Genetics of gallstone disease.B Mittal, R Mittal
Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow - 226014, India. , India
Correspondence Address: Source of Support: None, Conflict of Interest: None PMID: 12215703
Source of Support: None, Conflict of Interest: None
Gallstone disease is a complex disorder where both environmental and genetic factors contribute towards susceptibility to the disease. Epidemiological and family studies suggest a strong genetic component in the causation of this disease. Several genetically derived phenotypes in the population are responsible for variations in lipoprotein types, which in turn affect the amount of cholesterol available in the gall bladder. The genetic polymorphisms in various genes for apo E, apo B, apo A1, LDL receptor, cholesteryl ester transfer and LDL receptor-associated protein have been implicated in gallstone formation. However, presently available information on genetic differences is not able to account for a large number of gallstone patients. The molecular studies in the animal models have not only confirmed the present paradigm of gallstone formation but also helped in identification of novel genes in humans, which might play an important role in pathogenesis of the disease. Precise understanding of such genes and their molecular mechanisms may provide the basis of new targets for rational drug designs and dietary interventions.
Keywords: Cholelithiasis, epidemiology,genetics,Cholesterol, genetics,Female, Genetic Predisposition to Disease, Human, Incidence, Male, Pedigree, Polymorphism (Genetics), Prognosis, Risk Factors,
Gallstone disease is a major health problem worldwide, particularly in adult population. Incidence of the gallstone disease shows considerable geographical and regional variations. In United States, approximately 10-15% of adults have gallstones. In Latin-American countries; the prevalence of gallstone disease is even higher (up to 50% in the adult women). Its occurrence has been found to be at least 6% in the adult population of North India. The risk of gallstone formation is associated with sex, obesity, age, family history and ethnic background of the individuals in the population., Gallstones are more common in women than men at all ages. The reason for considerable variation in gallstone disease in the population is complex because it involves the interaction of multiple genes with varied environmental factors. The present review will mainly highlight the genetic factors that may be involved in the formation of cholesterol gallstones.
Several evidences suggest that genetic predisposition plays a major role in the development of cholesterol gallstones. Ultrasound studies confirm the increased prevalence of gallstones in the first-degree family members of patients with gallstone disease., Very high incidence of gallstones has been reported in certain ethnic groups like Pima-Indians. Cholesterol saturation index of bile which is a prerequisite for gallstone formation, has been found to be similar only in monozygotic twins. However, the disease does not follow simple Mendelian pattern of inheritance, because no single gene or gene family is primarily involved. Instead, a number of different genes with diverse gene products contribute towards gallstone formation.
Morphologically and chemically, gallstones are of two main types, cholesterol and pigment stones. Cholesterol gallstones are more common (90% of all gallstones) and contain more than 50% cholesterol by weight. Mechanisms of cholesterol lithogenesis include biliary cholesterol hyper secretion, super saturation of bile with cholesterol, nucleation of crystals and formation of gallstones. All these processes are under genetic control and/ or influenced through intermediate pathogenic steps linked to a variety of environmental factors. Hyper- secretion of cholesterol in the bile is considered to be an important prerequisite for gallstone formation., 
First step in gallstone formation is initiation of cholesterol monohydrate crystals, which grow in size and agglomerate to form macroscopic stones in the gall bladder. Bile cholesterol is mostly derived from preformed cholesterol circulating in the plasma and bound to different apo-lipoproteins. Cholesterol transporting molecules like apo E and apo B control the availability of cholesterol for bile secretion. Dietary cholesterol is absorbed in the intestine and transported in chylomicron remnant as part of low and high-density lipoproteins. After hepatic uptake, chylomicron remnant cholesterol becomes a substrate for bile acid synthesis and is secreted into bile both as bile salt and un-esterified cholesterol.
Genetic variation in cholesterol metabolism can be brought about by different isoforms of lipid transport and receptor molecules in the general population. Apo E and apo B-100 are prominent transporters that exist in polymorphic states in humans. Presence of certain isoforms of apo E and apo B-100 help in better uptake and delivery of cholesterol. Apo E, a component of very low density and high-density lipoproteins mediate the binding of lipoprotein particles to LDL and chylomicron receptors, thereby mediating plasma response to dietary cholesterol. Apo E acts as a ligand between triglyceride rich lipoprotein particles, hepatic low-density lipoproteins and chylomicron remnant receptors.A single gene in the chromosomal region 19q13 codes for apo E. Each individual possesses two apo E genes in either homozygous or heterozygous state. The gene has three common genetic variants- E2, E3 and E4 in humans. Therefore, six genotypes (E2/E2, E3/E3, E4/E4, E2/E3. E2/E4, E3/E4) are possible in the population. In E2 allele, the protein product has amino acid cysteine at positions 112 and 158 of the protein chain. In E4, the cysteine at both positions is substituted by arginine. The E3 is a hybrid with cysteine at 112th and arginine at 158th positions in the protein. These changes in amino acids cause differences in their receptor-binding affinities and catabolic rates of the resulting protein isoforms.The variations finally influence the serum levels and rate of clearance of circulating lipoprotein particles. Individuals with E2/E2 alleles have 11-14 mg/dl lower concentrations of total serum cholesterol as well as LDL cholesterol when compared with E3/E3 alleles. Persons with E4 allele in homozygous (E4/E4) or heterozygous (E4/ E2 or E3) state have still higher levels of both LDL and total cholesterol as compared to E3/E3 alleles. In fact, presence of an E4 allele in an individual has been associated with various diseases like coronary heart and Alzheimer’s disease.,
The support for the involvement of apo E in cholesterol lithogenesis has come from various sources. Several reports suggest that patients undergoing cholecystectomy have significantly higher E4 allele frequency than gallstone-free subjects. After ESWL for treatment of gallstones, persons with apo E4 allele have significantly higher rate of recurrence over a 5-year-period. The biliary lipid and gallstone cholesterol contents tend to increase in the sequence E4> E3> E2 in patients undergoing cholecystectomy. The difference is more prominent in women as compared to men. In contrast; apo E2 allele has been shown to provide protection against gallstone disease, particularly in women.
Although there are consistent reports of association between apo E4 and stone formation, a few other studies have not found any allelic differences between gallstone patients and controls. The main reason for the discrepancy has been attributed to control individuals enrolled in such studies. Asymptomatic benign gallstones are usually present in a large number of individuals in the population. Therefore, there is a need to screen all controls by ultrasonography before taking blood samples. The overall difference between the presence of apo E4 allele in homozygous or heterozygous state, in stone-formers versus normal population, has been reported in the range of 15-25%. Therefore, apo E4 allele alone may not be responsible in a majority of the cases even among those with strong familial tendency for stone-formation. Besides apo E, polymorphisms in other genes like apo A1, apo B, and LDL receptor and cholesterol ester transfer protein involved in lipid metabolism have also been implicated in gallstone disease.
Apo B-100 serves as a ligand for receptor-mediated endocytosis of LDL. The gene is located on chromosome 2 and several polymorphic alleles of apo B-100 have been associated with disorders like coronary heart disease and non-insulin dependent diabetes mellitus. The Xba1 polymorphic allele (X?) of the apo B gene is characterised by a high cholesterol concentration and a higher LDL-cholesterol in serum. High frequency of X+ allele in apo B-100 genes has been reported in stone patients from China. Heterozygosity (X+/X-) of apo B-100 may be associated with gallstone disease and gallbladder cancer in India. In an earlier study from Finland, the frequency of X+ alleles of apo B-100 was reported to be higher in patients having a calculus or cholesterolosis with stones as compared to those with simple cholesterol gallstones. It appears that a relative contribution of apo B polymorphism in gallstone disease is rather limited to certain populations. Another lipoprotein, apo AI has been shown to act as an anti-nucleating agent in stone formation in bile. It also helps in removal of lipids from the bile and acts in preventing gallstone formation.
In recent years, cholesteryl ester transfer protein (CETP) has emerged as an important plasma component for HDL cholesterol metabolism. CETP facilitates the exchange of neutral lipids among plasma lipoproteins and induces a transfer of cholesteryl ester from HDL to triglyceride-rich lipoprotein in exchange for triglyceride.,  Humans with high activity of cholesteryl ester transfer protein (which account for all transfer of cholesteryl ester from HDL to VLDL and LDL particles in plasma) might have low HDL cholesterol as well as high VLDL-triglyceride levels, a pattern of plasma lipids that has been shown to cause increased risk of gallstones in case control studies.
Members of LDL-receptor gene family participate in diverse biological processes including lipoprotein metabolism. Receptor-associated protein (RAP) serves as a molecular chaperone within the endoplasmic reticulum to assist the folding of certain LDL receptor members of the family. Till date, at least 23 mutations and polymor-phisms in the LRPAP1 gene coding for RAP have been reported. There is a large deletion polymorphism in intron 5, which has been linked to the regulation of cholesterol metabolism in coronary artery disease. It remains to be investigated whether it has any association with gallstone disease or not.
The risk of atherosclerosis is inversely related to plasma levels of HDL cholesterol. Kozarsky et al observed the disappearance of plasma HDL and substantial increase in biliary cholesterol of hepatocytes over-expressing SR-B1, a well-defined HDL receptor.Whether low HDL is also a risk factor for gallstone formation, is yet to be determined.
In the above discussion, certain polymorphisms in apo E, apo- B and many other genes involved in lipid metabolism have been associated with increased risk of gallstone formation. Various proteins encoded by these genes may act by different mechanisms. Some of them may enhance the hepatic uptake of chylomicron remnants while others might cause increased absorption of intestinal cholesterol or decrease the synthesis of bile. All these alterations result in supersaturation of cholesterol in the bile that ultimately lead to gallstone formation. Although the individual risk associated with each allele may be small, it considerably increases in conjunction with other susceptibility genes and/or environ-mental factors.
The pathophysiology of gallstone disease in humans seems to be complex and more genes are likely to be discovered. Due to the limitations of genetic manipulations in humans, important conclusions about disease related genes are being derived from the animal models. However, methods for studying complex traits like gallstone disease differ from those used to discover genes for simple Mendelian defects. Using a genetic technique called quantitative trait analysis (QTL) with the help of variable DNA sequences (micro satellites), mice genes relevant to gallstone susceptibility have been mapped. The genome-wide search has resulted in identification of several candidate genes, which are involved in gallstone formation in mice.  In humans, QTL analysis of affected sib-pairs or families is restricted due to multiple modes of inheritance of the trait, incomplete penetrance, genetic heterogeneity and a large variation in environmental conditions. In mice, different in-bred strains are available which differ in their susceptibility to gallstone formation when kept on synthetic diet containing 1% cholesterol and 0.5% colic acid. The inter-strain differences in gallstone formation in mice can be utilised for QTL analysis. The method involves experimental crosses between inbred strains with different susceptibility and quantification of gallstones in second-generation progeny. These data are statistically correlated with microsatellite genetic markers spaced at 15 cM interval in the entire mice genome. Using combined genomic strategies and phenotypic studies in mice; several QTL regions for gallstone susceptibility have been identified. As expected, some of the candidate genes in the QTL belong to classic rate-limiting enzymes of cholesterol metabolism. Other prominent candidate genes responsible for cholesterol gallstones in mice (lith genes) identified so far have been shown to code for hepatic regulatory enzymes (Hmgcr, Cypa1, Soat2), cholecystokinin receptor (Cckar), HDL receptor (Srb1), apolipoproteins (ApoE), basolateral transporters for organic cation (Slc22a1) and canaliculus export pump for bile salts (Abcb11).,, Presently, many of their human orthologs (LITH genes) are being evaluated for their roles in gallstone formation in humans. Therefore, the genetic study of lith genes in mice models not only proves classic paradigms in gallstone disease (super-saturation of biliary cholesterol), but also paves the way for the identification of novel genes, which may also play crucial role in pathophysiology of gallstone disease. These developments are certainly going to accelerate the discovery of new targets for rational drug design and dietary interventions.
The authors acknowledge Dr. G. Choudhury, Department of Gastroenterology, SGPGIMS, Lucknow, for the valuable suggestions.