Pathogenesis of gallstones: a genetic perspective


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Pathogenesis of gallstones: a genetic perspective

  1. 1. Best Practice & Research Clinical Gastroenterology Vol. 20, No. 6, pp. 997e1015, 2006 doi:10.1016/j.bpg.2006.05.007 available online at 2 Pathogenesis of gallstones: a genetic perspective Frank Grunhage ¨ Frank Lammert* MD, Dr med Professor 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 strategies. 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: (F. Lammert). 1521-6918/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.
  2. 2. 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
  3. 3. Pathogenesis: a genetic perspective 999 Synthesis CTP:phosphocholine HMG-CoA Cholesterol cytidylyltransferase reductase 7α-hydroxylase ABCB11 ABCB4 ABCG8 ABCG5 Secretion Phosphatidyl- Cholesterol (CH) Bile salts (BS) choline (PC) BS micelles PL-CH vesicles (60-80 nm) (~20 nm) Unilamellar Simple BS-CH PC-CH(-BS) Fusion micelles vesicles (2-3 nm) (40-100 nm) Bile Mixed BS-CH-PC Multilamellar micelles PC-CH(-BS) (4-6 nm) vesicles CH monohydrate (<500 nm) crystals Mucin gel Gallbladder Gallstone (CH) CSI 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.
  4. 4. 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 50,52,79 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 55,57,58,82 Apolipoprotein B APOB 107730 GC02M021135 þ þ RFLP e Hepatic (China) VLDL synthesis Y 74e78 Apolipoprotein E APOE 107741 GC19P050100 þ 34 þ Intestinal cholesterol absorption [, Chylomicron clearance [, Bile acid synthesis Y 18,61,62,64 Cholecystokinin-A CCKAR 118444 GC04M026159 þ þ Gallbladder receptor hypomotility 59 Cholesteryl CETP þ RFLP Gene HDL ester transfer (Finland) absent catabolism [ protein in mice 66,67 Cystic fibrosis CFTR Chloride 602421 GC07P116713 þ þ Bile pH Y, transmembrane (ABCC7) channel biliary regulator bilirubin secretion [, faecal bile salt excretion[
  5. 5. 54e56 Cytochrome CYP7A1 Cholesterol 118470 GC08M059565 þ þ (China) Promoter þ Bile acid P450 7A1 7a-hydroxylase rSNP synthesis Y (rate-limiting 204A > C À enzyme of bile acid synthesis) GBD, gallbladder disease 1; OMIM, Online Mendelian Inheritance in Men; RFLP, restriction fragment length polymorphism; SNP, single-nucleotide polymorphism. a b c 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 gallstone-susceptible. Pathogenesis: a genetic perspective 1001
  6. 6. 1002 F. Grunhage and F. Lammert ¨ Table 2. Classification of gallstones. Type Localization Prevalence Composition Colour Computed (%) tomography 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 (palmitate, stearate) þ cholesterol 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
  7. 7. Pathogenesis: a genetic perspective 1003 Table 3. Causes and risk factors for gallstone formation and potential mechanisms. Cholesterol gallbladder stones General factors Age Biliary cholesterol secretion [ Biliary bile salt secretion Y Gender Higher gallstone prevalence related to parity and oestrogens Pregnancy/oestrogens Hepatic cholesterol uptake and synthesis [ Cholesterol 7a-hydroxylase activity Y Biliary cholate/dexoycholate pool [ Gallbladder hypomotility 0 biliary sludge Nutritional/metabolic factors 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 Insulin resistance 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 [ Disease associations 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 absorption [ Gallbladder hypomotility Chronic haemolysis 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)
  8. 8. 1004 F. Grunhage and F. Lammert ¨ Table 3 (continued ). Anatomic abnormalities Biliary stricture Ascending cholangitis Duodenal diverticulum Ascending cholangitis Drugs 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
  9. 9. 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 RISK FACTORS 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
  10. 10. 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).
  11. 11. 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 Twin studies 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
  12. 12. 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 Monogenic cholelithiasis 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 mice.52 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- velop gallstones.53 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).
  13. 13. 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- mittent haemolysis.
  14. 14. 1010 F. Grunhage and F. Lammert ¨ Polygenic cholelithiasis 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 Resources website).73 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. SUMMARY 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
  15. 15. Pathogenesis: a genetic perspective 1011 Practice points 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 genetic analysis. 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). Research agenda3 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 characterized. Biomarkers in plasma or urine that indicate lithogenicity of bile should be identified. 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. ACKNOWLEDGEMENTS 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. REFERENCES 1. Lammert F Sauerbruch T. Mechanisms of disease: the genetic epidemiology of gallbladder stones. Nat Clin Pract Gastroenterol Hepatol 2005; 2: 423e433.
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