Frank Lammert
Department of Internal Medicine IUniversity of Bonn, Germany
Pathogenesis ofGallstone Formation
Gallstones - a Common Disease
• Gallstone prevalence in Europe andUnited States: 10 - 20%Everhart et al. Gastroenterology (1999), Völzke et al. Digestion (2005)
• > 190,000 cholecystectomies per year in Germany Lammert et al. S3-Guidelines (2007)
• Occlusion or transection of thecommon bile duct 0.15%BQS Germany (2005)
• Costs (United States): $10 billion / yearMaurer et al. Gastroenterology (2007)
Gallstones: Classification
Schafmayer et al. BMC Gastroenterol (2006)
Brownpigmentstones
Calciumbilirubinate 10%
Infectedbile
ducts
Cholesterolstones
Blackpigment stones
Cholesterol
Bilirubinpolymers
> 90%Gallbladder
(and bile ducts)
2%Gallbladder
(and bile ducts)N = 1025
Cholesterol Gallstones: Molecular Mechanisms
Portincasa et al. Lancet (2006)
Gallbladderhypomotility
Biliary cholesterolhypersecretion
Slow intestinaltransit
Increased deoxycholate levels
CCK + FGF19 Control Gallbladder Motility
Choi et al. Nat Med (2006)
Fgf15-/- Mice
T-Cells are Critical for Gallstone Formation
Maurer et al. Gastroenterology (2007)
Rag2-/- MiceExperimental design:
1. Lithogenic diet(1% cholesterol + 0.5% cholic acid) for 8 wks
2. Adoptive transfer of immune cells1 wk before initiation oflithogenic diet
(IL-1β, IFN-γ, TNF-α)
< 1%1 - 5%5 - 7.5%7.5 - 10%10 - 15%> 15%
Figge et al. Z Gastroenterol (2002)
Gallstone Prevalence in Human Populations
Gallstones - a Complex Disease
< 65 ≥ 65 < 65 ≥ 65 yrs
Swedish Twin Registry
30
10
20
Con
cord
ance
rate
(%)
Monozygotic twins N = 11,073Dizygotic twins N = 18,183
Katsika et al. Hepatology (2005)
Structural Equation Modelling
Genetic factors= Lith (lithogenic)
genes (25%)
Commonfactors
(13%)
Environmentalfactors(62%)
Lammert & SauerbruchNat Clin Pract (2005)
Environmental Factors: Recent Studies
• High caloric / high carbohydrate dietLeitzmann et al. Gastroenterology (2005)
• Low fiber dietTsai et al. Am J Gastroenterol (2004)
• Physical inactivityLeitzmann et al. NEJM (1999)
• Metabolic syndrome / abdominal obesityMéndez-Sánchez et al. AJG (2005), Tsai et al. Gut (2006)
• Rapid weight lossShiffman et al. Ann Intern Med (1995)
• "Weight cycling"Tsai et al. Arch Intern Med (2006)
• Estrogen substitutionCirillo et al. JAMA (2005)
Lithogenic diet(1% cholesterol, 0.5% cholic acid, 15% fat)
Inbred mouse strains= homozygous for all alleles
Genetic Factors: Inbred Mouse Model
Susceptible
C57LC57BL/6
SWR
Resistant
AKR129A/J
⇒ Genetic factors (Lith genes) determine the formation of cholesterol gallstones in mice
Paigen et al. Physiol Genomics (2000)
Quantitative Trait Locus (QTL)-Analysis
Inbred Mouse Strains
×
×
Lithlocus(QTL)
F2 (N > 300)
F1 F1
Gallstone-susceptible
Gallstone-resistant
Lammert et al. Gastroenterology (2001)Wittenburg & Carey Dig Liver DIs (2003)
Genetic linkage analysis
Gallstones - + + + + -
Lith Loci: The Murine Gallstone Map 2006
Lyons & Wittenburg Gastroenterology (2006)
Phosphatidylcholine
Bile salts
Cholesterol
ABCB4
ABC = ATP Binding Cassette
Lith Genes: ABC Transporters
ABCB11
ABCG5/G8
Small PNAS (2004)
Cholesterol ABCG5/G8
Wittenburg et al. Gastroenterology (2003)
Exons
ABCG5 Gene ABCG8 Gene
ABCG8 - a Lith gene in Mice ...
Lith9
Lith9 Locus
Cholesterol ABCG5/G8
ABCG8 - a Lith gene in Mice and Humans
Acalovschi et al. Eur J Intern Med (2006)
Affected Sib Pair (ASP) Analysis (N = 178):
Strategy: If the disease is linked to a locus, affected sib pairs are more likely to carry the same allele than is to be expected by chance.
Non-parametric linkage (NPL)Score for ABCG8 D19H= 7.1 (⇔ P = 4.6 ×10-13)
Grünhage et al. Hepatology (2007)
Cholesterol ABCG5/G8
ABCG8 - a Lith gene in Mice and Humans
• Genome wide association (GWA) scan
• Screening panel: 280 cases and 360 controls
• 464,585 ("500 K") single nucleotide polymorphisms
• Association with ABCG8 D19H(P = 7.7 × 10-9)
• Replication panels: 1,832 German and 167 Chilean patients
Buch et al. Nat Genet (2007)
Strategy: If the disease is associated with a locus, cases are more likely to carry the same allele than controls.
Cholesterol ABCG5/G8
Wittenburg et al. Gastroenterology (2003)Grünhage et al. Hepatology (2007)
Buch et al. Nat Genet (2007)
Exons
ABCG5 Gene ABCG8 GeneChromosome 2p21
Risk variant D19H
ABCG8 - a Lith gene in Mice and Humans
• Genome wide quantitative trait locus (QTL) analysis in mice
• Genome wide association (GWA) scan in humans• Affected sib pair (ASP) analysis in humans• Odds ratio for D19H 2.2 - 3.0 • Population attributable fraction 8 - 11%
Abcb4 (Mdr2) Knockout Mouse
Phosphatidyl-choline ABCB4
Bile salts ABCB11
→ No mixed micelles→ Chronic cholangitis→ Transient cholesterol intermediates
Smit et al. Cell (1993)Lammert et al. Hepatology (2004)
Weihs, Konikoff et al. J Lipid Res (2005)
Cholelithiasis
100%
50
9 12 15 18 9 12 15 18Age (wks)
Cholesterol ABCG5/G8
Gallstones: From Bench to Bedside
Gallstones: Case #1
37 y o female
• Bile duct stones ("string of pearls") → ERC and sphincterotomy
• Intrahepatic cholestasis of pregnancy (ICP) at 27 and 30 weeks of gestation, lowγ-GT → premature deliveries
• Liver biopsy: intrahepatic cholestasis, mild portal hepatitis and fibrosis
Mühlenberg et al. Z Gastroenterol (2007)
Gallstones: Case #1
37 y o female
• Bile duct stones ("string of pearls") → ERC and sphincterotomy
• Intrahepatic cholestasis of pregnancy (ICP) at 27 and 30 weeks of gestation, lowγ-GT → premature deliveries
• Liver biopsy: intrahepatic cholestasis, mild portal hepatitis and fibrosis
Mühlenberg et al. Z Gastroenterol (2007)Ropponen et al. Hepatology (2006)
ICP + Gallstones
Phosphatidyl-choline ABCB4
Bile salts ABCB11
Cholesterol ABCG5/G8
Rosmorduc et al. Gastroenterology (2003)
• Mutations of the phosphatidylcholine transporter ABCB4
• Age at onset of symptoms< 40 yrs
• Cholesterol gallbladder stones and intrahepatic sludge or microlithiasis(OR 6.1)
• Recurrence of biliary symptoms after cholecystectomy (OR 8.5)
• Mild chronic cholestasis • Association with
intrahepatic cholestasis of pregnancy
c.957C>T(stop
codon)
Meier et al. Hepatology (2006)
c.1331T>C (V444A)
Gallstones: Case #1ABCB4 Deficiency
Phosphatidyl-choline ABCB4
Bile salts ABCB11
Cholesterol ABCG5/G8
Rosmorduc et al. Gastroenterology (2003)
• Mutations of the phosphatidylcholine transporter ABCB4
• Age at onset of symptoms< 40 yrs
• Cholesterol gallbladder stones and intrahepatic sludge or microlithiasis(OR 6.1)
• Recurrence of biliary symptoms after cholecystectomy (OR 8.5)
• Mild chronic cholestasis • Association with
intrahepatic cholestasis of pregnancy
c.957C>T(stop
codon)
Meier et al. Hepatology (2006)
c.1331T>C (V444A)
ABCB4 Deficiency
Lithogenic Gene Signature: ABCB4 + ABCB11
Gallstones: Case #2
23 y o female
• Cystic fibrosis (CF)
• Focal biliary fibrosis
• Gallstones
• CFTR (ABCC7) mutations class IV
- Gallstone prevalence in CF: 4 - 27%
- Black pigment stones
↓'Hyperbilirubinbilia'
↓Bilirubin precipitation
Liver
Unconjugatedbilirubin
↓Enterohepatic
circulation of bilirubin
↓Absorption of
unconjugated bilirubin in the colon
Gallbladder
Colon
IleumBile salts
Cystic fibrosis (CF)Crohn's diseaseLiver cirrhosis
↓Bile salt loss
Vitek & CareyEur J Clin Invest (2003)
Black Pigment Stones: Pathophysiology
Genotype Bilirubin (mg/dl)
(TA)6 (TA)6 0.35 ± 0.03(TA)6 (TA)7 0.54 ± 0.05*(TA)7 (TA)7 0.72 ± 0.08*
1A1 2 3 4 5
A(TA)6TAA wild-typeA(TA)7TAA Gilbert polymorphism
UDP glucuronosyltransferase UGT1A1
Promoter
TATA box
Exons
Lithogenic Gene Signature: CF + Gilbert
100%
20
40
80
60
CF+ stones
*
CF- stones
(TA)6 (TA)7 (TA)6 (TA)7
N = 52Wasmuth & Lammert Hepatology (2006)
Pathogenesis of Gallstone Formation
Geneticfactors
Environmentalfactors
modified from H. Witt
Polygenic (common)cholelithiasis
Monogeniccholelithiasis
ABCG8...
DietPhysical inactivity
Estrogens...
ABCB4ABCB11CFTR (ABCC7)...
Individual "lithogenic signatures"• Genetic factors: ABCG8, ABCB4, ABCB11, UGT1A1 ...
• Environmental factors: estrogens, enterohepatic bacteria ...
Normalrisk
Definedhigh risk groups
Speculation: Future Developments
Primary preventionwithout drugs
Weight reduction,sports, diet
modification
UDCA, nuclear receptor ligands, statins ?,
ezetimibe ?
Prevention with drugs (or prophylactic
cholecystectomy ?)
Friedrich August WalterAnatomisches Museum (1796)
Friedrich Theodor FrerichsKlinik der Leberkrankheiten (1861)