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A Variant of the SLC10A2 Gene Encoding the Apical
Sodium-Dependent Bile Acid Transporter Is a Risk Factor
for Gallstone Disease
Olga Renner1, Simone Harsch1, Elke Schaeffeler1, Stefan Winter1, Matthias Schwab1,5, Marcin Krawczyk2,
Jonas Rosendahl3, Henning Wittenburg3, Frank Lammert2, Eduard F. Stange4*
1Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany, 2Department of Medicine II, Saarland University
Hospital, Homburg, Germany, 3Department of Gastroenterology and Hepatology, University of Leipzig, Leipzig, Germany, 4Department of Internal Medicine I, Robert
Bosch Hospital, Stuttgart, Germany, 5Department Clinical Pharmacology, University Hospital Tuebingen, Tuebingen, Germany
Abstract
Background: Cholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated.
As one of these mechanisms, a decreased expression of the ileal apical sodium-dependent bile acid transporter gene
SLC10A2 in gallstone carriers was described previously. In this study the SLC10A2 gene was investigated to identify novel
genetic variants and their association with gallstone formation.
Methodology/Principal Findings: Study subjects were selected with the presence or absence of gallstones confirmed by
ultrasound and medical history. Genomic DNA was obtained from blood leukocytes. Sequence analysis was performed of all
six exonic and flanking regions as well as of 2,400 base pairs of the SLC10A2 promoter in a cohort of gallstone carriers and
control subjects from Stuttgart, Germany. Genotype frequencies of newly identified genetic variants (n = 6) and known
single nucleotide polymorphisms (n = 24) were established using MALDI-TOF mass spectrometry. Six new genetic variants
were found within the SLC10A2 gene. Although none of the variants was linked to gallstone disease in the Stuttgart cohort
overall, the minor allele of SNP rs9514089 was more prevalent in male non-obese gallstone carriers (p= 0.06680, OR = 11.00).
In a separate population from Aachen, Germany, the occurrence of rs9514089 was two-fold higher in gallstone patients
(22%) than in corresponding controls (11%) (p= 0.00995, OR = 2.19). In the pooled Aachen/Stuttgart cohort rs9514089 was
highly significantly linked to cholelithiasis (p= 0.00767, OR = 2.04). A more frequent occurrence was observed for male
gallstone carriers (22%) compared to controls (9%) (p= 0.01017, OR = 2.99), for the total normal weight group (p= 0.00754,
OR = 2.90), and for male non-obese gallstone patients (p= 0.01410, OR= 6.85). Moreover, for the minor allele of rs9514089 an
association with low plasma cholesterol levels was found especially in gallstone carriers (p= 0.05).
Conclusions/Significance: We have identified SLC10A2 as a novel susceptibility gene for cholelithiasis in humans.
Comprehensive statistical analysis provides strong evidence that rs9514089 is a genetic determinant especially in male non-
obese gallstone carriers. The minor allele of rs9514089 is related to differences in plasma cholesterol levels among the
subjects.
Citation: Renner O, Harsch S, Schaeffeler E, Winter S, Schwab M, et al. (2009) A Variant of the SLC10A2 Gene Encoding the Apical Sodium-Dependent Bile Acid
Transporter Is a Risk Factor for Gallstone Disease. PLoS ONE 4(10): e7321. doi:10.1371/journal.pone.0007321
Editor: Iris Schrijver, Stanford University, United States of America
Received July 30, 2009; Accepted September 11, 2009; Published October 13, 2009
Copyright: � 2009 Renner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Robert Bosch Foundation (P4-1/03). Henning Wittenburg was supported by the Deutsche Forschungsgemeinschaft
(WI1905 3-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: eduard.stange@rbk.de
Introduction
Cholelithiasis is the most common gastrointestinal disease with a
prevalence of 15% in European adults [1–4]. Family and twin
studies in humans as well as inbred mice have clearly
demonstrated that a complex genetic basis determines the
individual risk to develop cholesterol gallstones in addition to
factors such as obesity and pregnancy [5–7].
Several chromosomal regions exhibit linkage to gallstone
disease. Complementary to the murine model [8,9], the detailed
analysis of human gallstone susceptibility genes in Mexican
families provided suggestive evidence of linkage on chromosome
1p [6]. Rosmorduc et al. [10] showed that rare loss of function
mutations in the ABCB4 gene encoding the hepatocanalicular
transporter required for biliary phospatidylcholine secretion may
lead to gallstone formation in certain patients. These findings in
humans are consistent with the spontaneous occurrence of
gallstones in Abcb4 knockout mice [11]. An increased gallstone
risk in humans was demonstrated for the ABCG8 19H allele by a
genomewide association scan with 500,000 SNPs in Caucasian
individuals [12]. These findings were also confirmed in the
combined linkage and association study by Gru¨nhage and co-
workers [13]. Additionally, the results are consistent with
supporting evidence from animal model that the ABCG5/G8
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transporter is a lithogenic risk factor [9]. Another recent
association study observed an Arg64-variant of the b3-adrenergic
receptor (ADRB3) more frequently in gallstone patients [14].
Moreover, variations of the gene encoding the nuclear bile salt
receptor FXR in an inbred mouse model and in three ethnically
distinct populations were described as risk factors for the
development of cholelithiasis [15].
In the current study we follow up on our previous work that
investigated a role of the apical sodium-dependent bile acid
transporter (ASBT, gene name SLC10A2) in gallstone formation.
We have shown that in non-obese gallstone patients, ASBT
expression is diminished, and that it is associated with low cytosolic
ileal lipid binding protein (ILBP) and basolateral organic solute
bile acid exporter (OSTa-OSTb) expression [16,17]. In line with
differences in cholesterol metabolism in lean and obese subjects
[18], the reduced expression of the transporter was weight-specific
and observed in normal weight individuals only [16]. Further-
more, subclinical inflammation was excluded as the molecular
pathomechanism of reduced ASBT expression in gallstone patients
[19]. The role of this transporter has been suggested to be the
major determinant of bile acid pool size [20]. The size of the
circulating pool of bile acids is an essential factor influencing
cholesterol homeostasis [21]. Moreover, an impaired intestinal bile
acid absorption, resulting in fecal bile acid loss and a decreased
bile acid pool size, was identified to represent the primary defect in
some patients with cholesterol gallstones [22]. Accordingly, a
reduction of the bile acid pool size and the cholesterol
supersaturation of gallbladder bile were identified as pivotal
pathophysiological defects that promote cholesterol gallstone
formation [23–27].
Based on these findings we hypothesized that mutations of the
SLC10A2 gene might result in gallstone formation by impairing the
bile acid pool size. The first human mutation rendering SLC10A2
dysfunctional was identified in a patient with Crohn’s disease [28].
Moreover, it was shown that inherited mutations within the
human SLC10A2 gene are responsible for the abolished bile acid
transport in a family with congenital primary bile acid malab-
sorption (PBAM) [29]. In addition, the SLC10A2 region was
suggested to be relevant in bilirubin metabolism in a genome wide
screening study [30]. Also, the frame-shift mutation 646insG in
SLC10A2, which was associated with the abolished bile acid
transport activity, was detected in a patient with familial
hypertriglyceridemia (FHTG) [31]. In addition to genetic
investigations, impaired intestinal bile acid absorption was
observed in patients with hypertriglyceridemia as a result of
diminished expression of apical bile acid transporter protein ASBT
[32]. Recently, we demonstrated a reduced expression of ASBT
that was linked to a haplotype block of SLC10A2 containing a total
of ten variants [33].
The aim of the present study was to systematically analyze the
SLC10A2 gene for genetic variants and to investigate whether the
variants are associated with the development of gallstones. We
established the genotype frequencies of a total of 30 sequence
variations within the SLC10A2 gene; six of these variants were
newly identified. Most importantly, an association of the SNP
rs9514089 with gallstone prevalence was observed in two distinct
human Caucasian populations suggesting that a genetic variant of
SLC10A2 confers an increased risk of gallstone formation.
Results
Sequence Analysis of SLC10A2
To identify novel genetic variants associated with cholelithiasis,
the SLC10A2 gene was screened by sequencing, using a total of 93
samples from the Stuttgart population. The mutational analysis of
60 gallstone carriers and 33 control individuals resulted in the
identification of six novel distinct variants in the untranslated
region (59-UTR) and in the coding regions of SLC10A2. These
genetic variants (two in the 59 UTR region, two in exon 1 and two
in exon 5) were found in female and in male gallstone carriers as
well as in one male control. All individuals carried the newly
identified variants in the heterozygous state. Detailed information
of the variants is listed in Table 1.
Genetic Variations of SLC10A2 in the Stuttgart Cohort
To investigate whether any of the variants were associated with
gallstones, we compared the genotype frequencies of all single
nucleotide polymorphisms (SNPs) in gallstone carriers and control
subjects in the Stuttgart cohort. In addition to the newly identified
genetic variants, based on frequency, 24 previously reported SNPs
of SLC10A2 were selected from the NCBI data bases (http://www.
ncbi.nlm.nih.gov/sites/entrez) to explore their association with
cholelithiasis. The main characteristics of all 30 SNPs examined in
the Stuttgart population are listed in Table 1. The study
population was subdivided into weight-, gender- and disease-
specific subgroups and genotype frequencies, odds ratios (ORs)
and confidence intervals (CI) for each subgroup were calculated to
obtain genotype-associated disease risk estimations. Moreover, the
dominant and recessive models of inheritance were tested by
analyzing wild type homozygous individuals (MM) versus all
carriers of the minor variant (mm) or by comparison of wild type
homozygous and heterozygous (Mm) individuals to the subjects
with the minor allele ((MM+Mm),.mm). Generally, in a total of
127 genotyped individuals the frequencies of most common SNPs
showed neither significant gender-specific nor weight-specific
differences. The overall minor allele frequencies (MAF) of the
SLC10A2 SNPs ranged from 0 to 0.366. The majority of these
SNPs displayed MAF lower than 10% in the population analyzed.
The polymorphisms of the promoter and untranslated regions
(UTR) (rs3759501, rs3759502, rs3759503, rs3759504, rs7990819,
rs16961281, rs41281682) were detected in gallstone carriers and
controls, and their distributions did not exhibit any significant
differences between both groups. The novel genetic variants
rs71653645/c.-548C.T and rs71653646/c.-332C.T, that were
identified in two separate gallstone carriers, were not found in any
additional subjects. The synonymous variant rs71640246/p.N52
in exon 1 was identified in two male gallstone carriers only.
Moreover, the female gallstone carrier with the nucleotide change
G-to-A leading to the premature stop codon at position 66
(rs71640247/p.W66X) represented a single case of individual
sequence alteration. Notably, in our cohort no variant alleles were
found for the following four sequence variants that were described
in the database: intron 1: rs1329516, exon 2: rs157381/Pro142,
exon 5: L243P, rs72547505/T262M. In addition, there were no
statistical differences between gallstone carriers and controls for
the various coding region variants: rs41281680/p.A43,
rs55971546/A98I rs60380298/V159I, rs41281678/L169,
rs188096/A171S. The mutation P290S (rs56398830) rendering
SLC10A2 dysfunctional, which was identified initially in a patient
diagnosed with Crohn’s disease [28], was present at higher
frequency in the control population (5.2%) compared to gallstone
carriers (2.6%, p=0.3534, OR=0.6393). Moreover, this SNP
occurred more frequently in overweight individuals (6.9%)
compared to non-obese subjects (1.7%) (p=0.0638, OR=3.082).
Furthermore, the newly identified sequence alteration p.F296L
was only present in the control group with a frequency of 1.4%.
On the other hand, the distribution of the variant p.F304L was
similar between the case (1.8%) and control (1.4%) groups.
SLC10A2 and Cholelithiasis
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Interestingly, for one of the sequence variants, rs9514089, we
observed a trend towards an association with cholelithiasis in this
Stuttgart population. However, this association was observed only
in male gallstone carriers with normal weight (20%) compared to
male normal weight controls (0%) (data shown as supplemental
Table S1).
SLC10A2 Polymorphism rs9514089 in the Aachen Cohort
To confirm the association of the rs9514089 polymorphism to
gallstones, a larger additional population from Aachen, comprising
matched pairs of gallstone carriers and controls, was analyzed.
Interestingly, the Aachen cohort expressed a clearly higher SNP
frequency of the rs9514089 variant in gallstone carriers of the total
population as well as in every distinct subgroup compared with
control subjects. In the total population, this association was
significant (p=0.00995, OR=2.19). In this population, the G
allele was more frequent in gallstone carriers (22%) compared to
controls (11%). Moreover, the significant association of the minor
allele with gallstone prevalence was also observed in male gallstone
carriers (p=0.03319, OR=2.79) as well as in normal weight
gallstone patients (p=0.03517, OR=2.59) (data shown as
supplemental Table S2). The p-values and risk estimations for
the Aachen cohort and the pooled population are listed for the
recessive model.
Table 1. Main characteristics of all 30 SNPs examined in the Stuttgart population.
Variant dbSNP Region cDNA a/Genotype Protein b MAF c HWE
rs/ss number (p-value) d
1 rs3759501 upstream of 59-UTR T.A 0.343 1.0
2 rs3759502 upstream of 59-UTR C.T 0.337 1.0
3 rs3759503 upstream of 59-UTR C.T 0.346 1.0
4 rs3759504 upstream of 59-UTR T.C 0.323 0.6092
5 rs7990819 e upstream of 59-UTR T.C 0.044 1.0
6 rs66994812 e upstream of 59-UTR C.T 0.037 1.0
7 rs66924010 e upstream of 59-UTR T.C 0.043 1.0
8 rs71653645 f 59-UTR c.-548C.T 0.0040 1.0
9 rs71653646 f 59-UTR c.-332C.T 0.0040 1.0
10 rs16961281 59-UTR c.-225C.T 0.067 0.8779
11 rs41281682 e 59-UTR c.-17C.G 0.043 1.0
12 rs41281680 e exon 1 c.129C.T p.A43 0.043 1.0
13 rs71640246 f exon 1 c.156C.T p.N52 0.0080 1.0
14 rs71640247 f exon 1 c.197G.A p.W66X 0.0040 1.0
15 rs55971546 exon 1 c.292G.A p.V98I 0.043 1.0
16 ss99307920 e, g exon 1/intron 1 boundary c.377+12T.C 0.043 1.0
17 rs1329516 exon 1/intron 1 boundary c.377+109G.T 0.0 1.0
18 rs9514089 intron 1/exon 2 boundary c.378-105A.G 0.366 1.0
19 ss99307921 e, g intron 1/exon 2 boundary c.378-97A.G 0.037 1.0
20 rs157381 exon 2 c.426G.A p.P142 0.0 1.0
21 rs60380298 e exon 2 c.475G.A p.V159I 0.043 1.0
22 rs67736127 e intron 2/exon 3 boundary c.497-74C.A 0.043 1.0
23 rs66842575 e intron 2/exon 3 boundary c.497-40A.T 0.043 1.0
24 rs41281678 exon 3 c.505C.T p.L169 0.031 0.2148
25 rs188096 exon 3 c.511G.T p.A171S 0.142 0.9408
26 - exon 4 c.728T.C p.L243P 0.0 1.0
27 rs72547505 exon 5 c.785C.T p.T262M 0.0 1.0
28 rs56398830 exon 5 c.868C.T p.P290S 0.02 1.0
29 rs71640248 f exon 5 c.886T.C p.F296L 0.0040 1.0
30 rs61966074 f exon 5 c.910T.C p.F304L 0.0080 1.0
SNPs are numbered 1–30 reflecting their sequential order on the physical map of SLC10A2 sequence.
59-UTR = untranslated region.
aNumbering according to transcript NM_00452 including the transition initiation codon.
bNumbering according to NP_00443.1 starting at translation initiation codon.
cMinor allele frequencies (MAF) are indicated in bold.
dHardy-Weinberg equilibrium.
ePolymorphism was previously described to be associated with reduced SLC10A2-expression [32].
fNewly identified genetic variant.
grs-number is available with the next dbSNP Build, B131 (planned for November 2009).
doi:10.1371/journal.pone.0007321.t001
SLC10A2 and Cholelithiasis
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SLC10A2 Polymorphism rs9514089 in the Pooled
Population
To increase the power of our analysis, the populations of
Stuttgart and Aachen were pooled. For this analysis, the
distribution of the SNP rs9514089 was determined in a total of
255 controls and 240 gallstone carriers (Table 2). In the entire
pooled cohort, we observed an association of rs9514089 with
gallstone carrier status (p=0.00767, OR=2.04). The increased
frequency of the homozygous GG genotype in the combined
analysis of all samples from both independent German cohorts was
observed for all gallstone patients (GG frequency 20%) in
comparison to the control group (GG frequency 11%). In
accordance with our results in the individual single cohorts, the
pooled population also demonstrated a more frequent occurrence
(p=0.01017, OR=2.99) of the genetic variant in male gallstone
carriers (22%) compared to male controls (9%). Furthermore, the
analysis of the different gallstone carrier subgroups revealed a
significant association of the minor variant with gallstone
susceptibility in the normal weight group (p=0.00754,
OR=2.90). In contrast to female normal weight gallstone carriers,
an increased SNP frequency was observed in the male non-obese
gallstone patients in comparison to the male non-obese control
group (p=0.01410, OR=6.85).
rs9514089 GG Genotype Frequencies in SLC10A2 and
Plasma Lipid Levels
The distributions of rs9514089 GG genotypes in both Stuttgart
and Aachen populations as well as in the pooled group were
analyzed with respect to age, BMI, plasma cholesterol and
triglyceride levels (Table S3). For the pooled group, a correction
term for the study centre was incorporated in the models. The
various genotypes were not associated with age, BMI or
triglycerides. However, patients carrying the G allele of
rs9514089 displayed lower total cholesterol levels compared to
patients with the A allele. In the association analysis, a significant
association of rs9514089 and total cholesterol levels was observed
in the Aachen cohort (p=0.04, data not shown). The Stuttgart
cohort demonstrated a non-significant trend towards lower
cholesterol plasma levels in gallstone patients with GG genotypes
than in patients with the major allele. In the pooled population, we
confirmed the association of the GG genotypes of rs9514089 in
gallstone carriers with low cholesterol levels. After correction by
study centre, this association was only observed to display a
borderline effect (p=0.05).
Discussion
In this candidate gene study the SCL10A2 gene was systemat-
ically sequenced, and a first cohort from Stuttgart was genotyped
for the association of a total of 30 SNPs with cholelithiasis. Among
the analyzed variants, the SNP rs9514089 was significantly more
prevalent in male non-obese gallstone carriers. Subsequently, this
association was verified in an additional larger replication cohort.
In addition this SNP was associated with lower plasma cholesterol
values.
This is the first positive genetic association study of a factor
involved in bile acid transport. Previous association studies have
reported a linkage of gallstone formation and point mutations in
the ABCB4 gene encoding the phospholipid transporter MDR3
[10,34]. This is consistent with the spontaneous occurrence of
cholelithiasis in Abcb4 knockout mice [11]. A genomewide
association scan has linked the coding variant D19H of the
ABCG8, the hepatocanalicular cholesterol transporter, to this
disease with an OR of 2.2 [12]. This association was confirmed by
Gru¨nhage et al. in a nonparametric linkage analysis of affected sib
pairs with the 19H allele compared to gallstone-free controls
(OR=3.0) [13]. The study of Klass et al. identified a gallstone-
associated polymorphism in the hepatic b3-adrenergic receptor
(ADRB3) suggested to affect gallbladder motility [14]. Kovacs et
al. described a linkage of variations of the FXR gene and gallstone
susceptibility in humans [15]. Notably, the linkage to cholelithiasis
was gender-specific (males: OR=3.09; females: OR=0.85). No
association of this haplotype with gallstone prevalence could be
found in a German cohort (OR=0.83) and, in a Chilean female
population a paradoxical trend towards a protective effect of
NR1H4_1 was observed (p=0.08, OR=0.69).
So far no gallstone-associated SNP has been described for bile
acid transporters in the intestine. For the SLC10A2 gene only a few
dysfunctional mutations were identified in single patients diag-
nosed with Crohn’s disease, hypertriglyceridemia and/or in
patients with different forms of bile acid malabsorption
[28,31,35,36]. However, these loss-of-function-mutations could
not be associated with specific diseases in general. In the current
Table 2. Prevalence of gallstones and rs9514089 polymorphism in the pooled Stuttgart and Aachen cohorts.
Subgroup Controls n (%) Gallstone carriers n (%) AA a, .gg c (AA a+Ag b), .gg c
Genotype A/A A/G G/G A/A A/G G/G p-value OR (95% CI) p-value OR (95% CI)
Total 93 (37) 129 (52) 27 (11) 91 (38) 98 (42) 47 (20) 0.05303 1.78 (0.99–3.23) 0.00767 2.04 (1.19–3.55)
Males 47 (40) 59 (51) 10 (9) 40 (42) 34 (36) 21 (22) 0.05864 2.45 (0.97–6.55) 0.01017 2.99 (1.26–7.56)
Females 50 (37) 70 (51) 17 (12) 51 (36) 64 (45) 26 (19) 0.36153 1.50 (0.69–3.33) 0.18639 1.59 (0.78–3.31)
Normal weight e 49 (38) 69 (53) 11 (9) 40 (39) 41 (40) 22 (21) 0.04184 2.43 (0.99–6.27) 0.00754 2.90 (1.27–7.01)
Males 22 (43) 27 (53) 2 (4) 17 (47) 11 (31) 8 (22) 0.07375 5.01 (0.85–54.42) 0.01410 6.85 (1.25–70.70)
Females 27 (35) 42 (54) 9 (11) 23 (34) 30 (45) 14 (21) 0.31514 1.81 (0.60–5.71) 0.17103 2.02 (0.75–5.71)
Overweight f 44 (37) 60 (50) 16 (13) 51 (38) 57 (43) 25 (19) 0.45777 1.35 (0.60–3.07) 0.30547 1.50 (0.72–3.20)
Males 25 (39) 32 (49) 8 (12) 23 (39) 23 (39) 13 (22) 0.30883 1.75 (0.55–5.85) 0.16034 2.00 (0.70–6.09)
Females 19 (35) 28 (51) 8 (14) 28 (38) 34 (46) 12 (16) 1.00000 1.02 (0.31–3.46) 1.00000 1.14 (0.39–3.48)
p,0.05 was regarded as statistically significant, odds ratio (OR) and 95% confidence interval (CI).
aAA =major allele.
bAg= heterozygote allele.
cgg =minor allele, the subjects were divided into subgroups with body mass index (BMI)#25 kg/m2 = e normal weight and BMI.25 kg/m2= f overweight.
doi:10.1371/journal.pone.0007321.t002
SLC10A2 and Cholelithiasis
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study, we identified the polymorphism rs9514089 of SLC10A2 as
the first intestinal bile acid related candidate for the development
of gallstones. In the pooled cohort from Stuttgart and Aachen we
found a significant association of the polymorphism rs9514089
with gallstone disease (p=0.00767, OR=2.04). In addition, we
analyzed the effect of the SNP rs9514089 in different gender- and
weight-specific groups. Herein, this sequence variant was signif-
icantly associated with cholelithiasis in the total male and in the
total normal weight subgroups. However, the most significant
linkage was found for male normal weight gallstone carriers
(p=0.01410, OR=6.85), indicating that the occurrence of the
rs9514089 polymorphism is gender- and weight-specific.
Although our study demonstrates, that the genetic gallstone
disease risk is attributable to the GG variant in the SLC10A2 gene,
it remains unclear how this genetic variant affects the function of
the ASBT transporter and how these functional differences lead to
gallstone disease. Several scenarios may possibly explain the
different prevalence of this polymorphism in gallstone carriers and
controls: First, in carriers of the G allele, the SNP rs9514089 could
be the relevant polymorphism, causing a decreased transcription
of the gene that could lead to a diminished epithelial bile acid
uptake. Second, it may be hypothesized that the genomic structure
of the SLC10A2 gene with the G allele is the major determinant of
the diminished ASBT expression though post-transcriptional
mechanisms could influence protein expression. Third, the GG
variant could alter the binding of several transcription factors
promoting gallstone formation. As mentioned above, we have
previously shown that expression of this transporter is diminished
in the ileum of normal weight gallstone patients [16]. Unfortu-
nately, our cohort of patients from which we were able to obtain
ileal biopsies is too small for genetic association studies.
To assess whether the association results for the GG variant of
the SLC10A2 gene were affected by confounding risk factors for
gallstones, we performed a multivariate analysis that included the
rs9514089 variant and lithogenic risk factors such as age, gender,
BMI and plasma cholesterol levels. The only significant association
relates to serum cholesterol in the Aachen population, where lower
cholesterol levels, were found in gallstone carriers of the GG
genotype of the rs9514089 variant compared with gallstone
carriers with the major allele. Although this trend was confirmed
in the Stuttgart cohort, in the pooled population the association of
the GG genotype in gallstone carriers with low cholesterol levels
was only borderline (p=0.05). Such an association with a lipid
transporter is in line with prior studies where the D19H and
T400K variants of ABCG8 and the ABCG5 Q604E polymorphism
were associated with altered plasma cholesterol levels [37–39]. On
the other hand, Acalovschi et al. found no link between ABCB4
and ABCB11 polymorphisms, lithogenic dyslipidemia, and gall-
stone risk [40]. Furthermore, in previous studies low total
cholesterol levels have been linked to the development of gallstones
[41] but in our cohorts this was not the case. Therefore, and also
because of the lack of significance in the total cohort, there is little
evidence for a confounder effect of serum cholesterol in the present
investigation. There was also no relevant association between the
rs9514089 variant and triglyceride levels, although patients with
hypertriglyceridemia have been demonstrated to exhibit dimin-
ished expression of apical sodium-dependent bile acid transporter
protein [32]. To assess the association of rs9514089 polymorphism
to plasma lipids further, future investigations in additional
populations are required.
In conclusion, we identified SLC10A2 as a novel intestinal
common susceptibility factor for gallstones in humans. The results
of this study may prompt investigations which will address more
comprehensive genotype-phenotype association. In the future,
additional genetic risk factors are likely to be identified, which we
envisage to provide novel means for the risk assessment and
possibly prevention of gallstones.
Materials and Methods
Ethics statement
The local ethics comittees (for the study population from
Aachen: ethics committees from the Departement of Medicine
University of Aachen and Departement of Medicine University of
Leipzig as well as for the study population from Stuttgart: ethics
committee of the University Hospital of Tuebingen and University
Tuebingen) approved these studies and all subjects gave written
informed consent prior to participation.
NCBI data statement
All new data have been deposited in GenBank (NCBI data bank
http://www.ncbi.nlm.nih.gov/BankIt), rs-numbers are already
available.
Study populations and clinical characteristics
The study population from Stuttgart (Germany), comprises 127
individuals, of which 71 are healthy individuals and 56 are
gallstone carriers. The recruitment criteria for the subjects
included in this study were described previously [16,17,33]. The
clinical characteristics of the study participants, including age,
BMI, triglyceride and cholesterol levels did not differ significantly
between the gallstone carriers and controls and are summarized in
Table 3.
Table 3. Clinical characteristics of study participants from Stuttgart and Aachen cohorts.
Variables
Stuttgart controls
(n =71)
Stuttgart gallstone carriers
(n=56)
Aachen controls
(n =184)
Aachen gallstone carriers
(n =184)
Gender (f/m) (33/38) (40/16) (103/81) (103/81)
Age (years) 60612 63612 63613 63613
BMI (kg/m2) 2664 2664 25.464.1 26.164.3
Triglyceride (mg/dl) (1–200) a 128682 132692 1466105 143679
Cholesterol (mg/dl) (140–240) a 200634 198637 202655 200653
Values are given as means6SD; BMI =body mass index;
a = normal range.
The characteristics of the Aachen population were, in part, published previously [42].
doi:10.1371/journal.pone.0007321.t003
SLC10A2 and Cholelithiasis
PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7321
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