country, and, in Saudi Arabia and Kuwait, figures as
high as 1 in 5,000 have been proposed for some areas. However,
consanguineous marriages in these latter countries may cause unusually
high local incidences (11).
Although the basic defect in CLD has been determined by physiological
studies that involve deficient
Cl
/HCO
3
exchange in the distal ileum and colon, the biochemical mechanism
remained totally unknown until the gene mutated in CLD was identified.
Taking advantage of the suggested genetic founder effect in the Finnish
population, Kere et al. (20) studied three candidate genes and
serendipitously mapped the CLD gene close to the cystic fibrosis
transmembrane conductance regulator
(CFTR) gene. More detailed genetic
and physical mapping studies (13, 15) suggested a candidate gene known
as DRA (for downregulated in adenoma;
Ref. 26). DRA was initially cloned as
a candidate tumor-suppressor gene, based on its abundant expression in
the colon and its strongly reduced expression in colon adenomas and
carcinomas. Later, it was observed to have high protein homology to a
newly cloned sulfate transport protein encoded by the diastrophic dysplasia gene DTDST (10). Thus the
genetic map position of DRA, its site
of expression, and its homology to a sulfate transporter made it a
passable candidate for the CLD gene. A
mutation search in Finnish CLD patients revealed homozygosity for a
single mutation, deletion of valine at position 317 (V317del) in all 32 Finnish patients studied, in accordance with the expected founder
effect. A nonsense mutation, 344delT, was found in two Polish patients, and a missense mutation, H124L, was found in another two. These results
implicated DRA as the
CLD gene (14).
 |
WORLDWIDE MUTATION SPECTRUM |
The identification of the gene mutated in CLD and determination of its
genomic structure (9) allowed molecular epidemiologic studies
worldwide. The studies of CLD patients from diverse populations have
revealed in all cases mutations in the
DRA gene; thus it appears that CLD as
a clinical entity is caused by mutations in a single gene (Table
1). So far, at least 20 different mutations have been identified in CLD patients (6, 11, 12,
14). Each of the three populations with an observed high incidence has
its own founder mutation: in Finland, the V317del mutation is present
in all but one chromosome studied to date (98%); in Saudi Arabia and
Kuwait, the G187X mutation is present in 17 of 18 chromosomes (94%);
and, in Poland, the most common mutation I675-676ins accounts for
16 of 34 disease chromosomes (47%). Thus the Polish founder mutation
gives rise to homozygotes but also compound heterozygotes together with
a set of other mutations that would only rarely result in the disease
in the absence of the founder mutation. In contrast, the rare patients
from other populations have been found to be compound heterozygotes
carrying different mutations in each chromosome. The fact that CLD has been observed in many populations worldwide and the wide spectrum of
mutations suggest that new mutations in the
CLD gene are not uncommon, but none of
the CLD mutations has undergone such
an enrichment as the F508del mutation of its chromosomal neighbor, the
CFTR gene.
 |
BIOCHEMISTRY, FUNCTION, AND REGULATION OF THE CLD PROTEIN |
The CLD (or DRA) protein is expressed in the epithelial cells,
particularly in the brush-border cells of normal ileum and colon, but
is also expressed in a few other tissues (4, 14) (Fig.
1). It is predicted to be a 10-, 12-, or
14-transmembrane segment integral membrane protein, and it is variably
N-glycosylated (4) (Fig.
2). There are no recognizable ATP-binding
domains. With the use of two different expression systems
(Xenopus laevis oocytes and Sf9 insect
cells), the protein has been shown to constitute an
Na+-independent sulfate
transporter that can be inhibited by the anion transporter inhibitor
DIDS (3, 27). Its anion specificity also includes at least oxalate and,
as shown recently in a Xenopus oocyte
model, Cl
(23). In this
cell model, Cl
transport
mediated by the CLD protein was also shown to have features compatible
with a
Cl
/OH
exchanger (23). The exact biochemical and physiological role of the CLD protein and its relationship to
Cl
/HCO
3
exchange in ileum and colon are still subject to study.
However, the known functions and the role of the mutated protein in the
pathogenesis of CLD provide several hypotheses that can now
be directly tested.

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Fig. 1.
Immunohistochemical staining of the congenital chloride diarrhea (CLD)
protein in normal colon (left).
Preimmune serum control is also shown
(right). Note the positively
staining epithelial cells along the surface but the scarcity of
positive cells in crypts.
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Fig. 2.
Predicted domain structures of the CLD protein and distribution of
mutations. Three alternative models with 10 (A), 12 (B), or 14 (C) transmembrane segments are
shown. Locations of known mutations are indicated with symbols:
circles, point mutations; triangles, insertions; inverted triangles,
deletions. Missense mutations are indicated with open symbols, nonsense
mutations with solid symbols, and splice site mutations with striped
symbols.
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1) The simplest and perhaps most
intriguing model, is that the CLD protein alone constitutes in
brush-border cells the apical Cl
/HCO
3
exchanger whose physiological role is to absorb up to 900 mmol of
Cl
daily and expel an equal
amount of HCO
3. This hypothesis, if
true, would add another structurally different protein family to the
known
Cl
/HCO
3
exchangers. In humans, there are three anion exchanger
(AE) genes designated as
AE1,
AE2, and
AE3, which encode for the erythrocyte
band 3 protein and two homologous anion transporters, respectively.
Mutations in the AE1 gene cause red blood cell abnormalities (29), and knockout mice show spherocytosis and
hemolytic anemia due to reduced membrane stability (28). The human
AE gene family members show ~20%
homology over ~100 amino acids to the CLD protein, whereas the
two other members of the human sulfate transporter gene family (see
below) are 32-48% homologous to the CLD protein over more than
700 amino acids. Thus the AE family
and sulfate transporter gene family are structurally distinct.
2) Other models are more complex.
The CLD protein may be part of a multiprotein structure with a
Cl
/HCO
3
exchanger function. In this hypothesis, the CLD protein must have a
critical regulatory role, to accommodate for the fact that mutations in
it cause CLD in vivo and a
Cl
transport defect in a
Xenopus oocyte model.
3) In yet another model, the
Cl
/HCO
3
exchange may require two (or more) transporter proteins having
functions that are tightly coupled by a common substrate. In this
model, mutation in just one of the proteins, CLD, would block the
functions of both transporters, causing the observed overall defect in
Cl
/HCO
3 exchange.
The availability of several naturally occurring mutations associated
with CLD disease will facilitate the testing of hypotheses about the
protein's functional role. Mutations seem to occur in all parts of the
protein, but at least two potentially interesting features emerge.
First, there seem to be three clusters of several tightly spaced
mutations (12). They may represent mutation-prone segments of DNA or,
alternatively, protein domains that are critical for intact function.
Second, there are several mutations that localize in the long
COOH-terminal tail in the 10- and 12-transmembrane segment models and
in a large intracellular loop in the 14-transmembrane segment model
(Fig. 2). It is less likely that these mutations would affect the
synthesis or integration of the protein in the cell membrane; rather,
this COOH-terminal, mutation-prone domain may have a distinct
regulatory function. This inference is supported also by the presence
of several protein kinase C and casein kinase II target sites in the
COOH-terminal segment.
So far, the biochemical effects of mutations on CLD mRNA or protein
processing and function remain largely unknown. Most data are available
on the V317del mutation common in Finland. Both CLD mRNA and protein
have been detected in colon epithelia of patients, whereas expression
of the protein in Xenopus oocytes showed deficient sulfate and
Cl
transport in comparison
to the wild-type protein (23). Further biochemical studies are needed
to understand the effect of different mutations on CLD protein
processing and function.
It is well established that the downregulation of the
CLD gene correlates with colon tumor
progression (2, 26). The wide spectrum of different mutations
associated with CLD suggests that the primary pathogenetic effect of
mutations is indeed in anion transport, and downregulation in cancer
cells may well be a secondary effect. The specific expression patterns
of the CLD gene in different tissues
and different sites in the gut epithelium and in normal and malignant
cells make its regulation a highly interesting subject for study.
 |
A FAMILY OF TISSUE-SPECIFIC ANION TRANSPORTERS |
In addition to the CLD and
DTDST genes mutated in a disorder of
gut anion exchange and in growth retardation syndromes, respectively, a
third closely related gene, PDS, was
recently found to be defective in Pendred syndrome, characterized by
congenital sensorineural deafness and goiter (7) (Table
2). Structurally, all three human genes
belong to the family of sulfate transporters, which, as we gather more
information about its members' physiological roles, may turn out to be
a misnomer. This protein family and the relationships of structurally
related proteins across all organisms are illustrated in a phylogenetic
tree (Fig. 3). We wish to emphasize that
the tree was not constructed to illustrate relationships between the
different organisms or between the functional roles of the different
proteins but rather to give a homology-based grouping of the protein
members of the family. Structurally related proteins with conserved
domains are present among bacteria, yeast, fungi, plants, and animals.
It is, however, striking that the animal branch has the smallest number
of known members. Furthermore, 7 of 13 animal entries represent
proteins from Caenorhabditis elegans
whose genes are almost exhaustively listed at present. In all other
animal species, only six members have been identified, three of them
from humans; each of the human genes has a distinct, tissue-specific
function that has been associated with a specific disorder by
positional cloning (Table 2).
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Table 2.
Three structurally related human genes with an anion transport function
and mutations in distinct diseases
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Fig. 3.
A phylogenetic tree illustrating structural relationships within the
sulfate transporter protein family. The three human genes are indicated
in bold; for other species, only the species name is given. The
National Center for Biotechnology Information Entrez protein accession
number is indicated after each entry.
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These observations suggest that numerous other members of this gene and
protein family may remain unidentified. Speculatively, it is possible
that additional family members would exist in humans, with perhaps
distinct, tissue-specific functions, as suggested by the known
CLD,
DTDST, and
PDS genes, proteins, and diseases. Their transport specificity may not be restricted to mostly sulfate in
vivo, but it is indeed possible that the transport of other anions is
physiologically more important. The functional study of the known
proteins and the identification of additional members of this gene
family provide rich sources for a student of the physiology of anion
transport and will promote the understanding of specific transport
mechanisms in different parts of the body.
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ACKNOWLEDGEMENTS |
We thank Albert de la Chapelle and Christer Holmberg for numerous
discussions, collaboration, and support; Siru Haila and Merja Nissinen
for excellent laboratory work; and our many clinical collaborators for
making mutation studies possible.
 |
FOOTNOTES |
*
Third in a series of invited articles on Genetic
Disorders of Membrane Transport.
Our research on CLD is supported by the Finnish Foundation for
Pediatric Research, Ulla Hjelt Fund, Helsinki University Central Hospital research funds, Sigrid Jusélius Foundation, Academy of
Finland, and Research and Science Foundation of Farmos.
Address for reprint requests: J. Kere, Finnish Genome Center, PO Box 21 (Tukholmankatu 2), Univ. of Helsinki, 00014 Helsinki, Finland.
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REFERENCES |
1.
Al-Abbad, A.,
H. Nazer,
S. Sanjad,
and
E. Al-Sabban.
Congenital chloride diarrhea: a single center experience with ten patients.
Ann. Saudi Med.
15:
466-469,
1995.
2.
Antalis, T. M.,
J. A. Reeder,
D. C. Gotley,
M. K. Byeon,
M. D. Walsh,
K. W. Henderson,
T. S. Papas,
and
C. W. Schweinfest.
Down-regulation of the down-regulated in adenoma (DRA) gene correlates with colon tumor progression.
Clin. Cancer Res.
4:
1857-1863,
1998[Abstract].
3.
Byeon, M. K.,
A. Frankel,
T. S. Papas,
K. W. Henderson,
and
C. W. Schweinfest.
Human DRA functions as a sulfate transporter in Sf9 insect cells.
Protein Expr. Purif.
12:
67-74,
1998[Medline].
4.
Byeon, M. K.,
M. A. Westerman,
I. G. Maroulakou,
K. W. Henderson,
S. Suster,
X.-K. Zhang,
T. S. Papas,
J. Vesely,
M. C. Willingham,
J. E. Green,
and
C. W. Schweinfest.
The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein.
Oncogene
12:
387-396,
1996[Medline].
5.
Darrow, D. C.
Congenital alkalosis with diarrhea.
J. Pediatr.
26:
519-532,
1945.
6.
Etani, Y.,
S. Mushiake,
H. Tajiri,
K. Miki,
K. Kozaiwa,
A. Sawada,
K. Tada,
K. Ozono,
and
S. Okada.
A novel mutation of the down-regulated in adenoma gene in a Japanese case with congenital chloride diarrhea.
Hum. Mutat.
12:
362,
1998[Medline].
7.
Everett, L. A.,
B. Glaser,
J. C. Beck,
J. R. Idol,
A. Buchs,
M. Heyman,
F. Adawi,
E. Hazani,
E. Nassir,
A. D. Baxevanis,
V. C. Sheffield,
and
E. D. Green.
Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).
Nat. Genet.
17:
411-422,
1997[Medline].
8.
Gamble, J. L.,
K. R. Fahey,
J. Appleton,
and
E. McLachlan.
Congenital alkalosis with diarrhea.
J. Pediatr.
26:
509-518,
1945.
9.
Haila, S.,
P. Höglund,
S. W. Scherer,
J. R. Lee,
P. Kristo,
B. Coyle,
R. Trembath,
C. Holmberg,
A. de la Chapelle,
and
J. Kere.
Genomic structure of the human chloride diarrhea (CLD) gene.
Gene
214:
87-93,
1998[Medline].
10.
Hästbacka, J.,
A. de la Chapelle,
M. M. Mahtani,
G. Clines,
M. P. Reeve-Daly,
M. Daly,
B. A. Hamilton,
K. Kusumi,
B. Trivedi,
A. Weaver,
A. Coloma,
M. Lovett,
A. Buckler,
I. Kaitila,
and
E. S. Lander.
The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.
Cell
78:
1073-1087,
1994[Medline].
11.
Höglund, P.,
M. Auranen,
J. Socha,
K. Popinska,
H. Nazer,
U. Rajaram,
A. Al Sanie,
M. Al-Ghanim,
C. Holmberg,
A. de la Chapelle,
and
J. Kere.
Genetic background of congenital chloride diarrhea in high incidence populations: Finland, Poland, Saudi Arabia and Kuwait.
Am. J. Hum. Genet.
63:
760-768,
1998[Medline].
12.
Höglund, P.,
S. Haila,
K.-H. Gustavson,
M. Taipale,
K. Hannula,
K. Popinska,
C. Holmberg,
J. Socha,
A. de la Chapelle,
and
J. Kere.
Clustering of private mutations in the congenital chloride diarrhea/down-regulated in adenoma gene.
Hum. Mutat.
11:
321-327,
1998[Medline].
13.
Höglund, P.,
S. Haila,
S. W. Scherer,
L.-C. Tsui,
E. D. Green,
J. Weissenbach,
C. Holmberg,
A. de la Chapelle,
and
J. Kere.
Positional candidate genes for congenital chloride diarrhea suggested by high-resolution physical mapping in chromosome region 7q31.
Genome Res.
6:
202-210,
1996[Abstract].
14.
Höglund, P.,
S. Haila,
J. Socha,
L. Tomaszewski,
U. Saarialho-Kere,
M.-L. Karjalainen-Lindsberg,
K. Airola,
C. Holmberg,
A. de la Chapelle,
and
J. Kere.
Mutations in the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhea.
Nat. Genet.
14:
316-319,
1996[Medline].
15.
Höglund, P.,
P. Sistonen,
R. Norio,
C. Holmberg,
A. Dimberg,
K.-H. Gustavson,
A. de la Chapelle,
and
J. Kere.
Fine mapping of the congenital chloride diarrhea gene by linkage disequilibrium.
Am. J. Hum. Genet.
57:
95-102,
1995[Medline].
16.
Holmberg, C.
Congenital chloride diarrhea.
Clin. Gastroenterol.
3:
583-602,
1986.
17.
Holmberg, C.,
J. Perheentupa,
and
K. Launiala.
Colonic electrolyte transport in health and in congenital chloride diarrhea.
J. Clin. Invest.
56:
302-310,
1975[Medline].
18.
Holmberg, C.,
J. Perheentupa,
K. Launiala,
and
N. Hallman.
Congenital chloride diarrhea. Clinical analysis of 21 Finnish patients.
Arch. Dis. Child.
52:
255-267,
1977[Abstract].
19.
Kagalwalla, A. F.
Congenital chloride diarrhea. A study in Arab children.
J. Clin. Gastroenterol.
19:
36-40,
1994[Medline].
20.
Kere, J.,
P. Sistonen,
C. Holmberg,
and
A. de la Chapelle.
The gene for congenital chloride diarrhea maps close to but is distinct from the gene for cystic fibrosis transmembrane conductance regulator.
Proc. Natl. Acad. Sci. USA
90:
10686-10689,
1993[Abstract].
21.
Kirkinen, P.,
and
P. Jouppila.
Prenatal ultrasonic findings in congenital chloride diarrhea.
Prenat. Diagn.
4:
457-461,
1984[Medline].
22.
Lubani, M. M.,
K. I. Doudin,
D. C. Sharda,
A. A. Shaltout,
T. Al-Shab,
Y. K. Abdul Al,
M. A. Said,
M. M. Salhi,
and
S. A. Ahmed.
Congenital chloride diarrhea in Kuwaiti children.
Eur. J. Pediatr.
148:
333-336,
1989[Medline].
23.
Moseley, R. H.,
P. Höglund,
G. D. Wu,
D. G. Silberg,
S. Haila,
A. de la Chapelle,
C. Holmberg,
and
J. Kere.
Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea.
Am. J. Physiol.
276 (Gastrointest. Liver Physiol. 39):
G185-G192,
1999[Abstract/Free Full Text].
24.
Norio, R.,
J. Perheentupa,
K. Launiala,
and
N. Hallman.
Congenital chloride diarrhea, an autosomal recessive disease.
Clin. Genet.
2:
182-192,
1971[Medline].
25.
OMIM Center for Medical Genetics.
Online Mendelian Inheritance in Man. Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), 1997. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim.
26.
Schweinfest, C. W.,
K. W. Henderson,
S. Suster,
N. Kondoh,
and
T. S. Papas.
Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.
Proc. Natl. Acad. Sci. USA
90:
4166-4170,
1993[Abstract].
27.
Silberg, D. G.,
W. Wang,
R. H. Moseley,
and
P. G. Traber.
The down regulated in adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.
J. Biol. Chem.
270:
11897-11902,
1995[Abstract/Free Full Text].
28.
Southgate, C. D.,
A. H. Chisti,
B. Mitchell,
S. J. Yi,
and
J. Palek.
Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton.
Nat. Genet.
14:
227-230,
1996[Medline].
29.
Tanner, M. J. A.
Molecular and cellular biology of the erythrocyte anion exchanger (AE1).
Semin. Hematol.
30:
34-57,
1993[Medline].
30.
Tomaszewski, L. M.,
E. Kulesza,
and
J. Socha.
Congenital chloride diarrhoea in Poland.
Mater. Med. Pol.
4:
271-277,
1987.
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