INVITED REVIEW

Congenital Sodium Diarrhea: A Form of Intractable

Diarrhea, With a Link to Inflammatory Bowel Disease
y

Andreas R. Janecke, Peter Heinz-Erian, and Thomas Muller

ABSTRACT
Congenital diarrheal disorders (CDDs) represent a group of challenging
clinical conditions for pediatricians because of the severity of the presentation and the broad range of possible differential diagnoses. CDDs arise
from alterations in the transport of nutrients and electrolytes across the
intestinal mucosa, from enterocyte and enteroendocrine cell differentiation
and/or polarization defects, and from the modulation of the intestinal
immune response. Advances were made recently in deciphering the etiology
and pathophysiology of one of these disorders, congenital sodium diarrhea
(CSD). CSD refers to an intractable diarrhea of intrauterine onset with high
fecal sodium loss. CSD is clinically and genetically heterogeneous. A
syndromic form of CSD features choanal and intestinal atresias as well
as recurrent corneal erosions. Small bowel histology frequently detects an
epithelial tufting dysplasia. It is autosomal recessively inherited, and
caused by SPINT2 mutations. The nonsyndromic form of CSD can be caused
by dominant activating mutations in GUCY2C, encoding intestinal receptor
guanylate cyclase C (GC-C), and by autosomal recessive SLC9A3 loss-offunction mutations. SLC9A3 encodes Na/H antiporter 3, the major
intestinal brush border Na/H exchanger, and a downstream target of
GC-C. A number of patients with GUCY2C and SLC9A3 mutations developed inflammatory bowel disease. Both the number of recognized CDD
forms as well as the number of underlying disease genes are gradually
increasing. Knowledge of these CDD genes enables noninvasive, nextgeneration gene panel-based testing to facilitate an early diagnosis in
CDD. Primary Na/H antiporter 3 and GC-C malfunction is implicated
as a predisposition for inflammatory bowel disease in subset of patients.
Key Words: chronic diarrhea, diarrhea, guanylate cyclase, GUCY2C,
inflammatory bowel disease, intestinal failure, microbiota, neonatal
diarrhea, NHE3, SLC9A3, sodium, sodium-proton-exchanger, SPINT2

(JPGN 2016;63: 170176)

ongenital diarrheal disorders (CDDs) generally result from

specific genetic defects inherited as autosomal recessive
traits. There is a broad range of disorders that can present with
diarrhea as the first and/or prominent symptom in neonates (1).

Received October 22, 2015; accepted January 27, 2016.

From the Department of Pediatrics I, Medical University of Innsbruck,
and the yDivision of Human Genetics, Medical University of Innsbruck,
Innsbruck, Austria.
Address correspondence and reprint requests to Andreas R. Janecke, MD,
Department of Pediatrics I, Innsbruck Medical University, Anichstrasse
35, 6020 Innsbruck, Austria (e-mail: Andreas.Janecke@i-med.ac.at).
sterreichischen
The present work was supported by Jubilaumsfonds der O
Nationalbank (Grant nos. 14496 and 15627) and by Else KronerFresenius-Stiftung (Grant no. 2013_A230).
The authors report no conflicts of interest.
Copyright # 2016 by European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition and North American Society for Pediatric
Gastroenterology, Hepatology, and Nutrition
DOI: 10.1097/MPG.0000000000001139

170

What Is Known





Congenital sodium diarrhea was first described in

2 sporadic cases in 1985, and <50 patients are
reported in the literature to date.
A syndromic and a nonsyndromic form of congenital
sodium diarrhea were distinguished in 2009.
Mutations in 3 genes, SPINT2, GUCY2C, and SLC9A3,
can cause congenital sodium diarrhea.

What Is New





Congenital sodium diarrhea is a disorder of sodium

absorption that affects the intestinal sodium-proton
exchanger Na/H antiporter 3 in the nonsyndromic
form and the epithelial sodium channel in the
syndromic form.
This review provides guidelines for clinical and molecular diagnosis of congenital sodium diarrhea.
This review should increase awareness of this disorder.

These include deficiencies of proteins necessary for nutrient

absorption such as lactase and Na/glucose cotransporter SLC5A1,
deficiencies for ion absorption and pH regulation such as chloride/
bicarbonate exchanger SLC26A3, for enterocyte polarization such
as the motor protein myosin Vb, for enteroendocrine cell differentiation such as the transcription factor NEUROG3, and epithelial
integrity such as cell adhesion molecule EpCAM. In addition,
early-onset diarrhea is a prominent symptom in systemic metabolic
diseases such as congenital disorder of glycosylation type 1B and
cystic fibrosis.
The abnormal intestinal fluid transport can already begin in
utero and manifests as maternal polyhydramnios. The clinical
picture is often life-threatening at onset because of severe
dehydration and serum electrolyte imbalances. Different diseases
can then take very variable courses later on, with weaning from total
parenteral nutrition within days to weeks and years after birth
to lifelong partial or total parenteral nutrition and small bowel
transplantation. The more severe forms of CDD show significant
mortality and morbidity. CDDs may exacerbate in episodes
of infectious diarrhea and present with additional extraintestinal
complications. Several types of CDD can be treated with dietary
modification. The exact prevalence of CDD remains to be
established among populations and geographic areas (2).
Diarrhea can result from secretory or osmotic mechanisms.
Secretory diarrhea is characterized by an increased electrolyte and
water flux toward intestinal lumen, resulting from the inhibition of
JPGN

Volume 63, Number 2, August 2016

Copyright ESPGHAL and NASPGHAN. All rights reserved.

JPGN

Volume 63, Number 2, August 2016

Congenital Sodium Diarrhea

NaCl absorption by villous enterocytes or an increase in Cl

secretion by secretory crypt cells. Osmotic diarrhea is caused by
the presence of nonabsorbed nutrients in the gut lumen. Nonabsorbed solutes drive fluids into the lumen through an osmotic force.
Evaluation of CDDs includes the understanding of these mechanisms, and both the mechanisms apply in some disorders (2).
Diarrhea is also a symptom in patients with inflammatory
bowel disease (IBD), occurring in about 50% of acute flare-ups of
CD and in nearly all patients with ulcerative colitis (UC). Both
abnormal epithelial ion transport and altered intestinal motility have
been implicated in the pathophysiology of diarrhea in IBD (3,4).
Most interestingly, IBD occurs in a subgroup of patients with
1 rare type of CDD, congenital sodium diarrhea (CSD), the subject
of the present review. CSD is caused by a primary disturbance in
epithelial ion transport and emphasizes the concept of abnormal
epithelial ion transport in the pathogenesis of common forms of
IBD. The group of monogenic forms of CSD reviewed here can thus
be added to an evolved list of approximately 50 monogenic defects
with IBD-like pathology; these comprise defects in intestinal
epithelial barrier and stress response, and a number of immunodeficiencies (5).
CSD is caused by impaired intestinal Na absorption. The
purpose of the present review is to delineate CSD clinically,

genetically, and pathophysiologically, in light of very recent developments in this field. The present review may help to diagnose and
treat such patients and compiles the present knowledge of this
clinically and genetically heterogeneous condition. CSD was first
described in 2 sporadic patients in 1985 (6,7), and subsequently
about 50 cases have been reported in the literature.
Classical CSD results from a loss of function of sodiumproton antiporter 3 (NHE3), an apical membrane Na/H exchanger that absorbs a major part of intestinal Na, and is important
for systemic volume and acid-base homeostasis. In 1 subset of
patients, primary NHE3 deficiency is caused by recessive
SLC9A3 (solute carrier family 9, subfamily A, member 3; Mendelian Inheritance in Man [MIM] no. 182307) mutations resulting in
absent or nonfunctional protein of the encoded NHE3 itself. In a
second subset of CSD patients, secondary NHE3 deficiency
results from downregulation of NHE3 by elevated intracellular
cyclic guanosine monophosphate (cGMP) levels, induced by
constitutively activating and hyperstimulating mutations in
receptor guanylate cyclase C (GC-C; MIM no. 601330), encoded
by the GUCY2C gene (Fig. 1A, B).
About one third of patients display a pattern of congenital
malformations and episodes of superficial punctate keratitis, that is,
a syndromic form of CSD that can be distinguished from a
C
ST14

SPINT2

B
Prostasin

STa
Uroguanylin

Guanylin

Cl
GC-C

Na+

H+

CFTR

NHE3

ENaC

Na+

PKA
cAMP
PDE3
cGMP
A

PKGII
B

FIGURE 1. (A) GC-C and (B) NHE3 mutations cause CSD; (C) SPINT2 deficiency causes syndromic CSD. GC-C is stimulated by binding
intraluminal guanylin, uroguanylin or Sta, and synthesizes intracellular cGMP. cGMP elevation increases Cl secretion by CFTR and inhibits Na
absorption by NHE3, via PKA and PKGII mediation, respectively. Dominant GC-C mutations increase intracellular cGMP without ligand binding.
Loss of NHE3 results in decreased Na uptake. In all situations, electrolytes and water remain intraluminal and diarrhea emerges. SPINT2 is part of a
reciprocal zymogen activation complex that results in the formation of active matriptase (ST14) and prostasin. Prostasin activates the
heterotrimeric epithelial sodium channel ENaC. ENaC is critical for sodium reabsorption in the distal colon. SPINT2 thus regulates prostasinmediated ENaC activation. SPINT2 deficiency is supposed to result in ENaC misregulation and likely in enterocyte tufting by excess serine protease
activity. CFTR cystic fibrosis transmembrane conductance regulator; cGMP cyclic guanosine monophosphate; CSD congenital sodium
diarrhea; ENaC epithelial Na channels; GC-C guanylate cyclase C; NHE3 Na/H antiporter 3; PKA protein kinase A; PKGII protein
kinase, cGMP-dependent, type II.

171

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.

Janecke et al

JPGN

Volume 63, Number 2, August 2016

TABLE 1. Major characteristic findings in CSD and relevant differential diagnoses of diarrheas not responding to dietary modifications
Classical
CSD

Syndromic CSD

CLD

MVID

Epithelial dysplasia
(tufting enteropathy)

Polyhydramnios
Abdominal distenstion
due to dilated fluidfilled loops of intestine
Extraintestinal
manifestations

Prominent
Prominent

Prominent
Prominent

Prominent
Prominent

Usually absent
Absent

Usually absent
Absent

Absent

Absent

Absent

Absent

Morphological findings

No specific
findings

Choanal and intestinal

atresias, hypertelorism,
corneal erosions, cleft
palate, polydactyly
Villus atrophy and
characteristic tufts of
extruding epithelium

No specific
findings

Villus atrophy and

loss of microvilli

Villus atrophy and

characteristic tufts
of extruding
epithelium

Mutated gene

SLC9A3
(NHE3)
GUCY2C
(GC-C)

SPINT2

SLC26A3
(DRA)

Microvillus inclusions
and secretory
granules on EM
MYO5B, STX3

EPCAM

CLD chloride diarrhea; CSD congenital sodium diarrhea; DRA downregulated in adenoma; GC-C guanylate cyclase C; MVID microvillus
inclusion disease; NHE3 Na/H antiporter 3.

nonsyndromic (classical) form of CSD. Syndromic CSD is caused

by loss-of-function mutations in SPINT2, encoding serine protease
inhibitor, Kunitz type 2. SPINT2 protein is involved in the
regulation of the epithelial sodium channel (ENaC), which is
critical for sodium reabsorption in the distal colon. The activity
of ENaC in the distal colon is dependent on its proteolytic activation
by a system consisting of 2 serine proteases, matriptase and
prostasin, and their inhibitor SPINT2.

CONGENITAL SODIUM DIARRHEA: CLINICAL

PRESENTATION
CSD (MIM no. 270420) is a rare disorder with about 50 cases
reported in the literature (811). CSD is clinically and genetically
very heterogeneous. A history of maternal polyhydramnios and
premature birth between 32 and 35 weeks of gestation with appropriate weight and length is, however, typically obtained for patients
with CSD. Patients typically present with prominent abdominal
distension after birth due to dilated fluid-filled loops of intestine,
demonstrating a secretory diarrhea that begins prenatally (Table 1).
A watery diarrhea is present after birth, independent of breastfeeding or formula initiation. The diarrhea can be described as
nonstopping and can be mistaken for urine. There are increased
bowel sounds at examination, and passing of meconium is never
reported. The infants become irritable, and eventually apathetic,
and develop moderate to severe dehydration. Rarely, there is
no watery diarrhea noticed either due to severe dehydration or
intestinal paralysis. This pseudo-obstruction is caused by dilated
fluid-filled loops of intestine, which may result in volvulus, and the
affected neonate typically undergoes abdominal surgery.
Low serum Na concentrations may be noted and indicate
CSD when this determination is made in serum samples taken
before initiation of fluid and electrolyte supplementation therapy.
Fecal Na concentrations are most often elevated in patients with
CSD, especially after the initiation of fluid and electrolyte supplementation therapy. Note that fecal Na concentrations can be normal

in CSD, when the depletion of body sodium has progressed for some
time. Urinary Na concentration, as a marker of body Na depletion,
is sometimes found to be low before and during inadequate fluid and
sodium supplementation. Fractional sodium excretion may be a better
marker of sodium status in these patients because it is independent
of urine flow (12,13). Other laboratory findings include metabolic
acidosis and alkaline fecal pH. Clinical findings in CSD and major
differential diagnoses are compiled in Table 1.
Classical CSD (also called the nonsyndromic phenotype)
resembles congenital chloride diarrhea (MIM no. 214700) (14), but
is differentiated by having high fecal loss of Na and metabolic
acidosis contrary to alkalosis. It is distinguished from enterocyte
differentiation and polarization disorders such as microvillus
inclusion disease (MIM no. 251850) (15,16) and nonsyndromic
tufting enteropathy (17) by histopathology.
Notably, IBD of both early and adolescent onset was
diagnosed in a subset of patients with the nonsyndromic form of
CSD. This observation adds to the hypothesis that changes in
electrolyte homeostasis may represent a primary contribution to
the development of IBD (see below). Intestinal anatomy, histology,
and transit functions were normal in the other patients with
nonsyndromic CSD.
After birth, all of the patients with CSD require total parenteral nutrition for treatment of dehydration for at least several
months. Sodium supplementation has been provided to all patients
and is needed in the treatment of severe dehydration and to maintain
normal body growth by preventing total body sodium depletion.

CONGENITAL SODIUM DIARRHEA: ETIOLOGY

AND PATHOGENESIS
Intestinal Sodium Absorption

Several interconnected mechanisms are responsible for Na

and water absorption along the gastrointestinal tract. Postprandial
Na absorption in the small intestine is driven by Na-dependent

172

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.

JPGN

Volume 63, Number 2, August 2016

glucose and amino acid symporters, whereas 2 other key

nonnutrient-dependent mechanisms in the small and large
intestines involve electroneutral apical Na/H exchange (NHE)
and ENaC. NHE, mediated primarily by the apical NHE3 isoform,
is responsible for NaCl and bicarbonate absorption through coupled
Na/H and Cl/HCO3 (or Cl/OH) exchange, the latter mechanism mediated by PAT1 (SLC26A6) and downregulated in adenoma (DRA; SLC26A3). Elelectrogenic Na absorption carried by
the epithelial Na channels (ENaC, comprised of a-,b-,g-ENaC
subunits) takes place in the surface epithelium and upper crypts
of the distal colon. All of these transport mechanisms require
a favorable electrochemical gradient maintained by the basolateral
Na/K-ATPase, Cl channels (apical CFTR and basolateral CLC2), and K channels (Kir 7.1). Water passively follows the ion
movement paracellularly, through the tight junctions, or transcellularly, through the cell membrane, to avoid the buildup of osmotic
gradients (18).

NHE3 Mutations in Classical CSD

Jejunal brush-border membrane transport studies suggested
a defect in sodium-proton exchange activity in 3 sporadic
patients diagnosed with classical CSD including 1 of the first
reported patients (6,19,20). Which NHE isoform was involved
and the reason for the compromised function, however, had
not been identified at that time. Recently, 1 study showed that
loss-of-function mutations in SLC9A3 cause classical CSD in
approximately 40% of families, and that the disease is autosomal
recessively inherited in these families (11). A whole-gene deletion
as well as truncating and missense mutations were identified in
9 patients with classical CSD in SLC9A3 (solute carrier family 9,
subfamily A, member 3; MIM no. 182307), the gene encoding
sodium-proton antiporter 3 (NHE3). A combination of wholeexome sequencing, chromosomal microarray analyses and direct
Sanger sequencing was applied to identify the cause of CSD in a
cohort of unrelated patients. The whole-gene deletion and the
truncating mutations are expected to abolish protein production
from these alleles, and reduced Na/H exchange activity of all
investigated SLC9A3 missense mutants was observed following in
vitro expression. Missense mutations showed either a reduced
transport function or a reduced surface expression of NHE3.
The explanation for the reduced surface expression could be either
abnormal trafficking to the membrane or reduced brush border
membrane stability.
The identified SLC9A3 mutations were all private and not
listed in public databases (dbSNP, the 1000 Genomes Project, the
National Heart, Lung, and Blood Institute Exome Sequencing
Project, the exome aggregation consortium server), with the exception of the observation of 1 particular SLC9A3 missense mutation
that occurred in 2 seemingly unrelated patients. Two unrelated
patients were heterozygous for 1 SLC9A3 mutation with no allelic
mutations identified after sequencing the complete coding region
and multiplex ligation-dependent probe amplification analysis of
SLC9A3 exons, most likely missing the 2 allelic mutations in deep
intronic or in promoter sequences.
The apical membrane Na/H exchanger NHE3 absorbs a
major part of intestinal Na, and it is important for systemic volume
and acid-base homeostasis. Its surface expression and activity are
mainly regulated by its trafficking to and from the brush border
(21). Increases in intracellular cGMP, Ca2, or cAMP inhibit
electroneutral NaCl absorption by NHE3 and can result in diarrhea
(21). NHE3 consists of 12 N-terminal transmembrane domains that
carry out Na/H exchange, followed by a long cytoplasmic
domain that interacts with the cytoskeleton and interacts with a
number of proteins that regulate NHE3 (21).

Congenital Sodium Diarrhea

No. of families
Nonsyndromic
CSD unsolved

Syndromic CSD
with SPINT2
mutations

n=8
n = 14
n=8

Nonsyndromic
CSD with NHE3
mutations

n=4

Nonsyndromic CSD with

GUCY2C mutations

FIGURE 2. Distribution of mutations identified in nonsyndromic and

syndromic CSD. Of 34 unrelated families with 43 patients with CSD,
SPINT2 mutations were identified in all families with syndromic CSD.
GUCY2C (GC-C) and SLC9A3 (NHE3) mutations account for 4 and 8
families with nonsyndromic CSD, respectively. CSD congenital
sodium diarrhea; GC-C guanylate cyclase C; NHE3 Na/H antiporter 3.

GUCY2C Mutations in Classical CSD

Dominant activating mutations in receptor GC-C (MIM no.
601330), encoded by the GUCY2C gene, were found to cause
classical CSD in 4 patients (10) corresponding to 20% of families
with this disorder (Fig. 2). These mutations were identified
by whole-exome sequencing of unrelated patients and all 4 heterozygous mutations had occurred de-novo in the patients. The mode of
transmission of GC-C mutations to the next generation would be
autosomal dominant.
GC-C is a transmembrane guanylate cyclase with the highest
expression in the intestinal tract. Uroguanylin, guanylin, and
heat-stable toxin produced by enterotoxigenic Escherichia coli
(ETEC) are luminal ligands that can stimulate intracellular cGMP
production upon binding, as part of one of several signaling
pathways that exist in enterocytes (22). Patient GC-C mutations
were shown to cause elevated basal and stimulated intracellular
cGMP levels (10), 1 effect of which is the inhibition of NHE3 via its
phosphorylation by cGMP kinase II (MIM no. 601591) as shown
in PS120 fibroblasts and Caco-2/Bbe cells (23,24), thereby providing an explanation for the secretory diarrhea by abrogated Na
absorption (22).
Enhanced activity of CSD-associated mutations was higher
compared with wild-type GC-C activity and to the activity seen with
1 activating GC-C mutation reported previously as the cause of a
dominantly inherited form of secretory diarrhea (familial diarrhea
syndrome) in 32 individuals of a Norwegian family (25). Familial
diarrhea syndrome is characterized by early onset chronic diarrhea,
associated to meteorism, abdominal pain and dysmotility, and with
IBD present in about 25% of patients.

Loss of NHE3 Function as a Result of SLC9A3

or GUCY2C Mutations
Classical CSD thus results from a loss of NHE3 function
resulting in abrogated Na absorption, enhanced fluid secretion,
and diarrhea. In 1 subset of patients, primary NHE3 deficiency is
caused by recessive SLC9A3 mutations resulting in absent or
nonfunctional protein. In a second subset of patients with CSD,
secondary NHE3 deficiency results from downregulation
by elevated intracellular cGMP levels, induced by constitutively
activating and hyperstimulating GUCY2C mutations (Fig. 1A, B).
Similar to NHE3-deficient patients, Nhe3/ mice exhibit diarrhea,
metabolic acidosis, low blood pressure, and reduced body fat and

173

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.

Janecke et al

JPGN

die rapidly when placed on a low Na diet (26,27). To date, CSD

phenotypes caused by activating GC-C mutations or NHE3 lossof-function mutations cannot be distinguished clinically because
of the small number of reported patients (10,11). Interestingly,
inactivating mutations of GUCY2C resulted in meconium ileus
in 3 Bedouin families (28,29) as a consequence of diminished fluid
and ion secretion that is normally maintained by the action of
guanylin and uroguanylin on GC-C.

IBD as a Consequence of Loss of NHE3 Function

Most interestingly, IBD developed in 6 of 36 patients with
dominant GC-C mutations, and in 2 of 9 patients with recessive
SLC9A3 mutations, implicating NHE3 in the pathogenesis of IBD,
at least in a subset of patients (10,11,25). Although it has been
shown that diarrhea associated with UC occurs primarily as the
result of inhibition of NHE-mediated Na absorption in UC (30),
the observations made in patients with CSD suggest that inhibition
of NHE3-mediated Na absorption may also predispose to IBD.
Evidence for dominant GUCY2C mutations predisposing to IBD
was inferred from the observation of increased expression of
GUCA2B, encoding uroguanylin, in colonic mucosa from patients
with diarrhea-predominant irritable bowel syndrome (31), and from
large-scale IBD genome-wide association studies that showed a
strong replicated association between UC and the SLC9A3 locus
(32,33). This association between loss of NHE3 function and IBD
is further supported by a number of rodent and human studies.
Nhe3/ mice develop spontaneous distal chronic colitis and have
increased susceptibility to dextran sulfate-induced mucosal injury
(34). An abnormal microbiome contributes to the Nhe3/ colitis
because no colitis was reported in a very clean facility (3436).
NHE3 activity is reduced in both UC and Crohn disease with
activity reduced in areas of inactive colitis as well as in the areas
of active inflammation (37,38). Reduced NHE3 activity was
reported both with reduced message and protein (37) and with
normal message, amount of protein, and surface expression (38).
Further support of the role of reduced sodium absorption in
the pathogenesis of IBD comes from studies that report changes in
the intestinal microbial composition due to alterations in electrolyte
transport and in mucosal pH such as demonstrated in Nhe3/ mice
(35), also in the event of an acute Clostridium difficile infection
resulting in decreased mucosal NHE3 expression and an altered
microbiota composition compared with healthy subjects (39).
The intestinal barrier is necessary to maintain a physical
separation between commensal bacteria and the mammalian
immune system, and a breakdown in this barrier through multiple
distinct pathways can directly promote chronic intestinal inflammation. Monogenic defects have been found to alter intestinal
immune homeostasis via several mechanisms and these can result
in the disruption of the epithelial barrier and the epithelial response.
NHE3 may be considered to play a critical role in the composition
of the gut microbiota and its deficiency may thus contribute to
dysbiosis observed in patients with IBD (35). In patients with IBD,
host genetic, environmental, and microbial influences converge and
result in a dysregulated mucosal immune response against the
commensal intestinal microbiota.

SYNDROMIC CSD: ETIOLOGY AND

PATHOGENESIS
About one third of the reported patients with CSD have a
syndromic form of CSD; characteristically, these patients show
uni- or bilateral, membraneous or bony choanal atresia at birth, and
present with episodes of painful corneal epithelial erosions throughout life. Less frequently, anal and jejunal atresias, hypertelorism,
cleft palate, and polydactyly were encountered. This form of CSD is

Volume 63, Number 2, August 2016

also referred to as a syndromic form of congenital tufting enteropathy or intestinal epithelial dysplasia, because it often shows
clustered enterocytes that form tufts with branching crypts on
histology (17,40).
In 2000, Muller et al identified 5 patients with CSD originating from 1 large consanguineous Austrian kindred and demonstrating autosomal recessive inheritance in that family. All known
sodium-proton exchanger genes including SLC9A3, the gene encoding NHE3, were excluded as sites of the mutation in that family
by linkage analyses (8). The positional candidate approach
subsequently identified a homozygous splice-site mutation in
SPINT2 (serine peptidase inhibitor, Kunitz type, 2; also referred
to as hepatocyte growth factor activator inhibitor 2 (HAI-2) and
placental bikunin; MIM no. 605124) in this family (9). A decreased
amount of mRNA and abnormal splicing of the residual mRNA
was demonstrated, which indicated that the mutation caused a lossof-function. Pathogenic biallelic SPINT2 mutations were identified
in 19 additional families (9,40,41), who were all clinically characterized as displaying the syndromic form of CSD. Identified
SPINT2 mutations were mainly of the nonsense and splice-site
type, and 1 was a point mutation of the first nucleotide of the start
codon (c.1A>T), further indicating SPINT2 deficiency in syndromic CSD. One point mutation (c.488A>G) predicting a missense
change from an invariantly conserved tyrosine within the catalytical
domain of the Kunitz type serine protease inhibitor to a cysteine
residue (p.Tyr163Cys) was particularly common among the
majority of seemingly unrelated families with syndromic CSD.
Haplotype analyses show that this mutation represents a founder
mutation (our unpublished data). This missense mutation conferred
reduced inhibitor activity on the prototype serine protease trypsin in
vitro. Both the functionally analyzed missense mutation and the
splice-site mutation may represent hypomorphic disease alleles, and
to date, all reported SPINT2 genotypes of patients may include at
least 1 hypomorphic mutation, and residual SPINT2 activity may be
required for normal human development as was shown for mouse
development. A complete Spint2 knockout was associated with
severe clefting of the embryonic ectoderm and embryonic death
in mice (42).
Spint2 mRNA is found along the entire gastrointestinal tract
in man (43) and mouse (44). SPINT2 targets the serine proteases
matriptase and prostasin, which form a reciprocal zymogen
activation complex that results in the formation of active matriptase
and prostasin. Matriptase (encoded by St14) mRNA has an expression pattern similar to Spint2, and prostasin (encoded by Prss8)
mRNA is present in the small and large intestines with a higher
abundance in the distal part (45).
Xenopus laevis oocytes were used as a heterologous expression system to functionally assess the activity of intestinal candidate
serine proteases and their inhibition by SPINT2 and its mutant
associated with CSD (p.Tyr163Cys). This assay used the epithelial
sodium channel ENaC as the readout protein for the proteolytic
activity of intestinal membrane-bound serine proteases. The
p.Tyr163Cys mutation induced a loss of inhibitory activity toward
prostasin and a partial loss on the inhibition of matriptase by
SPINT2 emphasizing matriptase and prostasin as the physiological
targets of SPINT2 (45). ENaC is critical for sodium reabsorption in
the distal colon, and the activity of ENaC, at least in the distal colon,
is dependent on its proteolytic activation by the matriptase-prostasin system. SPINT2 forms a complex with matriptase-prostasin
and thereby regulates prostasin-mediated ENaC activation (Fig. 1C)
(46). Loss of SPINT2 from intestinal epithelium of small and large
intestine causes a dramatic decrease in cell surface expression of
matriptase in intestinal epithelia (47). A model of SPINT2
deficiency-induced consumptive depletion of matriptase from
colonic epithelium predicts a deficiency of activated prostasin at the

174

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.

JPGN

Volume 63, Number 2, August 2016

apical membrane and is compatible with decreased sodium absorption by ENaC (47) and the development of CSD. The etiology of
syndromic features listed above occurring in patients with SPINT2
mutations has not been clarified.

CSD: SUMMARY AND FUTURE DIRECTIONS

About 40% of the patients with classical CSD did not harbor
either SLC9A3 or GUCY2C mutations indicating other genes are
responsible for the disease in these patients and point to considerable genetic locus heterogeneity in this disorder (Fig. 2).
Recent progress stimulates further search for mutations in
CSD genes as well as future research on drugs and peptides to
restore or enhance Na absorption of diarrheal disorders.
GUCY2C and SLC9A3 mutations represent monogenetic
variants that provide a high susceptibility to develop early and
late onset IBD and provide support for the importance of the
micromilieu at the brush border for disease pathogenesis.

REFERENCES
1. Canani BR, Terrin G, Cardillo G, et al. Congenital diarrheal disorders:
improved understanding of gene defects is leading to advances in
intestinal physiology and clinical management. J Pediatr Gastroenterol
Nutr 2010;50:3606.
2. Field M. Intestinal ion transport and the pathophysiology of diarrhea.
J Clin Invest 2003;111:93143.
3. Priyamvada S, Gomes R, Gill RK, et al. Mechanisms underlying
dysregulation of electrolyte absorption in inflammatory bowel disease-associated diarrhea. Inflamm Bowel Dis 2015;21:292635.
4. Ghishan FK, Kiela PR. Epithelial transport in inflammatory bowel
diseases. Inflamm Bowel Dis 2014;20:1099109.
5. Uhlig HH, Schwerd T, Koletzko S, et al. The diagnostic approach to
monogenic very early onset inflammatory bowel disease. Gastroenterology 2014;147:9901007e3.
6. Booth IW, Stange G, Murer H, et al. Defective jejunal brush-border
Na/H exchange: a cause of congenital secretory diarrhoea. Lancet
1985;1:10669.
7. Holmberg C, Perheentupa J. Congenital Na diarrhea: a new type of
secretory diarrhea. J Pediatr 1985;106:5661.
8. Muller T, Wijmenga C, Phillips AD, et al. Congenital sodium diarrhea is
an autosomal recessive disorder of sodium/proton exchange but unrelated
to known candidate genes. Gastroenterology 2000;119:150613.
9. Heinz-Erian P, Muller T, Krabichler B, et al. Mutations in SPINT2 cause
a syndromic form of congenital sodium diarrhea. Am J Hum Genet
2009;84:18896.
10. Muller T, Rasool I, Heinz-Erian P, et al. Congenital secretory diarrhea
caused by activating germline mutations in GUCY2C. Gut 2015
[Epub ahead of print].
11. Janecke AR, Heinz-Erian P, Yin J, et al. Reduced sodium-proton
exchanger NHE3 activity causes congenital sodium diarrhea. Hum
Mol Genet 2015;24:661422.
12. Heinz-Erian P, Akdar Z, Haerter B, et al. Decreased urinary sodium-tourinary creatinine ratio identifies sodium depletion in pediatric acute
gastroenteritis. Klin Padiatr 2016;228:248.
13. Coates AJ, Crofton PM, Marshall T. Evaluation of salt supplementation
in CF infants. J Cyst Fibros 2009;8:3825.
14. Hoglund P, Haila S, Socha J, et al. Mutations of the down-regulated in
adenoma (DRA) gene cause congenital chloride diarrhoea. Nat Genet
1996;14:3169.
15. Muller T, Hess MW, Schiefermeier N, et al. MYO5B mutations cause
microvillus inclusion disease and disrupt epithelial cell polarity. Nat
Genet 2008;40:11635.
16. Wiegerinck CL, Janecke AR, Schneeberger K, et al. Loss of syntaxin 3
causes variant microvillus inclusion disease. Gastroenterology
2014;147:658.
17. Sivagnanam M, Mueller JL, Lee H, et al. Identification of EpCAM as the
gene for congenital tufting enteropathy. Gastroenterology 2008;135:
42937.
18. Ghishan FK, Kiela PR. Small intestinal ion transport. Curr Opin
Gastroenterol 2012;28:1304.

Congenital Sodium Diarrhea

19. Keller KM, Wirth S, Baumann W, et al. Defective jejunal brush border
membrane sodium/proton exchange in association with lethal familial
protracted diarrhoea. Gut 1990;31:11568.
20. Fell JM, Miller MP, Finkel Y, et al. Congenital sodium diarrhea with a
partial defect in jejunal brush border membrane sodium transport,
normal rectal transport, and resolving diarrhea. J Pediatr Gastroenterol
Nutr 1992;15:1126.
21. Donowitz M, Mohan S, Zhu CX, et al. NHE3 regulatory complexes.
J Exp Biol 2009;212:163846.
22. Arshad N, Visweswariah SS. The multiple and enigmatic roles of
guanylyl cyclase C in intestinal homeostasis. FEBS Lett 2012;586:
283540.
23. Cha B, Kim JH, Hut H, et al. cGMP inhibition of Na/H antiporter 3
(NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein. J Biol Chem 2005;280:
16642 50.
24. Chen T, Kocinsky HS, Cha B, et al. Cyclic GMP Kinase II (cGKII)
inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at
three required sites: identification of a multifunctional phosphorylation
site. J Biol Chem 2015;290:195265.
25. Fiskerstrand T, Arshad N, Haukanes BI, et al. Familial diarrhea syndrome caused by an activating GUCY2C mutation. N Engl J Med
2012;366:158695.
26. Schultheis PJ, Clarke LL, Meneton P, et al. Renal and intestinal
absorptive defects in mice lacking the NHE3 Na/H exchanger.
Nat Genet 1998;19:2825.
27. Noonan WT, Woo AL, Nieman ML, et al. Blood pressure maintenance
in NHE3-deficient mice with transgenic expression of NHE3 in small
intestine. Am J Physiol Regul Integr Comp Physiol 2005;288:R685
91.
28. Romi H, Cohen I, Landau D, et al. Meconium ileus caused by mutations
in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C. Am J
Hum Genet 2012;90:8939.
29. Smith A, Bulman DE, Goldsmith C, et al. Meconium ileus in a Lebanese
family secondary to mutations in the GUCY2C gene. Eur J Hum Genet
2015;23:9902.
30. Rajendran VM, Nanda Kumar NS, Tse CM, et al. Na-H exchanger
isoform-2 (NHE2) mediates butyrate-dependent Na absorption in
dextran sulfate sodium (DSS)-induced colitis. J Biol Chem 2015;290:
2548796.
31. Camilleri M, Carlson P, Acosta A, et al. RNA sequencing shows
transcriptomic changes in rectosigmoid mucosa in patients with irritable
bowel syndrome-diarrhea: a pilot case-control study. Am J Physiol
Gastrointest Liver Physiol 2014;306:G108998.
32. Anderson CA, Boucher G, Lees CW, et al. Meta-analysis identifies 29
additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 2011;43:24652.
33. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have
shaped the genetic architecture of inflammatory bowel disease. Nature
2012;491:11924.
34. Laubitz D, Larmonier CB, Bai A, et al. Colonic gene expression profile
in NHE3-deficient mice: evidence for spontaneous distal colitis. Am J
Physiol Gastrointest Liver Physiol 2008;295:G6377.
35. Larmonier CB, Laubitz D, Hill FM, et al. Reduced colonic microbial
diversity is associated with colitis in NHE3-deficient mice. Am J Physiol
Gastrointest Liver Physiol 2013;305:G66777.
36. Kiela PR, Laubitz D, Larmonier CB, et al. Changes in mucosal
homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury. Gastroenterology 2009;137:
96575.
37. Sullivan S, Alex P, Dassopoulos T, et al. Downregulation of sodium
transporters and NHERF proteins in IBD patients and mouse colitis
models: potential contributors to IBD-associated diarrhea. Inflamm
Bowel Dis 2009;15:26174.
38. Yeruva S, Farkas K, Hubricht J, et al. Preserved Na()/H() exchanger
isoform 3 expression and localization, but decreased NHE3 function
indicate regulatory sodium transport defect in ulcerative colitis. Inflamm
Bowel Dis 2010;16:114961.
39. Engevik MA, Engevik KA, Yacyshyn MB, et al. Human Clostridium
difficile infection: inhibition of NHE3 and microbiota profile. Am J
Physiol Gastrointest Liver Physiol 2014;308:G497509.

175

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.

Janecke et al

JPGN

40. Salomon J, Goulet O, Canioni D, et al. Genetic characterization of

congenital tufting enteropathy: epcam associated phenotype and
involvement of SPINT2 in the syndromic form. Hum Genet
2013;133:299310.
41. Sivagnanam M, Janecke AR, Muller T, et al. Case of syndromic tufting
enteropathy harbors SPINT2 mutation seen in congenital sodium diarrhea. Clin Dysmorphol 2010;19:48.
42. Mitchell KJ, Pinson KI, Kelly OG, et al. Functional analysis of secreted
and transmembrane proteins critical to mouse development. Nat Genet
2001;28:2419.
43. Kawaguchi T, Qin L, Shimomura T, et al. Purification and
cloning of hepatocyte growth factor activator inhibitor type 2, a
Kunitz-type serine protease inhibitor. J Biol Chem 1997;272:27558
64.

Volume 63, Number 2, August 2016

44. Itoh H, Kataoka H, Hamasuna R, et al. Hepatocyte growth factor

activator inhibitor type 2 lacking the first Kunitz-type serine proteinase
inhibitor domain is a predominant product in mouse but not in human.
Biochem Biophys Res Commun 1999;255:7408.
45. Faller N, Gautschi I, Schild L. Functional analysis of a missense
mutation in the serine protease inhibitor SPINT2 associated with
congenital sodium diarrhea. PLoS One 2014;9:e94267.
46. Szabo R, Uzzun Sales K, Kosa P, et al. Reduced prostasin (CAP1/
PRSS8) activity eliminates HAI-1 and HAI-2 deficiency-associated
developmental defects by preventing matriptase activation. PLoS Genet
2012;8:e1002937.
47. Friis S, Sales KU, Schafer JM, et al. The protease inhibitor HAI-2,
but not HAI-1, regulates matriptase activation and shedding through
prostasin. J Biol Chem 2014;289:2231932.

176

www.jpgn.org

Copyright ESPGHAL and NASPGHAN. All rights reserved.