Best Practice & Research Clinical Gastroenterology
Vol. 20, No. 6, pp. 997e1015, 2006
available online at http://www.sciencedirect.com
Pathogenesis of gallstones:
a genetic perspective
Frank Lammert* MD, Dr med
Department of Internal Medicine I, University Hospital Bonn, University of Bonn, Sigmund-Freud-Str. 25,
53127 Bonn, Germany
Cholelithiasis is one of the most prevalent gastroenterological diseases, imposing a huge
economic burden on health-care systems. Gallbladder stones form when the concentration
of cholesterol or bilirubin exceeds the solubility in the bile salt and phospholipid-rich bile.
The physiology of biliary lipid secretion by a number of specialized transport proteins has re-
cently been elucidated, and underlying genetic defects in these proteins have been identiﬁed
as susceptibility factors for gallstone disease. Recent studies of identical twins and family strongly
support the idea of a genetic component to gallstone disease. Epidemiological studies in high-risk
populations indicate that gallstone formation is caused by multiple environmental inﬂuences and
common genetic factors and their interactions. Monogenic subtypes of cholelithiasis, such as bil-
iary lipid transporter deﬁciencies, appear to be rare. The characterization of lithogenic genes in
knockout and transgenic mice, and the identiﬁcation of many gallstone susceptibility loci in in-
bred mice, provide the basis for studies of the corresponding genes in patients with gallstones.
The transfer of ﬁndings from mouse genetics to the bedside might lead to new strategies for
individual risk assessment and reveal molecular targets for the development of new treatment
Key words: ABC transporters; cholelithiasis; gallstones; genetic studies; lithogenic genes.
Gallbladder disease is one of the major digestive diseases: 10e20% of Europeans and
Americans carry gallbladder stones,1,2 and the prevalence of gallstone disease seems
to be rising as a result of longer life expectancy. Although the majority of gallstones
are silent, symptoms and severe complications ensue in around 25% of cases, neces-
sitating surgical removal of the gallbladder. Each year, an estimated 700,000
* Corresponding author. Tel.: þ49 228 1209; Fax: þ49 228 4698.
E-mail address: firstname.lastname@example.org (F. Lammert).
1521-6918/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.
998 F. Grunhage and F. Lammert
cholecystectomies are performed in the US.3 Cholelithiasis incurs annual medical ex-
penses of $6.5 billion in the US and is the second most expensive digestive disease,
currently exceeded only by gastro-oesophageal reﬂux disease.4
MOLECULAR MECHANISMS OF BILE FORMATION
Bile formation enables the removal of excess cholesterol, either directly or after ca-
tabolism to bile salts, and it is a key function of the liver. Bile is an aqueous solution
of lipids, with bile salts (67% of solutes by weight), phospholipids (22%) and cholesterol
(4%) representing the three main lipid species.5 Hepatocytes express speciﬁc ATP-
dependent transport proteins e known as ABC transporters e for each of these
three lipids at the canalicular membrane domain (Figure 1). The ABCB11 transporter
is the bile salt export pump, ABCB4 is the transporter for the major biliary phospho-
lipid phosphatidylcholine (lecithin) (Table 1), and ABCG5/ABCG8 form obligate heter-
odimers for biliary cholesterol secretion (Figure 1).6,7
Bile salts are pumped into the canalicular lumen by ABCB11 and reach sufﬁciently
high concentrations to form simple micelles. After biliary secretion, phosphatidylcho-
line and cholesterol form metastable unilamellar vesicles (Figure 1), most likely directly
after acceptance of the lipids from their transporters by bile-salt micelles in the prox-
imity of the membrane.6,7 The unilamellar vesicles are converted into mixed micelles
during their passage through the biliary tree (Figure 1).5,8 The composition of hepatic
bile is further modiﬁed by the bicarbonate- and chloride-rich secretions of cholangio-
cytes, which is associated with a net inﬂux of water into bile through aquaporin chan-
nels. An important chloride channel in cholangiocytes is the cystic ﬁbrosis
transmembrane conductance regulator (CFTR), the gene for which is mutated in cystic
ﬁbrosis (Table 1).
MOLECULAR PATHOMECHANISMS OF GALLSTONE FORMATION
Cholesterol gallbladder stones
Gallstones are classiﬁed as cholesterol stones or pigment stones (Table 2). More than
80% of gallstones consist mainly of cholesterol crystals and are formed within the gall-
bladder.8 Three key mechanisms contribute to the formation of cholesterol gallbladder
stones: cholesterol supersaturation of bile, gallbladder hypomotility, and destabilization
of bile by kinetic protein factors. Cholesterol is virtually insoluble in water, and its sol-
ubility in bile depends on the detergent properties of bile salts and phospholipids.9
Cholesterol-supersaturated bile contains more cholesterol than can be solubilized
by mixed micelles at equilibrium (cholesterol saturation index (CSI) > 1) (Figure 1).
The CSI is deﬁned as the ratio of the actual biliary cholesterol concentration and
the maximal concentration that would be soluble at phase equilibrium in model bile
with equal lipid composition.10 Cholesterol-supersaturated bile contains multilamellar
vesicles (liquid crystals), fusion and aggregation of which precede the formation of
solid cholesterol crystals (Figure 1). As illustrated in the ternary phase diagram,9,10
solid crystals occur in bile at high relative bile salt and low phospholipid concentra-
tions, and at cholesterol:phospholipid ratios >1.
An excess of biliary cholesterol in relation to bile salts and phospholipids can result
from hypersecretion of cholesterol, or from hyposecretion of bile salts or phospho-
lipids (Table 3). Cholesterol hypersecretion, which is the most common cause of
Pathogenesis: a genetic perspective 999
CTP:phosphocholine HMG-CoA Cholesterol
cytidylyltransferase reductase 7α-hydroxylase
Phosphatidyl- Cholesterol (CH) Bile salts (BS)
PL-CH vesicles (60-80 nm) (~20 nm)
PC-CH(-BS) (4-6 nm)
vesicles CH monohydrate
(<500 nm) crystals
2 1 0
Figure 1. Pathogenesis of cholesterol gallstone formation. Schematic diagram of rate-limiting enzymes for
synthesis and hepatocanalicular transport proteins of hepatobiliary lipids, cholesterol carriers in bile, and
cholesterol saturation index (CSI). The biliary lipids (phospholipids, cholesterol and bile salts) are secreted
into bile by ATP-dependent transport proteins (ABC transporters). The cholesterol transporter is a hetero-
dimer (ABCG5/G8). On biliary secretion, phosphatidylcholine and cholesterol form metastable unilamellar
vesicles and bile salts simple micelles. These lipid aggregates are converted into mixed micelles during their
passage through the biliary tract into the gallbladder. If bile contains more cholesterol than can be solubilized
by mixed micelles, it is supersaturated with cholesterol, and cholesterol-rich multilamellar vesicles form,
fuse, and nucleate solid cholesterol crystals. Thus, conditions that increase the ratio of cholesterol to bile
salts and phospholipids favour gallstone formation. Cholesterol supersaturation is indicated by a cholesterol
saturation index (CSI) >1. The gallbladder secretes mucous glycoproteins, which form the matrix for pre-
cipitation of crystal aggregates and gallstones.
1000 F. Grunhage and F. Lammert
Table 1. Summary of gallstone (Lith) genes.
Gene Symbol Function Databases Frequency Gene Mouse Potential References
variant model mechanisms
OMIMa Gene-cardsb Rare Several Common
monogenic families polygenic
ATP binding ABCB4 Hepatocanalicular 171060 GC07M086676 þ þ Biliary
cassette (GBD1) phosphatidylcholine and phospholipid
transporter B4 (lecithin) ﬂoppase 600803 secretion Y
ATP binding ABCB11 Hepatocanalicular 603201 GC02M169604 þ þc Biliary bile 53,80,81
cassette bile-salt export salt
transporter B11 pump secretion Y
Apolipoprotein B APOB 107730 GC02M021135 þ þ RFLP e Hepatic
Apolipoprotein E APOE 107741 GC19P050100 þ 34 þ Intestinal
Cholecystokinin-A CCKAR 118444 GC04M026159 þ þ Gallbladder
Cholesteryl CETP þ RFLP Gene HDL
ester transfer (Finland) absent catabolism [
protein in mice
Cystic ﬁbrosis CFTR Chloride 602421 GC07P116713 þ þ Bile pH Y,
transmembrane (ABCC7) channel biliary
faecal bile salt
Cytochrome CYP7A1 Cholesterol 118470 GC08M059565 þ þ (China) Promoter þ Bile acid
P450 7A1 7a-hydroxylase rSNP synthesis Y
(rate-limiting 204A > C À
GBD, gallbladder disease 1; OMIM, Online Mendelian Inheritance in Men; RFLP, restriction fragment length polymorphism; SNP, single-nucleotide polymorphism.
The gene was identiﬁed to co-localize with the murine lithogenic locus on chromosome 2. However, in contrast to humans, mice over-expressing Abcb11 are
Pathogenesis: a genetic perspective 1001
1002 F. Grunhage and F. Lammert
Table 2. Classiﬁcation of gallstones.
Type Localization Prevalence Composition Colour Computed
Pure cholesterol Gallbladder 75 Cholesterol Yellow Iso- or hypo-dense
stone monohydrate compared to bile
Black pigment Gallbladder 5 Polymerized Black Mainly hyper-dense
stone calcium bilirubinate
Brown pigment Bile ducts 20 Calcium salts of Brown Partly hyper-dense
stone long-chain fatty acids
supersaturation,8 might be caused by increased hepatic uptake or synthesis of choles-
terol, decreased hepatic synthesis of bile salts, or decreased hepatic synthesis of
cholesteryl esters for incorporation in very-low-density lipoproteins (VLDL). In non-
obese individuals who form cholesterol-rich gallbladder stones, gallstones were associ-
ated with a small bile salt pool cycling at a normal frequency within the enterohepatic
circulation.11 Furthermore, it has been suggested that slow intestinal transit increases
bacterial degradation of primary to secondary bile salts in the colon.12 Bile then contains
a greater proportion of deoxycholate conjugates which, in turn, increases biliary choles-
terol secretion and saturation, thereby enhancing gallstone formation.13
In humans, most cholesterol present in gallstones is of dietary origin, consistent
with the observation that hepatic biosynthesis contributes <20% to biliary choles-
terol.14 The hepatic uptake of cholesterol is mediated by the scavenger receptor B-I
(SRB1) for high-density lipoproteins (HDL), which contribute most of the biliary cho-
lesterol under physiological conditions, the apolipoprotein (Apo) B/E receptor for
low-density lipoproteins (LDL), and the LDL receptor-related protein for chylomicron
remnants, which carry exogenous cholesterol from the intestine to the liver. The in-
verse correlation between serum HDL levels and gallstones suggests that cholesterol
cholelithiasis is associated with an induced reverse cholesterol transport and hepatic
catabolism of HDL (Table 3).14 The rate-limiting enzymes of hepatic cholesterol and
bile-salt synthesis are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase
and cholesterol 7a-hydroxylase (CYP7A1), respectively (Table 1, Figure 1). These
enzymes are regulated by the sterol regulatory element-binding protein (SREBP)
and nuclear receptor (NR) signalling pathways. Bile salts serve as natural ligands of
the nuclear receptor FXR, which represents the bona ﬁde hepatic bile-salt sensor
of the liver. FXR represses CYP7A1 expression, but stimulates the expression of the
ABC transporters for bile salts (ABCB11) and phospholipids (ABCB4),15 whereas the
cholesterol transporter ABCG5/G8 is induced by the nuclear receptor for oxysterols
(LXR). Studies in knockout and transgenic mice have demonstrated that many of the
genes involved in hepatic cholesterol metabolism affect cholesterol gallstone forma-
tion in vivo,15e17 but with few exceptions variants of these genes have yet to be inves-
tigated in patients with gallstones.
Stasis of bile in the gallbladder favours stone formation, as indicated by stone forma-
tion during pregnancy, rapid weight loss, or total parenteral nutrition (Table 3). Gallblad-
der emptying in response to either a test meal or a cholecystokinetic stimulus e such as
intravenous cholecystokinin (CCK) or caerulin or intraduodenal fat or amino acids e is
impaired in patients with gallstones.8 Both the fasting and the residual postprandial
Pathogenesis: a genetic perspective 1003
Table 3. Causes and risk factors for gallstone formation and potential mechanisms.
Cholesterol gallbladder stones
Age Biliary cholesterol secretion [
Biliary bile salt secretion Y
Gender Higher gallstone prevalence related to parity
Pregnancy/oestrogens Hepatic cholesterol uptake and synthesis [
Cholesterol 7a-hydroxylase activity Y
Biliary cholate/dexoycholate pool [
Gallbladder hypomotility 0 biliary sludge
High-caloric low-ﬁbre diet Biliary cholesterol secretion [
Intestinal transit time [ 0 bacterial bile salt
catabolism [ 0 biliary deoxycholate pool [
High-carbohydrate diet, dietary Hepatic cholesterol synthesis [
glycaemic load Bile salt malabsorption
Obesity Cholesterol synthesis [ 0 biliary cholester
ol secretion [
Rapid weight loss/surgery for obesity Hepatic cholesterol uptake [
Bile salt synthesis Y
Mucin secretion [ 0 nucleation
Low fat diet 0 gallbladder hypomotility
Hypertriglyceridaemia/low HDL cholesterol Biliary cholesterol secretion [
Pancreatic insufﬁciency CCK production Y 0 gallbladder hypomotility
Spinal cord injury Gallbladder hypomotility (neuronal)
Black pigment gallbladder stones
Crohn disease with severe ileal Bile salt malabsorption 0 intestinal bilirubin
manifestation or ileal resection absorption [
Vitamin B12 malabsorption
Total parenteral nutrition Gallbladder hypomotility
Enterohepatic cycling of bilirubin [
Vitamin B12 or folic acid deﬁciency Ineffective erythropoiesis
Chronic haemolysis (e.g. liver cirrhosis, malaria Bilirubin load [
sickle-cell disease, thalassaemia, spherocytosis)
Liver cirrhosis Bile salt synthesis Y
Bile salt malabsorption 0 intestinal bilirubin
Cystic ﬁbrosis Enterohepatic cycling of bilirubin [
Bile pH Y 0 biliary b-glucuronidase activity [
Formation of thick mucus layer
Brown pigment bile-duct stones
Cholangitis Deconjugation of bilirubin by bacterial enzymes
(continued on next page)
1004 F. Grunhage and F. Lammert
Table 3 (continued ).
Biliary stricture Ascending cholangitis
Duodenal diverticulum Ascending cholangitis
Oestrogens Biliary cholesterol secretion [
Octreotide Large intestinal transit time [ 0 deoxycholate
pool [ 0 biliary cholesterol secretion [
CCK release Y 0 gallbladder hypomotility
Cloﬁbrate Biliary cholesterol secretion [
Ceftriaxone Biliary secretion of drug 0 precipitation
gallbladder volumes are greater in most patients with cholesterol gallbladder stones
than in controls. Gallbladder hypomotility is probably due to absorption of cholesterol
from supersaturated bile by the gallbladder wall. Excess cholesterol in smooth muscle
cells might stiffen sarcolemmal membranes and decouple the G-protein-mediated signal
transduction that usually ensues when CCK-A binds to its receptor, thereby paralyzing
gallbladder contractile function.18
Nucleation and growth of cholesterol crystals in model biles in vitro are modulated
by promoter and inhibitor proteins, which interact with vesicles and solid crystals, re-
spectively.19,20 However, only gallbladder mucin e the main component of biliary
sludge e has been shown to promote stone formation in vivo (Figure 1).21 Mucin is
a mixture of sparingly soluble high-molecular-weight mucous glycoproteins that are
secreted by biliary epithelial cells. In animals, aspirin and other non-steroidal anti-
inﬂammatory drugs (NSAIDs) prevent the increase in biliary mucus glycoprotein in-
duced by a lithogenic diet and stone formation,22 but the effects of aspirin or NSAIDs
on bile composition in humans are controversial, and there is scant evidence that
chronic aspirin/NSAID ingestion affects the incidence of gallstones. Emerging experi-
mental evidence indicates that enterohepatic bacteria and bacterial bioﬁlms promote
cholesterol crystallization, as well as formation of sludge and gallstones.23
Black pigment gallbladder stones
A small proportion of gallbladder stones are black pigment stones. These consist pre-
dominantly of polymerized calcium bilirubinate, which precipitates if the ion product of
calcium and unconjugated bilirubin exceeds its solubility product and polymerizes
slowly in biliary sludge.
Haemolytic anaemias or ineffective erythropoiesis are the most conspicuous sour-
ces of excess unconjugated bilirubin (Table 3). Another pathway involves ileal disease
or resection causing spillage of bile salts into the colon; this promotes solubilization
and absorption of unconjugated bilirubin and results in increased enterohepatic cycling
and biliary secretion of bilirubin.24
Brown pigment bile duct stones
Brown pigment stones are mostly formed within the bile ducts as a consequence of
bacterial infection and hydrolysis of glucuronic acid from bilirubin by bacterial
Pathogenesis: a genetic perspective 1005
b-glucuronidase. This results in a decreased solubility of deconjugated bilirubin, ulti-
mately leading to the formation of stones consisting of calcium salts of unconjugated
bilirubin, deconjugated bile salts, varying amounts of cholesterol, and saturated long-
chain fatty acids. Many bile duct stones are mixed stones (cholesterol stones originat-
ing from the gallbladder with a pigment shell).
Intrahepatic brown pigment stones are related to infestation with the parasites Clo-
norchis sinensis and Ascaris lumbricoides.25 In Western countries, intrahepatic gallstones
tend to be associated with periampullary diverticula,26 Caroli syndrome, strictures,
tumours, and other ductal abnormalities causing biliary stasis and infection (Table 3).
Bacteria are found in the bile in almost all patients with intrahepatic stones.
EPIDEMIOLOGY OF GALLSTONES: EVIDENCE FOR GENETIC
Prevalence of gallstones in the Western world is 10e20%, with considerable variation
between different ethnic groups.1,2 Gallstones are more frequent with increasing age
and in women.2 As determined by cross-sectional ultrasound surveys,1,27 the preva-
lence rates of gallbladder stones show remarkable geographic variation. Gallbladder
stones are common in most European countries, as well as in North and South
America, but the prevalence is low in Asia and Africa. Environmental factors are likely
to contribute to these marked differences. Data from several large epidemiological
studies in the US, Europe, China and Japan implicate chronic overnutrition with carbo-
hydrates and triglycerides, as well as depletion of dietary ﬁbre, as environmental trig-
gers for cholesterol gallstone formation.14 Gallstone disease phenotypes are likely to
result from the complex interaction of genetic factors, high-carbohydrate, high-fat and
low-ﬁbre diets,28 and other not fully deﬁned environmental factors, including low
physical activity (Table 3).2,29 This hypothesis is supported by the profound increases
of cholesterol gallstone prevalence rates in Native Americans, post-war European
countries, and current urban centres in East Asia, all of which were associated with
the introduction of high-calorie ‘Westernized’ diets.14,28 Gallbladder disease is strongly
related to the metabolic syndrome and/or its major components, such as hyperinsu-
linism, dyslipidaemia, and abdominal adiposity.30,31 However, a cholesterol-rich diet
signiﬁcantly induces lithogenic bile only in gallstone carriers but not in stone-free
controls,32 indicating that intestinal cholesterol absorption33 and/or biliary cholesterol
secretion are in part genetically determined. When dietary conditions are controlled,
biliary factors such as outputs of biliary lipids (bile salts, cholesterol, and phospho-
lipids), as well as size, molecular composition, and hydrophilic/hydrophobic balance
of the bile salt pool, could together exert a major inﬂuence on the efﬁcacy of intestinal
cholesterol absorption. Any of these could explain, in part, the inter-individual differ-
ences in cholesterol absorption. Studies in Abcb4 deﬁcient mice suggest that physiolog-
ical levels of biliary phospholipid outputs are necessary for normal intestinal
cholesterol absorption.33 In Cyp7a1 knockout mice, biliary bile salt secretion and
bile salt pool size are markedly reduced, and the animals absorb only trace amounts
of cholesterol because of bile salt deﬁciency in bile.34 Similarly, mice with homozygous
disruption of the sterol 27-hydroxylase (Cyp27) gene, which encodes the key enzyme
of the alternative pathway of bile salt synthesis, display signiﬁcantly reduced bile salt
synthesis and pool size, and intestinal cholesterol absorption decreases from 54% to
4% in knockout mice.35
Recently cholesterol absorption has been shown to depend on the expression of
cholesterol transport protein NPC1L1 (Niemann-Pick C1-like protein 1) at the apical
1006 F. Grunhage and F. Lammert
surface of enterocytes.36 Enterocytes also efﬂux cholesterol back into the intestinal
lumen via the apical ABCG5/G8 transport system.33 Of note, rare variants of the
NPC1L1 gene are associated with reduced cholesterol absorption in African Ameri-
cans,37 indicating that a fraction of the genetic variance in cholesterol absorption is
due to multiple alleles with modest effects that are present at low frequencies in
the general population (Figure 2).
Major genes or oligogenes with stronger allele effects are probably contributing to
the extraordinarily high prevalence of gallstones in American Indian populations
in both North and South America. A recent ultrasound survey in 13 American Indian
tribes in Arizona, Oklahoma, and South and North Dakota38 found cumulative stone
prevalence rates of 64% in American Indian women and 30% in men, even though their
total caloric intake is comparable to that of white Americans. The Pima Indians of Ari-
zona have the highest recorded prevalence rate for gallstones.39 Pima women develop
supersaturated bile around the age of puberty; by the age of 25e30 years most have
born several children, and up to 80% have developed gallstones. It has been speculated
that the wide distribution of genes conferring gallstone susceptibility in American In-
dians might be related to ‘thrifty’ genes that conferred survival advantages when Paleo-
Indians migrated to the Americas during the last Great Ice Age (50,000e10,000 years
ago).40 This speculation is driven by the epidemiological associations of gallstones with
the metabolic syndrome, obesity and type 2 diabetes mellitus, all of which might be
caused by ‘thrifty’ genes.14,40 The main pathophysiological link appears to be biliary
hypersecretion of cholesterol owing to persistently increased cholesterol synthesis in
obese and hyperinsulinaemic patients (Table 3).1
Figure 2. Inverse relationship between allele frequency and effect. The major genes that confer large effects
that are the basis of classical genetics are mostly rare. Polygenes with extremely small effects account for
nearly all alleles subject to selection and are currently beyond cost-effective study. Between them are oligo-
genes with effects large enough to be detected in a feasible study, thus perhaps elucidating clinically impor-
tant metabolic patterns or even representing useful targets for pharmacogenetics. Adapted from Morton
(2005, Journal of Clinical Investigation 115: 1425e1430).
Pathogenesis: a genetic perspective 1007
In the Third National Health and Nutrition Examination Survey,41 the highest age-
standardized gallstone prevalence was seen in Mexican-American women (27%), fol-
lowed by white (17%) and African American women (14%). These differences can
be attributed to the American Indian heritage of Mexican-Americans. This ‘admixture
hypothesis’ is supported by a genetic study from Chile,42 which assessed the degree of
admixture by mitochondrial DNA (mtDNA) in Mapuche Indians, Easter Island Maoris,
and Hispanics. The prevalence of gallstone disease was highest (35%) in Mapuches,
who migrated from North America (100% American Indian mtDNA), intermediate
(27%) in Hispanics (88% American Indian mtDNA), and still 21% in Maoris, who orig-
inate from Polynesia (0% American Indian mtDNA) and in whom obesity appears to be
a major environmental factor causing gallstones. Furthermore, a follow-up study43
showed that the plasma ratios of lathosterol to cholesterol and 7a-hydroxy-
4-cholest-3-one to cholesterol e which are surrogate markers for cholesterol and
bile salt synthesis, respectively e are increased in gallstone-susceptible Mapuche
Indian women; whether these inductions represent a primary defect or a secondary
response to increased intestinal bile salt loss is unknown. The latter hypothesis is
supported by the recent observation of decreased expression of ileal bile salt trans-
porters in normal-weight female gallstone carriers.44
Genetic factors determining gallstone formation
A large study in Swedish twins provided strong evidence for a role of genetic factors in
gallstone pathogenesis.45 The Swedish Twin Registry was linked with the inpatient-
discharge and causes-of-death registries for symptomatic gallstone disease and gall-
stone surgery-related diagnoses in 43,141 pairs of twins born between 1900 and
1958. Concordance rates were signiﬁcantly higher in monozygotic compared with
dizygotic twins for both genders. Genetic factors accounted for 25% (95% conﬁdence
interval (CI) 9e40%), shared environmental effects (e.g. diet in childhood) for 13% (CI
1e25%), and individual environmental effects for 62% (CI 56e68%) of the phenotypic
variation among twins, as estimated by structural equation modelling.45
To investigate biliary lipid compositions in twins, 35 male pairs were randomly
selected from the Finnish Twin Cohort.46 Serum levels of methylsterols, which reﬂect
hepatic cholesterol synthesis, and biliary concentrations of deoxycholate showed sig-
niﬁcant correlations in monozygotic but not dizygotic twins. These ﬁndings are consis-
tent with the concept that genetic factors determine biliary lipid secretion.
Family and linkage studies
Ultrasound surveys have documented that gallstones are two to four times more com-
mon in ﬁrst-degree relatives of gallstone patients compared with age-matched stone-free
controls.14 The San Antonio Family Diabetes/Gallbladder Study (SAFGS)47,48 employed
variance component analysis in Mexican-American families to assess the genetic deter-
minants of symptomatic gallbladder disease. In non-diabetic individuals, heritability for
gallbladder disease was high (53e77%) and comparable to the heritability of obesity
and type 2 diabetes.47,48 The ﬁrst genome-wide scan for gallstone susceptibility loci in
715 individuals from 39 SAFGS families48 employed genetic markers at an average dis-
tance of 10 cM and detected two major susceptibility loci (GBD2 and GBD3) for symp-
tomatic gallbladder disease on chromosome 1p.48 Another variance component
1008 F. Grunhage and F. Lammert
analysis in 358 families in Wisconsin, each of which contained at least two obese siblings,
determined that the heritability of symptomatic gallstones is 29 Æ 14%,49 which is similar
to the ﬁgure obtained in the Swedish twin study.45
Despite accumulating evidence that gallstone formation is genetically determined in
humans, direct conﬁrmation of the role of individual genes in human gallstone disease
is sparse. In small groups of patients with gallstones, monogenic predisposition has
been ascribed to mutations in the genes that encode the ABC transporters for phos-
phatidylcholine (ABCB4) or bile salts (ABCB11), cholesterol 7a-hydroxylase (CYP7A1),
the CCK-A receptor (CCKAR), and the cystic ﬁbrosis gene (CFTR) (Table 1).
The ﬁrst evidence that a single-gene defect causes gallstone formation in a deﬁned
subgroup e young patients with a recurring form of cholelithiasis e was provided by
Rosmorduc et al.50 The group identiﬁed mutations in the ABCB4 gene in patients with
cholesterol gallbladder stones and intrahepatic sludge or microlithiasis, recurrence
of biliary symptoms after cholecystectomy, positive family history, and mild chronic
cholestasis and/or intrahepatic cholestasis of pregnancy (Table 4).50 Microlithiasis
might increase the risk of biliary pancreatitis.51 The ﬁndings by Rosmorduc et al are
clinically relevant, since asymptomatic carriers might beneﬁt from prevention with ur-
sodeoxycholic acid (UDCA), which inhibits hepatic cholesterol synthesis and secre-
tion, induces bile ﬂow, and promotes the solubility of biliary cholesterol in liquid
crystals.50 The pathophysiological basis of the ABCB4 deﬁciency syndrome is consis-
tent with the spontaneous occurrence of cholesterol gallstones in Abcb4 knockout
Homozygous ABCB4 mutations that lead to complete absence of the phospho-
lipid transporter and virtually no secretion of phospholipids into bile result in pro-
gressive familial intrahepatic cholestasis (PFIC) type 3 and early liver cirrhosis in
childhood. PFIC type 2 is caused by mutations in the bile salt export pump gene
ABCB11. Cholelithiasis is also frequently observed in patients with PFIC type 2.7
A subgroup of patients with benign recurrent intrahepatic cholestasis (BRIC),
who have intermittent attacks of cholestasis without progression to liver cirrhosis,
have ABCB11 mutations.53 The majority of ABCB11-affected BRIC patients (65%) de-
Pullinger et al54 proposed an association between another single-gene defect and gall-
stone formation. Patients with homozygous deﬁciency of cholesterol 7a-hydroxylase
(CYPA1) display hypertriglyceridaemia and hypercholesterolaemia, but faecal bile salt ex-
cretion is markedly deﬁcient.54 Recently a Chinese association study55 demonstrated
Table 4. Low phospholipid-associated cholelithiasis (LPAC).
Age at onset of symptoms <40 years
Cholesterol gallbladder stones and intrahepatic sludge or microlithiasis (hyperechoic foci)
Recurrence of biliary symptoms after cholecystectomy
Positive family history
Mild chronic cholestasis
Association with intrahepatic cholestasis of pregnancy (33%)
ABCB4 mutations (56%)
Prevention by ursodeoxycholic acid (?)
Modiﬁed from Rosmorduc et al (2003, Gastroenterology 125: 452e459).
Pathogenesis: a genetic perspective 1009
that a common single-nucleotide polymorphism (SNP) within the CYP7A1 promoter is
associated with increased LDL cholesterol levels and gallstones (Table 1). Furthermore,
similar to ABCB4 and ABCB11 deﬁciency, the relevance of CYP7A1 for gallstone patho-
genesis has been demonstrated in CYP7A1 transgenic mice, which are resistant to stone
induction by a high-fat/high-cholesterol diet.56
Familial hypobetalipoproteinaemia due to mutations of the APOB gene might also
be associated with cholesterol gallstone formation, which could result from increased
cholesterol secretion as a compensatory mechanism to eliminate cholesterol not in-
corporated into ApoB-containing VLDL.57 Common APOB gene polymorphisms have
been associated with other complex diseases, such as coronary heart disease and
type 2 diabetes mellitus. Of note, signiﬁcant associations between a common poly-
morphism in exon 26 and cholesterol gallstone prevalence, as well as increased total
cholesterol, LDL cholesterol and ApoB concentrations in serum, have been detected
in two Chinese studies.55,58 This ﬁnding might be related to decreased LDLR afﬁnity
to its ligand APOB and underscores the putative importance of all hepatic choles-
terol uptake mechanisms for cholesterol gallstone formation. However, the lipid pro-
ﬁle is not typical for cholesterol stone patients,14 and the association could not be
detected either in a Finnish population59 or in a study from India.60 The Finnish
study59 found an association between stone prevalence and a common polymorphism
of the cholesteryl ester transfer protein (CETP), which is absent in mice. CETP
transfers cholesteryl esters and phospholipids from HDL to triglyceride-rich lipopro-
teins in exchange for triglycerides. This transfer lowers HDL cholesterol and in-
creases VLDL triglyceride levels, a lipid pattern that is associated with an
increased risk for gallbladder stones.1
Altered CCKAR structure has been associated with gallstone formation in isolated
cases of gallstone patients.61,62 Although further screening did not detect mutations in
the coding region of the CCKAR gene in patients with gallstones,63,64 the link between
CCKAR and gallstones is supported by Cckar knockout mice.18,64 These mice display
profound gallbladder hypomotility and prolonged small-intestinal transit times,18 re-
sulting in increased cholesterol absorption.
Single-gene mutations causing haemolytic anaemias (hereditary spherocytosis,
ANK1, EPB42, SPTA1, SPTB, SLC4A1; sickle-cell disease, HBB; thalassaemia major and
intermedia, HBB; and erythrocyte enzyme deﬁciencies, AK1, G6PD, GPI, GSR, PGK1,
PKLR, TPI1) and thus increased biliary bilirubin concentrations are well documented
(for gene abbreviations see OMIM database).65 In addition to these gene defects,
where black pigment stones are to be expected, there are also genes that predispose
to increased enterohepatic cycling of bilirubin, another cause of pigment stone forma-
tion.24 An example of such a gene defect associated with pigment stone formation is
cystic ﬁbrosis. Gallstone prevalence in cystic ﬁbrosis is 10e30%, compared with <5%
in age-matched controls, but biliary cholesterol saturation does not differ between pa-
tients with and without gallstones.66 Experimental ﬁndings in mice with mutations in
the CFTR gene indicate that enterohepatic cycling, as well as biliary secretion and de-
conjugation of bilirubin, is increased in cystic ﬁbrosis. Accordingly, we have recently
shown that patients with cystic ﬁbrosis and gallstones are signiﬁcantly more likely to
carry the Gilbert-syndrome-associated UGT1A1 mutation compared to stone-free pa-
tients.67 In support of this concept, an increased prevalence of the Gilbert mutation
has also been detected in stone patients with hereditary spherocytosis,68 sickle-cell
anaemia69 and thalassaemia,70 as compared to stone-free patients with the same con-
ditions, all of which increase the biliary supply of unconjugated bilirubin due to inter-
1010 F. Grunhage and F. Lammert
In humans, the identiﬁcation of lithogenic genes is hampered by the multifactorial patho-
genesis of gallstones, so cross-breeding experiments in inbred mouse strains that differ
in genetic susceptibility to cholesterol gallstone formation have been used to identify
the genetic factors that contribute to gallstone formation. The mouse model is based
on a lithogenic diet that contains 15% fat, 1% cholesterol and 0.5% cholic acid, which
promotes intestinal absorption and biliary secretion of cholesterol.14,17,71 Using quan-
titative trait locus (QTL) analysis14,17,71 and in silico association mapping,72 more than
20 murine gene loci for gallstone susceptibility (Lith genes) and several candidate genes
(ABC transporters, NRs, mucins) have been identiﬁed (for updates see QTL
However, detailed analyses of human Lith genes have yet to be performed. Only APOE
polymorphisms have been extensively investigated in human gallstone disease (Table 1).
ApoE is the high-afﬁnity ligand for the hepatic LDL receptor and the LDL-receptor-re-
lated protein. There are three common codominant APOE alleles (32, 33 and 34), and
the six resulting ApoE isoforms (E2/E2, E3/E3, E4/E4, E2/E3, E2/E4, E3/E4) can be distin-
guished by isoelectric focusing. These isoforms cause differences in receptor-binding af-
ﬁnities and clearance rates of circulating lipoproteins. Presence of the 34 allele has been
associated with conditions such as coronary heart disease and Alzheimer disease, and
Bertomeu et al74 showed that the 34 allele is also associated with cholelithiasis. The
ApoE2 isoform, by contrast, was found less frequently in women with gallstones than
in controls.75 In line with these ﬁndings, Apoe knockout mice show a markedly lower fre-
quency of gallstones than wild-type controls upon challenge with a high-cholesterol
diet.76 The association between gallstones and APOE could be due to increased hepatic
cholesterol uptake via chylomicrons in patients carrying the isoform ApoE4; alternatively,
biliary ApoE might have a role in the destabilization of bile.1,74 However, recent studies
failed to conﬁrm the association between gallstones and ApoE isoforms (reviewed by
Lammert and Sauerbruch).1 Different ethnicities with lower gallstone prevalence rates
and the inclusion of younger control probands might explain the discrepancies, since
the 34 allele frequency is low in Asian populations and decreases with age. Other studies
showed a higher 34 allele frequency in patients with cholesterol stones than in patients
with pigment stones,77 and a higher stone recurrence rate within 7 years after extracor-
poreal shock wave lithotripsy (ESWL) in 34 carriers (73% versus 50%).78 Screening for
carriers of common ApoE variants cannot be recommended on the basis of the current
association studies and does not guide therapy.
During the past decade, epidemiological, family and twin studies in humans, as well as
genetic studies in mice, have shown that cholelithiasis is a complex multifactorial dis-
order inﬂuenced by both genetic and environmental factors. However, in the future,
whole-genome association studies and reﬁned haplotype mapping in gallstone patients
are likely to identify the whole set of common lithogenic genes, eventually enabling us
to specify the individual risk for the development of gallstone disease. Since the disease
phenotype results from the manifestation of genetic susceptibility factors under the
inﬂuence of environmental factors, discovery of lithogenic genes would also open
avenues to control the inﬂuence of speciﬁc environmental factors. This might lead
to the design of new interventions, which extend our currently limited strategies
for prevention of this exceptionally prevalent digestive disease.79e82
Pathogenesis: a genetic perspective 1011
Particularly with advances in genomics of cholelithiasis, the family history will
be even more helpful in diagnosing, preventing, and treating this exceptionally
common gastroenterological disease.
In patients with gallbladder stones, additional hepatobiliary manifestations, such
as intermittent cholestasis or intrahepatic sludge with recurrence of symptoms
after cholecystectomy, point to rare monogenic forms of cholelithiasis e for
example, ABC transporter deﬁciencies e for which research laboratories offer
Screening for carriers of common gene variants (e.g. ApoE4) cannot be recom-
mended on the basis of current association studies and does not guide therapy.
Speciﬁc clinical settings (e.g. young age, association with diarrhoea) should trig-
ger further aetiological investigations in gallstone disease (e.g. exclusion of hae-
molytic anaemia, bile salt loss).
There is a need to further characterize the role of the murine Lith genes and
their products in causing gallstones.
The knowledge of murine Lith genes should be applied to the identiﬁcation of
homologous genes in humans associated with susceptibility to form gallstones.
The role of the enterohepatic bacteria, speciﬁcally the genus Helicobacter, in
gallstone formation in both animal models and human patients needs to be
Biomarkers in plasma or urine that indicate lithogenicity of bile should be
Practical and effective approaches to prevention of gallstones in high-risk pop-
ulations are needed.
Cross-sectional and longitudinal cohort studies of subjects with biliary pain are
necessary to allow for the analysis of potential risk factors such as genetics,
microlithiasis, nucleation factors, and gallbladder motility, and pilot studies of
prevention and treatment.
The authors’ experimental work relating to gallstone formation has been supported by
research grants from the Deutsche Forschungsgemeinschaft and the Ministry of
Science and Research of North-Rhine-Westphalia.
1. Lammert F Sauerbruch T. Mechanisms of disease: the genetic epidemiology of gallbladder stones.
Nat Clin Pract Gastroenterol Hepatol 2005; 2: 423e433.
1012 F. Grunhage and F. Lammert
2. Volzke H, Baumeister SE, Alte D et al. Independent risk factors for gallstone formation in a region with
high cholelithiasis prevalence. Digestion 2005; 71: 97e105.
3. Liver Disease Subcommittee of the Digestive Diseases Interagency Coordinating Committee. Gallblad-
der and biliary disease. In National Institutes of Health (ed.) Action Plan for Liver Disease Research.
Bethesda: National Institutes of Health, 2004, pp. 145e150.
4. Sandler RS, Everhart JE, Donowitz M et al. The burden of selected digestive diseases in the United
States. Gastroenterology 2002; 122: 1500e1511.
5. Carey MC LaMont JT. Cholesterol gallstone formation. 1. Physical-chemistry of bile and biliary lipid
secretion. Prog Liver Dis 1992; 10: 136e163.
6. Small DM. Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci
U S A 2003; 100: 4e6.
7. Oude Elferink RP, Paulusma CC Groen AK. Hepatocanalicular transport defects: pathophysiologic
mechanisms of rare diseases. Gastroenterology 2006; 130: 908e925.
8. Paumgartner G Sauerbruch T. Gallstones: pathogenesis. Lancet 1991; 338: 1117e1121.
9. Admirand WH Small DM. The physicochemical basis of cholesterol gallstone formation in man. J Clin
Invest 1968; 47: 1045e1052.
10. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978; 19:
11. Vlahcevic ZR, Bell CC, Buhac I et al. Diminished bile acid pool size in patients with gallstones.
Gastroenterology 1970; 59: 165e173.
12. Thomas LA, Veysey MJ, Murphy GM et al. Octreotide induced prolongation of colonic transit increases
faecal anaerobic bacteria, bile acid metabolising enzymes, and serum deoxycholic acid in patients with
acromegaly. Gut 2005; 54: 630e635.
13. Berr F, Kullak-Ublick GA, Paumgartner G et al. 7alpha-hydroxylating bacteria enhance deoxycholic acid
input and cholesterol saturation of bile in patients with gallstones. Gastroenterology 1986; 111: 1611e1620.
14. Paigen B Carey MC. Gallstones. In King RA, Rotter JF Motulsky AG (eds.) The Genetic Basis of
Common Diseases. London: Oxford University Press, 2002, pp. 298e335.
*15. Moschetta A, Bookout AL Mangelsdorf DJ. Prevention of cholesterol gallstone diseases by FXR
agonists in a mouse model. Nat Med 2004; 10: 1352e1358.
16. Buhman KK, Accad M, Novak S et al. Resistance to diet-induced hypercholesterolemia and gallstone
formation in ACAT2-deﬁcient mice. Nat Med 2000; 6: 1341e1347.
*17. Lammert F, Carey MC Paigen B. Chromosomal organization of candidate genes involved in choles-
terol gallstone formation: a murine gallstone map. Gastroenterology 2001; 120: 221e238.
*18. Wang DQ, Schmitz F, Kopin AS et al. Targeted disruption of the murine cholecystokinin-1 receptor
promotes intestinal cholesterol absorption and susceptibility to cholesterol cholelithiasis. J Clin Invest
2004; 114: 521e528.
19. Holzbach RT Busch N. Nucleation and growth of cholesterol crystals. Kinetic determinants in super-
saturated native bile. Gastroenterol Clin North Am 1991; 20: 67e84.
20. Jirsa M Groen AK. Role of biliary proteins and non-protein factors in kinetics of cholesterol crystal-
lization and gallstone growth. Front Biosci 2001; 6: E154eE167.
21. Ko CW, Sekijima JH Lee SP. Biliary sludge. Ann Intern Med 1999; 130: 301e311.
22. Lee SP, Carey MC LaMont JT. Aspirin prevention of cholesterol gallstone formation in prairie dogs.
Science 1981; 211: 1429e1431.
23. Maurer KJ, Ihrig MM, Rodgers AB et al. Identiﬁcation of cholelithogenic enterohepatic Helicobacter
species and their role in murine cholesterol gallstone formation. Gastroenterology 2005; 128:
*24. Brink MA, Slors JF, Keulemans YC et al. Enterohepatic cycling of bilirubin: a putative mechanism for
pigment gallstone formation in ileal Crohn’s disease. Gastroenterology 1999; 116: 1420e1427.
25. Huang MH, Chen CH, Yen CM et al. Relation of hepatolithiasis to helminthic infestation. J Gastroenterol
Hepatol 2005; 20: 141e146.
26. Tham TC Kelly M. Association of periampullary duodenal diverticula with bile duct stones and with
technical success of endoscopic retrograde cholangiopancreatography. Endoscopy 2004; 36:
27. Kratzer W, Mason RA Kachele V. Prevalence of gallstones in sonographic surveys worldwide. J Clin
Ultrasound 1999; 27: 1e7.
Pathogenesis: a genetic perspective 1013
28. Tsai CJ, Leitzmann MF, Willett WC et al. Glycemic load, glycemic index, and carbohydrate intake in
relation to risk of cholecystectomy in women. Gastroenterology 2005; 129: 105e112.
29. Leitzmann MF, Rimm EB, Willett WC et al. Recreational physical activity and the risk of cholecystec-
tomy in women. N Engl J Med 1999; 341: 777e784.
30. Boland LL, Folsom AR Rosamond WD. Hyperinsulinemia, dyslipidemia, and obesity as risk factors for
hospitalized gallbladder disease. A prospective study. Ann Epidemiol 2002; 12: 131e140.
31. Tsai CJ, Leitzmann MF, Willett WC et al. Prospective study of abdominal adiposity and gallstone disease
in US men. Am J Clin Nutr 2004; 80: 38e44.
32. Kern F. Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with choles-
terol gallstones. J Clin Invest 1994; 93: 1186e1194.
33. Lammert F Wang DQ. New insights into the genetic regulation of intestinal cholesterol absorption.
Gastroenterology 2005; 129: 718e734.
34. Schwarz M, Russell DW, Dietschy JM et al. Alternate pathways of bile acid synthesis in the cholesterol
7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feed-
ing. J Lipid Res 2001; 42: 1594e1603.
35. Repa JJ, Lund EG, Horton JD et al. Disruption of the sterol 27-hydroxylase gene in mice results in he-
patomegaly and hypertriglyceridemia. Reversal by cholic acid feeding. J Biol Chem 2000; 275:
*36. Altmann SW, Davis HR, Zhu L et al. Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol
absorption. Science 2004; 303: 1201e1204.
37. Cohen JC, Pertsemlidis A, Fahmi S et al. Multiple rare variants in NPC1L1 associated with reduced ste-
rol absorption and plasma low-density lipoprotein levels. Proc Natl Acad Sci U S A 2006; 103:
38. Everhart JE, Yeh F, Lee ET et al. Prevalence of gallbladder disease in American Indian populations: ﬁnd-
ings from the Strong Heart Study. Hepatology 2002; 35: 1507e1512.
39. Sampliner RE, Bennett PH, Comess LJ et al. Gallbladder disease in Pima indians. Demonstration of high
prevalence and early onset by cholecystography. N Engl J Med 1970; 283: 1358e1364.
40. Carey MC Paigen B. Epidemiology of the American Indians’ burden and its likely genetic origins.
Hepatology 2002; 36: 781e791.
41. Everhart JE, Khare M, Hill M et al. Prevalence and ethnic differences in gallbladder disease in the Unites
States. Gastroenterology 1999; 117: 632e639.
*42. Miquel JF, Covarrubias C, Villaroel L et al. Genetic epidemiology of cholesterol cholelithiasis among
Chilean Hispanics, Amerindians, and Maoris. Gastroenterology 1998; 115: 937e946.
43. Galman C, Miquel JF, Perez RM et al. Bile acid synthesis is increased in Chilean Hispanics with gallstones
and in gallstone high-risk Mapuche Indians. Gastroenterology 2004; 126: 741e748.
44. Bergheim I, Harsch S, Muller O et al. Apical sodium bile acid transporter and ileal lipid binding protein
in gallstone carriers. J Lipid Res 2006; 47: 42e50.
45. Katsika D, Grjibovski A, Einarsson C et al. Genetic and environmental inﬂuences on symptomatic gall-
stone disease: a Swedish study of 43,141 twin pairs. Hepatology 2005; 41: 1138e1143.
*46. Kesaniemi YA, Koskenvuo M, Vuoristo M et al. Biliary lipid composition in monozygotic and dizygotic
pairs of twins. Gut 1989; 30: 1750e1756.
47. Duggirala R, Mitchell BD, Blangero J et al. Genetic determinants of variation in gallbladder disease in the
Mexican-American population. Genet Epidemiol 1999; 16: 191e204.
*48. Puppala S, Dodd GD, Fowler S et al. A genomewide search ﬁnds major susceptibility loci for gallbladder
disease on chromosome 1 in Mexican Americans. Am J Hum Genet 2006; 78: 377e392.
49. Nakeeb A, Comuzzie AG, Martin L et al. Gallstones: genetics versus environment. Ann Surg 2002; 235:
*50. Rosmorduc O, Hermelin B, Boelle PY et al. ABCB4 gene mutation-associated cholelithiasis in adults.
Gastroenterology 2003; 125: 452e459.
51. Venneman NG, Renooij W, Rehfeld JF et al. Small gallstones, preserved gallbladder motility, and fast
crystallization are associated with pancreatitis. Hepatology 2005; 41: 738e746.
52. Lammert F, Wang DQ, Hillebrandt S et al. Spontaneous cholecysto- and hepatolithiasis in Mdr2À/À
mice: a model for low phospholipid-associated cholelithiasis. Hepatology 2004; 39: 117e128.
53. Van Mil SW, van der Woerd WL, van der Brugge G et al. Benign recurrent intrahepatic cholestasis type
2 is caused by mutations in ABCB11. Gastroenterology 2004; 127: 379e384.
1014 F. Grunhage and F. Lammert
54. Pullinger CR, Eng C, Salen G et al. Human cholesterol 7a-hydroxylase (CYP7A1) deﬁciency has a hyper-
cholesterolemic phenotype. J Clin Invest 2002; 110: 109e117.
55. Jiang ZY, Han TQ, Suo GJ et al. Polymorphisms at cholesterol 7alpha-hydroxylase, apolipoproteins B
and E and low density lipoprotein receptor genes in patients with gallbladder stone disease. World J
Gastroenterol 2004; 10: 1508e1512.
56. Miyake JH, Duong-Polk XT, Taylor JM et al. Transgenic expression of cholesterol 7a-hydroxylase pre-
vents atherosclerosis in C57BL/6J mice. Arterioscler Thromb Vasc Biol 2002; 22: 121e126.
57. Lancellotti S, Zaffanello M, Di Leo E et al. Pediatric gallstone disease in familial hypobetalipoproteinemia.
J Hepatol 2005; 43: 188e191.
58. Han T, Jiang Z, Suo G et al. Apolipoprotein B-100 gene XbaI polymorphism and cholesterol gallstone
disease. Clin Genet 2000; 57: 304e308.
59. Juvonen T, Savolainen MJ, Kairaluoma MI et al. Polymorphisms at the apoB, apoA-I, and choles-
teryl ester transfer protein gene loci in patients with gallbladder disease. J Lipid Res 1995; 36:
60. Singh MK, Pandey UB, Ghoshal UC et al. Apolipoprotein B-100 XbaI gene polymorphism in gallbladder
cancer. Hum Genet 2004; 114: 280e283.
61. Miller LJ, Holicky EL, Ulrich CD et al. Abnormal processing of the human cholecystokinin receptor gene
in association with gallstones and obesity. Gastroenterology 1995; 109: 1375e1380.
62. Schneider H, Sanger P Hanisch E. In vitro effects of cholecystokinin fragments on human gallbladders.
Evidence for an altered CCK-receptor structure in a subgroup of patients with gallstones. J Hepatol
1997; 26: 1063e1068.
63. Nardone G, Ferber IA Miller LJ. The integrity of the cholecystokinin receptor gene in gallbladder
disease and obesity. Hepatology 1995; 22: 1751e1753.
64. Miyasaka K, Takata Y Funakoshi A. Association of cholecystokinin A receptor gene polymorphism
with cholelithiasis and the molecular mechanisms of this polymorphism. J Gastroenterol 2002; 37:
65. OMIM database. http://www.ncbi.nlm.nih.gov/entrez/.
66. Angelico M, Gandin C, Canuzzi P et al. Gallstones in cystic ﬁbrosis: a critical reappraisal. Hepatology
1991; 14: 768e775.
67. Wasmuth HE, Keppeler H, Herrmann U et al. Coinheritance of Gilbert syndrome-associated UGT1A1
mutation increases gallstone risk in cystic ﬁbrosis. Hepatology 2006; 43: 738e741.
68. Del Giudice EM, Perrotta S, Nobili B et al. Coinheritance of Gilbert syndrome increases the risk for
developing gallstones in patients with hereditary spherocytosis. Blood 1999; 94: 2259e2262.
69. Chaar V, Keclard L, Diara JP et al. Association of UGT1A1 polymorphism with prevalence and age at
onset of cholelithiasis in sickle cell anemia. Haematologica 2005; 90: 188e199.
70. Premawardhena A, Fisher CA, Fathiu F et al. Genetic determinants of jaundice and gallstones in hae-
moglobin E beta thalassaemia. Lancet 2001; 357: 1945e1946.
71. Wittenburg H, Lyons MA, Li R et al. FXR and ABCG5/ABCG8 as determinants of cholesterol gall-
stone formation from quantitative trait locus mapping in mice. Gastroenterology 2003; 125:
72. Pletcher MT, McClurg P, Batalov S et al. Use of a dense single nucleotide polymorphism map for in silico
mapping in the mouse. PLoS Biol 2004; 2: e393.
73. QTL Resources. http://pga.jax.org/qtl.
*74. Bertomeu A, Ros E, Zambon D et al. Apolipoprotein E polymorphism and gallstones. Gastroenterology
1996; 111: 1603e1610.
75. Niemi M, Kervinen K, Rantala A et al. The role of apolipoprotein E and glucose intolerance in gallstone
disease in middle aged subjects. Gut 1999; 44: 557e562.
76. Amigo L, Quinones V, Mardones P et al. Impaired biliary cholesterol secretion and decreased gallstone
formation in apolipoprotein E-deﬁcient mice fed a high-cholesterol diet. Gastroenterology 2000; 118:
77. Van Erpecum KJ, Portincasa P, Dohlu MH et al. Biliary pronucleating proteins and apolipoprotein E in
cholesterol and pigment stone patients. J Hepatol 2003; 39: 7e11.
78. Venneman NG, van Berge-Henegouwen GP, Portincasa P et al. Absence of apolipoprotein E4 genotype,
good gallbladder motility and presence of solitary stones delay rather than prevent gallstone recurrence
after extracorporeal shock wave lithotripsy. J Hepatol 2001; 35: 10e16.
Pathogenesis: a genetic perspective 1015
79. Lucena JF, Herrero JI, Quiroga J et al. A multidrug resistance 3 gene mutation causing cholelithiasis,
cholestasis of pregnancy, and adulthood biliary cirrhosis. Gastroenterology 2003; 124: 1037e1042.
80. Figge A, Lammert F, Paigen B et al. Hepatic overexpression of murine Abcb11 increases hepatobiliary
lipid secretion and reduces hepatic steatosis. J Biol Chem 2004; 279: 2790e2799.
81. Henkel A, Wei Z, Cohen DE et al. Mice overexpressing hepatic Abcb11 rapidly develop cholesterol
gallstones. Mamm Genome 2005; 16: 903e908.
82. Wang HH Wang DQ. Reduced susceptibility to cholesterol gallstone formation in mice that do not
produce apolipoprotein B48 in the intestine. Hepatology 2005; 42: 894e904.