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)